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C & E Publishing, Inc.

202
 C&E
Publishing, Inc.
C & E Publishing, Inc. was
established in 1993, and is a
member of ABAP, PBAI,
NBDB, and PEPA.

Microbiology and Parasitology:

A Textbook and Laboratory Manual for the Health Sciences
Second Edition
Published in 2020 by C & E Publishing, Inc.
839 EDSA, South Triangle, Quezon City
Tel. No.: (02) 8929 5088
E mail: [email protected]
Copyright © 2020 by C & E Publishing, Inc.,
Fe A. Bartolome, and Elizabeth P. Quiles

ALL RIGHTS RESERVED. No part of this publication may be reproduced,

stored in a retrieval system, or transmitted in any form or by any
mechanical, photocopying, recording, or otherwise—without means—electronic,

the prior written permission of the publisher.

Cataloguing in Publication Data

QR Bartolome, Fe A.
65 Microbiology and parasitology: a textbook and laboratory
.B37 manual for the health sciences/Fe A. Bartolome and Elizabeth
2020 P. Quiles.—2nd ed.—Quezon City: C & E Publishing, Inc., ©2020.
xi, 530 p. : ill. ; cm.
Includes bibliography and index.
eISBN: 978 971 98 1596 9
1. Microbiology—Handbooks,manuals, etc. 2.
manuals, etc. I. Quiles, Elizabeth P. II. Titles
Parasitology—Handbooks,

Book Design: Paul Andrew L. Pagunsan

Cover Design: Ruth Anne D. Ellorin
Table of Contents

Foreword vii
Preface ix
Acknowledgments xi
Part I Textbook
Chapter 1: The Science of Microbiology
1
3
Chapter 2: Prokaryotic and Eukaryotic Cells 19
Chapter 3: Bacterial Morphology 29
Chapter 4: Bacterial Growth Requirements 41
Chapter 5: Normal Flora of the Human Body 51
Chapter 6: Medical and Surgical Asepsis 61
Chapter 7: Physical and Chemical Methods of Sterilization 77
Chapter 8: Antimicrobial Agents 91
Chapter 9: Host Response to Infection 101
Chapter 10: Bacteria and Disease 133
Chapter 11: Introduction to Parasitology 151
Chapter 12: Protozoa 169
Chapter 13: Cestodes 207
Chapter 14: Trematodes 223
Chapter 15: Nematodes 239
Chapter 16: Infections of the Skin 269
Chapter 17: Infections of the Respiratory Tract 287
Chapter 18: Infections of Gastrointestinal Tract 313
Chapter 19: Sexually Transmitted Infections 345
Chapter 20: Infections of the Urinary Tract 36
Chapter 21: Infections of the Eyes 371
Chapter 22: Infections of the Nervous System 379
Chapter 23: Viral Exanthems 397
Chapter 24: Other Systemic Infections 411

Part II Laboratory Exercises 429

Exercise No. 1: The Microscope 433
Exercise No. 2: The Cell 437
Exercise No. 3: Gram staining 441
Exercise No. 4: Acid fast Staining 445
Exercise No. 5: Sterilization and Disinfection 449
Exercise No. 6: Bacterial Structures 455
Exercise No. 7: Antimicrobial Susceptibility Testing 459
Exercise No. 8: Bacteria and Disease 463
Exercise No. 9: Immunology 467
Exercise No. 10: Protozoans 469
Exercise No. 11: Cestodes 473
Exercise No. 12: Trematodes 477
Exercise No. 13: Nematodes 481
Exercise No. 14: Infections of the Skin 485
Exercise No. 15: Infections of the Respiratory Tract 489
Exercise No. 16: Infections of the Gastrointestinal Tract 493
Exercise No. 17: UTI and Sexually transmitted Infections 497
Exercise No. 18: Infections of the Eyes and Central Nervous System 501
Exercise No. 19: Viral Exanthems 505
Exercise No. 20: Other Systemic Infections 509

References 513

Index 519
The Author
Foreword

It is health that is real wealth,

not pieces ofgold and silver.
Mahatma Gandhi

It is in being healthy that an individual can truly feel being wealthy An individual who is
healthy can function maximally He or she can perform his or her work more efficiently than
one who is always ill A healthy individual is more productive than one who is not Attaining
good health is one of the important goals everyone must have in order to live life to the fullest
healthcare professionals have an inherent duty to provide health services to the public
All

it is also an implied duty of health care professionals to serve as health educators However,

the public is equipped with the basic knowledge about diseases, particularly their mode If

of transmission, people will know the steps to take to prevent them If people know how to
prevent the occurrence of disease, they increase their chances of containing them An individual
will have more gains when he or she does not lose hours at work because of illness Expenses
on doctors’ fees, hospitalization costs, and procurement of medicines will be minimized Money
saved can be channeled to other needs such as food, clothing, and education of one’s children
a country where majority of people are below the poverty line, the maintenance of health is a In

major concern
students of the health sciences, it is expected that you do your share in educating people
As

about the value of being healthy This book is intended to help the students in this effort
aims to increase the understanding of students regarding common infectious and parasitic It

diseases so that they will be better equipped with the necessary knowledge that would help
them promote health and health awareness among the public
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Preface

This book provides discussions on various topics related to microbiology and includes
self assessment questions as well as laboratory exercises It is designed to help students enrolled
in the allied health and other health related courses gain better understanding of microbiology
and parasitology The topics included are in line with the topics recommended by the
Commission on Higher Education (CHED)
I constitutes the textbook component and is divided into 24 chapters Chapters 1 to 5
Part

deal with the scope of microbiology, including its evolution and branches Also included are
the principles of microscopy as well as the principles in staining and culturing of organisms
growth requirements of bacteria and the concept of normal or indigenous flora are also The

discussed
6 to 8 deal with a discussion of the concepts involved in microbial control,
Chapters

which includes medical and surgical asepsis, physical and chemical methods of sterilization,
and antibiotics The very important issue of drug resistance is also discussed
9 and 10 are concerned with infection and the body’s responses to infection
Chapters

regarding bacteria and how they produce disease are discussed Definitions of the Concepts

various types of infection are given and events occurring in the different stages of an infectious
disease process are explained These chapters also include the body’s defense mechanisms
against infectious agents, our immune response to these agents as well as hypersensitivity
reactions
11 to 15 are devoted to the discussion of parasitic diseases that affect humans
Chapters

classification of medically important parasites is presented The characteristics of these The

classes of parasites are discussed including their sources, mode of transmission, specific diseases
they produce, treatment and prevention
chapters 16 to 24 are concerned with the different infectious diseases that affect the
Finally,

different organ systems of the body, from the skin to the central nervous system It covers areas
such as characteristics of the etiologic agents, modes of transmission, clinical manifestations,
diagnosis, treatment and prevention Each chapter ends with a set of self assessment questions
designed to assess the student’s understanding of the different concepts discussed
II consists of laboratory exercises that are designed to reinforce the understanding of
Part

the students of the specific concepts discussed It consists of 20 exercises, the last few of which
involve case scenarios that aim to develop the students’ analytical thinking
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Acknowledgments

We, the authors, wish to thank all those who have given us support and encouragement in
making this material a reality We would like to thank God almighty for giving us the gift of
words, the Administration of Our Lady of Fatima University for allowing us to write this book
and supporting us throughout this journey, the late Dean Lurceli Santos for encouraging us to
pursue this endeavor, Dr Anthony Nicanor for believing in us and introducing us to the world
of writing, our friends and co faculty for being there for us and encouraging us to finish this
project, and our families for being behind us all the way, for putting up with us, and for just
being there for us You are our constant source of love, strength, and inspiration
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 P art
TEXTBOOK
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 CHAPTER
The Science
1 of Microbiology

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define Microbiology and give the importance of the study of Microbiology;
2. name important persons with significant contributions to the field of Microbiology;
3. differentiate the various types of microscopes and their uses;
4. compare the various staining methods used to visualize microorganisms; and
5. classify the different types of culture media based on their physical state, chemical
composition, and functional type.

Microbiology is derived from the Greek words mikros (“small”), bios (“life”), and logia or
logos (“study of”). It is therefore the study of organisms that are so small they cannot be seen
with the naked eye. These organisms are called microorganisms or microbes and are categorized
into two: (1) cellular, which may either be prokaryotes (bacteria, cyanobacteria, and archeans) or
eukaryotes (fungi, protozoa, and algae); and (2) acellular, which includes viruses. Microbiology
is further classified into different fields of study, namely: (1) bacteriology, the study of bacteria;
(2) virology, the study of viruses; (3) mycology, the study of fungi; (4) parasitology, the study
of protozoa and parasitic worms; (5) phycology, the study of algae; and, (6) immunology,
the study of the immune system and the immune response.
Why study microbiology? The study of microbiology is important for the following reasons:
1. Microbiology has an impact in the daily lives of humans. Microorganisms are
everywhere—in the air one breathes, in the environment, and even in one’s body.
About a thousand or more organisms inhabit the human body. These are collectively
called normal flora or indigenous flora which only produce disease in persons with
compromised immune systems
4 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. Some microorganisms are essential in biotechnologyand a wide range of industries

which include food and beverage, pharmaceuticals, mining, genetics, and many more.
Much of the knowledge available in the study of genetics and biochemistry utilize
microorganisms as model organisms.
3. Some microorganisms, especially bacteria and fungi, are important sources of
antimicrobial agents. For example, penicillin was derived from the fungus Penicillium.
4. Some microorganisms act as saprophytes or decomposers of waste products and dead
organisms, making them essential in maintaining a balanced ecosystem.
5. The study of microorganisms has led to a better understanding of how
microorganisms produce disease, paving the way to better disease management and
control. This was further improved through the discovery of vaccines that helped
prevent sickness from infections diseases. By knowing the sources of disease producing
microbes, sanitation practices improved immensely, leading to better mitigation of
infectious diseases.
6. Certain diseases which were thought to have been eradicated are now re emerging.
Some have the potential as biological warfare agents. At the same time, there are now
a number of pathogens that are developing resistance to antibiotics. In this context,
the study of microbiology is relevant for better understanding of the negative instances
in which science can be used.

Evolution of Microbiology
Archaeologists and evolutionists have uncovered evidence demonstrating the existence of
primitive microorganisms. In Western Australia, as many as eleven different types of fossils of
primitive microorganisms have been found in ancient rock formations, dating back to as early as
3.5 billion years ago, long before the existence of animals and humans.
Infectious diseases have existed for thousands of years. In 3180 BC, an epidemic known
as the “plague” broke out in Egypt. In 1122 BC, an outbreak of a smallpox like disease that
originated in China spread worldwide. The exhumed mummified remains of Rameses V
showed skin lesions resembling smallpox.
In the mid 1600s, the microscope was discovered and with the use of this instrument,
Robert Hooke was able to discover the cell—the basic unit of living organisms. His discovery
heralded the cell theory that stated living organisms are made up of cells. Then in the 1670s,
Anton von Leeuwenhoek, a Dutch merchant, created a single lens microscope that he used
to make observations of microorganisms which he then called animalcules. Through his
observations, he became known as the “Father of Microbiology” and was the one who first
provided accurate descriptions of bacteria, protozoa, and fungi
 The Science of Microbiology

In the middle and late 1800s, Louis Pasteur performed countless experiments that led
to his germ theory of disease. He postulated that microorganisms were in the environment and
could cause infectious diseases. He also developed the process of pasteurization, which kills
microorganisms in different types of liquids, and which became the basis for aseptic techniques.
He also introduced the terms aerobes and anaerobes and developed the fermentation process.
Pasteur’s attempts to prove his germ theory of disease were unsuccessful. It took Robert
Koch to prove that microorganisms caused certain diseases through a series of scientific steps
which led to his formulation of the Koch’s postulates. This led to an increased effort by other
scientists to prove and illustrate further the germ theory that was initially formulated by
Louis Pasteur. Thus, the late 1800s and the first decade of the 1900s came to be known as the
Golden Age ofMicrobiology. Since then, numerous scientists have made significant contributions
to the field of Microbiology. Edward Jenner discovered the vaccine for smallpox. Joseph Lister
applied the theory to medical procedures paving the way for the development of aseptic surgery.
After World War II, antibiotics were introduced to the medical world. Paul Ehrlich
discovered Salvarsan for the treatment of syphilis. This drug was heralded the “magic bullet” of
chemotherapy, which is treatment of disease by using chemical substances. Alexander Fleming
discovered the antibiotic penicillin from the mold Penicillium notatum. With the discovery of
antibiotics, the incidence of infectious diseases like tuberculosis, pneumonia, meningitis, and
others was significantly reduced.
Most of the experiments conducted in the field of microbiology during the early 20th
century involved the study of bacteria. During this time scientists were not yet equipped
with advanced technology in their study of microorganisms. It was only in the 1930s when
the electron microscope was developed that experimentations in microbiology became more
complex. It was also during that time when viral culture was introduced paving the way for
rapid discoveries on viruses. The vast knowledge gained from the experiments performed by
microbiologists together with the discovery of other vaccines in the 1940s and 1950s have led to
better prevention and control of numerous potentially fatal infectious diseases.

Microscopy
Microorganisms are miniscule organisms that cannot be seen with the naked eye. The
discovery of the microscope has led to their close observation, allowing microbiologists and
other scientists to study them further.
A microscope is an optical instrument that can magnify organisms a hundredfold or even a
thousand fold. From the time of its initial discovery in the 1600s, the microscope has undergone
great revolutionary changes. Making it more advanced and complex throughout time. The
following are the different types of microscopes that have evolved from von Leeuwenhoek’s
simple prototype.
6 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Compound Microscope
The compound microscope is a type of microscope that contains more than one magnifying
lens. It can magnify objects approximately a thousand times their original size. Visible light is
its main source of illumination. As such, it is also known as the compound light microscope.
The compound microscope utilized today consists of two magnifying lens systems.
The eyepiece (or ocular) contains what is called the ocular lens that has a magnifying power
of 10x. The second lens system is located in the objective that is positioned directly above the
organism to be viewed.

Eyepiece (ocular lens)

Head
Diopter adjustment
Locking screw
Revolving nose piece
Arm
Objectives
Stage
Slide holder Coarse focus
Condenser
Fine focus
Iris diaphragm
Stage controls
Built in light source
Brightness adjustment
On/off switch Base
With built in light source

Eyepiece (ocular lens)

Body tube
Coarse focus
Fine focus
Revolving nose piece
Arm
Objectives
Stage clips
Stage
Condenser
Iris diaphragm

Mirror Base

With mirror to direct an external light source

Figure 1.1 Two compound light microscopes which differ in their light sourc
 The Science of Microbiology 7

Table 1.1 Components of the compound light microscope

Component Function
Ocular lens or Topmost part of the microscope which is the lens the viewer looks
eyepiece through to see the specimen.
Revolving nose piece Located above the stage, it holds the objective lenses.
Diopter adjustment It is used to change focus on one eyepiece in order to correct
any difference in vision between the two eyes.
Body tube or head It connects the eyepiece to the objective lenses.
Arm It connects the body tube to the base of the microscope.
Coarse adjustment It brings the specimen into general focus.
Fine adjustment It fine tunes the focus and increases the details of the specimen.
Objective lenses This is held in place above the stage by the revolving nosepiece and are
the lenses that are closest to the specimen. It contains 3 to 5 objectives
ranging in power from 4X to 100X.
Stage Located beneath the revolving nosepiece, it is the flat platform
on which the specimen is placed.
Stage clips Situated above the stage, these are metal clips that hold the slide
in place.
Stage control Found beneath the stage, these knobs move the stage either left
or right or forward and backward.
Aperture The hole in the middle of the stage that allows light from the illuminator
to reach the slide containing the specimen.
On/off switch The switch located at the base of the microscope that turns
the illuminator on or off.
Illuminator The light source of the microscope.
Iris diaphragm Found on the condenser, it is used to adjust the amount of light coming
through the condenser.
Condenser It is found beneath the stage and contains a lens system that focuses
light onto the specimen. It gathers and focuses light onto the specimen.
Base It supports the microscope and it is where the illuminator is found.

Brightfield Microscope
Made up of a series of lenses and utilizing visible light as its source of illumination, the
brightfield microscope can magnify an object 1,000 to 1,500 times. This is used to visualize
bacteria and fungi. Objects less than or thinner than 0.2 μm cannot be visualized by this type
of microscope. The term “brightfield” is derived from the fact that the specimen appears dark
against the surrounding bright viewer field of this microscope. However, it has very low contrast
and most of the cells need to be stained to be properly viewed
8 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Darkfield Microscope
This microscope utilizes reflected light instead of transmitted light, with a special
condenser that has an opaque disc that blocks the light, such that only the specimen is
illuminated. The specimen to be studied appears bright against a dark background. This type of
microscope is ideal for studying specimens that are unstained or transparent and absorb little or
no light. It is also useful in examining the external details of the specimen such as its outline or
surface. This type of microscope is used to view spirochetes.

Phase contrast Microscope

Phase contrast microscopy is based on the principle that differences in refractive
indices and light waves passing through transparent objects assume different phases.
This type of microscopy was first introduced by Frits Zernike, a Dutch physicist, in 1934.
The phase contrast microscope has a contrast enhancing optical technique in order to produce
high contrast images of specimens that are transparent which include thin tissue slices, living
cells in culture, and subcellular particles (such as nuclei and organelles).

Image Plane Digital

Camera
Diffracted System
Direct Light
Transmitted
(Surround) Observation Light
Light Biological
Microscope

Objective

Specimen
Phase
Plate
Condenser

Condenser
Annulus

Figure 1.2 Phase contrast microscope and its part

 The Science of Microbiology 9

Living Cells in Brightfield and Phase Contrast

Figure 1.3 a Appearance
of cells under brightfield
microscope where cells appear
semi transparent. The only
visible structures are the highly
refractive regions, such as
the membrane, nucleus, and
unattached cells (rounded or
spherical). b Same specimen
viewed using phase contrast
a b microscope showing significantly
more structural detail.

Differential Interference Contrast Microscope

The differential interference contrast microscope is similar to the phase contrast
microscope except that it utilizes two beams of light instead of one and therefore has higher
resolution. The resulting contrasting colors of the specimen being studied are due to the
prisms that split the light beam. It was developed by Georges Nomarski in 1952 as an
improvement to the phase contrast microscope. It is useful in examining living specimens
when normal biological processes might be inhibited by standard staining procedures. However,
the three dimensional image of the specimen produced may not be accurate since the enhanced
areas of light and shadow may distort the appearance of the image.

Fluorescence Microscope
The fluorescence microscope makes use of ultraviolet light and fluorescent dyes called
fluorochromes. The specimen under study fluoresces or appears to shine against a dark
background. Fluorescence microscopy is based on the principle that certain materials emit
energy that is detectable as visible light when they are irradiated with the light of a given
wavelength. It uses a higher intensity of light source and this in turn excites a fluorescent
species. The fluorescent species then emits a lower energy light of a longer wavelength which
produces the magnified image instead of the original light source. Fluorescence microscopy
can be used to visualize structural components of small specimens such as cells and to detect
the viability of cell populations. It may also be used to visualize the genetic material of the
cell (DNA and RNA)
10 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Confocal Microscope
Also known as the confocal laser scanning microscope (CLSM) or laser confocal scanning
microscope (LCSM), the confocal microscope uses an optical imaging technique that
increases optical resolution and contrast of the micrograph by using a spatial pin hole to
block out of focus light in image formation. The specimen is stained with a fluorescent dye
to make it emit or return light. The object is scanned with a laser into planes and regions. This
is used, together with computers, to produce a three dimensional image. It is also useful in the
study of cell physiology.

Electron Microscope
The electron microscope utilizes a beam of electrons to create an image of the specimen.
The electron beams serve as the source of illumination and magnets are used to focus the beam.
The first prototype of this microscope was built by the German Engineer Ernst Ruska in 1933,
which had a resolution power of up to 50 nm. Modern electron microscopes are capable of
magnifying objects up to 2 million times. It is used to visualize viruses and subcellular structures
of the cell. There are two types of electron microscopes—transmissionelectron microscope and
scanning electron microscope. The transmission electron microscope (TEM) is the original form of
the electron microscope. It produces two dimensional, black and white images, and magnifies
objects up to 200,000 times. The scanning electron microscope (SEM) relies on interactions at the
surface rather than transmission. It can magnify bulk samples with greater depth of view so that
the image produced represents the 3 D structure of the sample, but the image is still only black
and white. Generally, it can magnify the object 10,000 times.

Scanning Probe Microscope

The scanning probe microscope was developed in the 1980s by the Swiss scientists
Dr. Gerd Binnig and Dr. Heinrich Rohrer. It is used to study the molecular and atomic shapes
of organisms on a nanoscale. A physical probe is used to scan back and forth over the surface
of a sample. A computer then gathers data that are used to generate an image of the surface.
It can also be used to determine the variations in temperature inside the cell as well as its
chemical properties.

Staining
Most microorganisms besides being very tiny are also devoid of any color and are thus
difficult to see, even with the use of the microscope. To facilitate visualization, staining
procedures have been developed by various scientists. These staining procedures are meant to
give color to the organisms, making them easier to see under the microscope
 The Science of Microbiology 11

Simple Stains
Simple stains make use of a single dye which can either be aqueous (water based) or
alcohol based. This method of staining is a quick and easy way to visualize cell shape, size,
and arrangement of bacteria. It uses basic dyes such as safranin, methylene blue, or crystal violet.
These stains give up or accept hydrogen ion, leaving the stain positively charged. Most bacterial
cells and cytoplasm are negatively charged and since the dye is positively charged, it adheres
readily to the cell surface enabling the visualization of bacterial cell morphology.

a b

Figure 1.4 a Cocci in clusters and b Bacilli

Differential Stains
Differential stains are used to differentiate one group of bacteria from another. There are
two types of differential staining procedures commonly used, namely:
1. Gram stain – distinguishes gram positive bacteria from gram negative bacteria.
gram positive bacteria stain blue or purple, while gram negative bacteria stain red
or pink. As a general rule, all cocci are gram positive except Neisseria, Veilonella, and
Branhamella. On the other hand, all bacilli are gram negative except Corynebacterium,
Clostridium, Bacillus, and Mycobacterium.

Table 1.2 Reagents used in Gram staining and expected results

Reagent Function Result if Gram positive Result if Gram negative
Crystal violet Primary stain
Purple or blue Purple or blue
Gram’s iodine Mordant* Purple or blue Purple or blue
Acetone or 95% Decolorizer Purple or blue Colorless
alcohol
Safranin Counterstain or Purple or blue Red or pin
secondary stain
*A mordant enhances the uptake of the primary stain.
12 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. Acid fast stain – stain used for bacteria with high lipid content in their cell wall,
hence cannot be stained using Gram stain. Two methods are used, namely:
a. Ziehl Neelsen stain – also known as the “hot method” because it requires steam
bathing the prepared smear after addition of the primary dye. This is because the
primary stain used is aqueous and will not bind to the cell wall of the organism.
Acid fast organisms will appear red on a blue background.
b. Kinyoun stain – also known as the “cold method” as it does not utilize
heat after addition of the primary stain, which is oil based. The acid fast
organisms will appear red on a green background.

Table 1.3 Reagents used in acid fast staining and the expected results
Reagent Result
Function
Ziehl Neelsen Kinyoun Acid fast Non acid fast
Carbol fuchsin Carbol fuchsin Primary stain Red or pink Red or pink
Acid alcohol Acid alcohol Decolorizer Red Colorless
Methylene Malachite Counterstain Ziehl Neelsen: Ziehl Neelsen:
blue green or secondary red organism/ blue organism/
stain blue background blue background
Kinyoun: red organism/ Kinyoun: green organism/
green background green background

Special Stains
These are used to demonstrate specific structures in a bacterial cell. For instance,
metachromatic granules can be visualized using the LAMB (Loeffler Alkaline Methylene Blue)
stain. Other special stains include Hiss stain (capsule or slime layer); Dyer stain (cell wall),
Fischer Conn stain (flagella), Dorner and Schaeffer Fulton stain (spores), and India ink
or nigrosine (capsule of the fungus Cryptococcus neoformans).

Capsule Staining
Capsules
Background Rods Flagella

a b

Figure 1.5 a Demonstration of the capsule using India ink and b flagella surrounding the
bacteria demonstrated using the Leifson method of stainin
 The Science of Microbiology 1

Culture Media
Staining procedures only give clues as to the probable organism being studied. To identify
a specific organism, culture using specific culture media is the most ideal. Media (sing. medium)
are used to grow microorganisms. A culture medium is basically an aqueous solution to which
all the necessary nutrients essential for the growth of organisms are added. These are classified
into three primary levels: physical state, chemical composition, and functional type.

According to Physical State

1. Liquid media – commonly called broths, milk, or infusions, these are water based solutions
that do not solidify at temperatures above the freezing point. These contain specific
amounts of nutrients but do not contain gelling agents such as gelatin or agar. Liquid
media are suited for the propagation of a large number of organisms, fermentation
studies, and other tests.
2. Semi solid media – exhibit a clot like consistency at ordinary room temperature and
contain agar at concentrations of 0.5% or less that allows thickening of the media without
producing a firm substance. They have a soft consistency similar to custard and are best
suited for culture of microaerophilic bacteria or for the study of bacterial motility.
3. Solid media – contain a solidifying agent such as 1.5%–2% agar, giving them a
firm surface on which cells can form discrete colonies. They are used for isolation
of bacteria and fungi or for determining the colony characteristics of the organism
under study. Solid media come in two forms: (a) liquefiable (or reversible) solid
media and (b) non liquefiable (or non reversible) solid media.

According to Chemical Composition

1. Synthetic media – contain chemically defined substances which are pure organic and/or
inorganic compounds. The precise chemical composition of a synthetic medium is known.
They may be simple or complex, depending on what supplement is added to it.
2. Non synthetic media – complex media that contain at least one ingredient that is not
chemically defined, which means that it is neither a simple or pure compound. It is not
representable by an exact chemical formula. Most are extracts of animals, plants, or yeasts.
Non synthetic media can support the growth of more fastidious organisms.
14 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

According to Functional Type

1. General Purpose media – are designed for primary isolation of a broad spectrum
of microbes and contain a mixture of nutrients that support the growth of both
pathogenic and non pathogenic organisms. Examples are peptone water, nutrient broth,
and nutrient agar.
2. Enrichment media – contain complex organic substances such as blood, serum, or special
growth factors, and are designed to increase the number of desired microorganisms
without stimulating the rest of the bacterial population. These are used to grow
fastidious or nutritionally exacting bacteria. There are two commonly used enrichment
media, namely:
a. Blood agar – contains general nutrients with 5%–10% (by volume) blood added
to a blood agar base. Certain gram positive bacteria produce exotoxins that cause
hemolysis of red blood cells contained in the blood agar. Their hemolytic reaction
is categorized into three, which is useful in the classification of these bacteria.
The hemolytic patterns are:
i. Beta hemolysis – shows complete lysis of red blood cells resulting in complete
clearing around the colonies.
ii. Alpha hemolysis – shows incomplete lysis of red blood cells, producing a
greenish discoloration of the blood agar around the colonies.
iii. Gamma hemolysis – shows no hemolysis, resulting in no change in the
medium.

a b c

Figure 1.6 Three types of hemolytic reactions seen in the culture: a beta hemolysis or complete
hemolysis; b alpha hemolysis or incomplete hemolysis; and c gamma hemolysis or no hemolysis

b. Chocolate agar – a type of nutrient medium that is used for the culture of fastidious
organisms such as Haemophilus sp. Heat is applied to lyse the red blood cells, causing
the medium to turn brown
 The Science of Microbiology 1

3. Selective media – contain one or more substances that encourage the growth of only a
specific target microorganism and inhibit the growth of others. It is designed to prevent
the growth of unwanted contaminating bacteria or commensals so only the target bacteria
will grow. Examples of approaches that will make the medium selective include changing
the pH of the culture medium or adding substances such as antibiotics, dyes, or other
chemicals. These are usually agar based solid media that allow isolation of individual
bacterial colonies. Examples of this type of culture medium include the following:
a. Thayer Martin agar – contains the antibiotics trimethroprim, nystatin, vancomycin,
and colistin. It is used for the isolation of Neisseria.
b. Mannitol Salt agar – contains 10% NaCl and used for the isolation of Staphylococcus
aureus.

c. MacConkey’s agar – promotes the growth of gram negative bacteria, primarily

those belonging to the family Enterobacteriaceae, and inhibits the growth of gram
positive bacteria through the addition of bile salts. It is both selective and differential.
d. Löwenstein Jensen medium – a selective medium used to recover Mycobacterium
tuberculosis. It is made selective by the incorporation of malachite green.

e. Saboraud’s dextrose agar – used for the isolation of fungi.

4. Differential media – allow the growth of several types of microorganisms. These are
designed to show visible differences among certain groups of microorganisms. The
differences may be in the form of variations in colony size or color, changes in color of
culture media, or formation of precipitates or gas bubbles. Differential media allow the
growth of more than one target microorganism that demonstrate morphologic variations
in colony morphology. Examples include MacConkey’s agar and Triple Sugar Iron agar.
5. Transport media – used for clinical specimens that need to be transported to the
laboratory immediately after collection. These media prevent the drying of specimen
and inhibit the overgrowth of commensals and contaminating organisms. Charcoal
is added to neutralize inhibitory factors. Examples are the Cary Blair transport medium
for transport of feces of suspected cholera patients and Pike’s medium which is used to
transport throat specimens of patients with streptococcal infection.
6. Anaerobic media – media used specifically for organisms that cannot survive in the
presence of oxygen and require reduced oxidation reduction potential and other nutrients.
These are supplemented with nutrients such as vitamin K and hemin. They undergo
boiling to remove dissolved oxygen. To reduce the oxidation reduction potential,
substances such as 1% glucose, 0.1% ascorbic acid, 0.1% thioglycolate, or 0.05% cysteine
are added. Methylene blue or resazurin is added as an indicator of the oxidation
reduction potential. Examples are chopped cooked meat and thioglycolate broth.
16 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• Microbiology is the study of small, living microorganisms or microbes that cannot be seen
with the naked eye. These organisms may be cellular (prokaryotes, eukaryotes, and the
like) or acellular such as viruses.

• Microbiology is divided into several fields that deal with the study of bacteria
(bacteriology), viruses (virology), fungi (mycology), protozoa and parasitic worms
(parasitology), algae (phycology), and the immune system (immunology).

• Microorganisms may be beneficial or harmful. Some microorganisms are used in

different industries such as in food and beverage. Some microorganisms are sources of
antibiotics while some are used in the field of biotechnology and genetic engineering.
Microorganisms are also important in maintaining a balanced ecosystem.

• While some microorganisms are essential and have beneficial uses, there are also
numerous microorganisms that produce disease in humans, some of which are
potentially fatal.

• Some microorganisms have the potential to be used as biological warfare agents.

• Microorganisms are so miniscule that for them to be visualized, they need to be
stained and studied using the microscope. Several types of microscopes have been
developed for this purpose—from the compound microscope to the more sophisticated
electron microscopes.

• The use of various staining procedures has made visualization of microorganisms easier.
These stains may be classified into simple, differential, and special stains.
› Simple stains make use of a single water or alcohol based dye that is used to
demonstrate the shape and basic structures of the organism.
› Differential stains are used to distinguish one group of bacteria from another group.
These include the Gram stain and the acid fast stain.
› Special stains are mainly used to demonstrate specific bacterial structures such as the
spores (Dorner or Schaeffer Fulton), flagella (Fischer & Conn), capsule (Hiss
stain),
or the metachromatic granules (LAMB stain).

• Specific culture media are the most ideal in identifying specific organisms. Several
classes of culture media have been developed and these culture media can be classified
into three primary levels: physical state (liquid, semi solid, solid), chemical composition
(synthetic and non synthetic), and functional type (general purpose, enrichment, selective,
differential, transport, and anaerobic)
 The Science of Microbiology 17

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. Which among the following groups of organisms are not considered cells?
a. Bacteria c. Viruses
b. Fungi d. Algae
2. Which among the following types of microscopes is used together with computers
to produce a three dimensional image and is also useful in the study of
cell physiology?
a. Phase contrast microscope c. Fluorescent microscope
b. Scanned probe microscope d. Confocal microscope
3. Which among the following parts of the microscope is used to gather and focus
light onto the specimen?
a. Coarse adjustment c. Eye piece
b. Fine adjustment d. Condenser
4. Who among the following scientists made the initial postulates regarding the germ
theory of disease?
a. Louis Pasteur c. Edward Jenner
b. Alexander Fleming d. Robert Koch
5. You discovered a new organism and you want to study its molecular and atomic
properties. Which among the following types of microscopes would be suited for
this purpose?
a. Electron microscope c. Scanned probe microscope
b. Fluorescent microscope d. Confocal microscope
18 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. You are given a new slide to study in the laboratory. Which part of the microscope
will you use to put the specimen into general focus?

a. Iris diaphragm c. Objective lenses

b. Coarse adjustment d. Fine adjustment
7. Which among the following classes of culture media is used to isolate fungi?
a. Thayer Martin agar c. Saboraud dextrose agar
b. Löwenstein Jensen agar d. Chocolate agar
8. Which among the following reagents used in Gram staining will enhance the
uptake of the primary stain?
a. Crystal violet c. 95% alcohol
b. Gram’s iodine d. Safranin
9. You culture an organism using blood agar and after 24 hours of incubation you
noted complete hemolysis of red blood cells surrounding the colonies. This is
classified as what type of hemolytic reaction?
a. Alpha hemolysis c. Delta hemolysis
b. Beta hemolysis d. Gamma hemolysis
10. Which among the following reagents is used as the counterstain in the
Ziehl Neelsen method of acid fast staining?
a. Safranin c. Malachite green
b. Carbol fuchsin d. Methylene blu
 CHAPTER
Prokaryotic and
2 Eukaryotic Cells

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. differentiate prokaryotes from eukaryotes; and
2. characterize the different medically important microorganisms.

Living Cells can be classified into two general categories—prokaryotesand eukaryotes.

Prokaryotes are organisms that do not possess a true nucleus and membrane bound
organelles (e.g., bacteria). Eukaryotic organisms are those that possess a true nucleus and
membrane bound organelles. They are usually multicellular organisms and include plants,
animals, fungi, parasites, and algae. Viruses are acellular organisms that possess only DNA or
RNA. They are dependent on host cells for their replication and are considered as obligate
intracellular parasites
20 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Comparison between Prokaryotes and Eukaryotes

1–10 μm
10–100 μm

chloroplast

mitochondrion

circle of DNA
nucleus linear DNA

Typical Prokaryotic Cell Typical Eukaryotic Cell

Figure 2.1 Diagrammatic representation showing the difference between a prokaryotic cell and a
eukaryotic cell

Table 2.1 Comparison between prokaryotic and eukaryotic cells

Feature Prokaryotic Eukaryotic
Genetic material Not enclosed within a membrane; Enclosed within a membrane;
not associated with histones; usually associated with histones; usually
circular linear
Size Smaller (1–2 μm by 1–4 μm or less) Greater than 5 μm in diameter
Cell type Mostly unicellular Mostly multicellular
Nucleus No true nucleus and nuclear With true nucleus enclosed by
membrane; called nucleoid nuclear membrane
Cell wall Simple Complex
Cell division Budding or binary fission Mitosis
Sexual No meiosis; transfer of DNA only Meiosis
reproduction
Cytoskeleton Absent Present
Mesosome Functions as mitochondria and Golgi Absen
complex
 Prokaryotic and Eukaryotic Cells 21

Feature Prokaryotic Eukaryotic

Ribosomes 70S; located in cytoplasm 80S; located in membranes such
as in the endoplasmic reticulum
70S; found in organelles such as
mitochondria or chloroplast
Membrane bound Absent Present
organelles
Extrachromosomal Present Absent
plasmid
Duration of cell Short (20–60 minutes) Long (12–24 hours)
cycle
Adapted from http://www.microbiologynotes.com/differencesbetween prokaryotic and eukaryotic cells

Medically Important Microorganisms

Organisms that are considered medically important are those that have the potential or the
ability to produce significant clinical disease in humans. They may be part of the normal flora
of the body or are true pathogenic organisms. These may be categorized into bacteria, viruses,
fungi, algae, and parasites (protozoa and helminths).
Viruses are acellular organisms. Their outer surface is called capsid, which is composed of
repeating sub units called capsomeres. Viruses possess only a single nucleic acid, either DNA
or RNA, but never both. In addition, viruses lack the necessary cellular parts that can allow
them to replicate independent of the host cell. They also lack the genes and enzymes that are
necessary for energy production. They rely on the cellular machinery of the host cell for protein
and energy production. Hence, viruses are considered obligate intracellular parasites.
Viruses are classified based on the following: (1) type of nucleic acid they possess;
(2) shape of the capsid (icosahedral, helical, polyhedral, or complex); (3) number of capsomeres;
(4) size of the capsid; (5) presence or absence of an envelope; (6) type of host they infect
(humans, plants, or animals); (7) type of disease they produce; (8) target cell or tropism
(e.g., T helper cells for HIV); and (9) immunologic or antigenic properties
22 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Herpesvirus Orf virus

Vaccinia virus Paramyxovirus (mumps)

Adenovirus

Rhabdovirus
T even coliphage Influenza virus
Flexuous
tailed phage

Polyomavirus Picornavirus ΦX174 phage Tubulovirus

1 μm

Figure 2.2 Diagrammatic representation of various forms and sizes of viruses

Bacteriophages are a special type of viruses that primarily infect bacteria. They are similar
to other viruses in that: (1) they are obligate intracellular parasites; (2) they are similarly
shaped like other viruses; and (3) they may also be classified based on the type of nucleic
acid they possess. They play a role in the acquisition of virulence factors of certain bacteria
(e.g., diphtheria toxin of Corynebacterium diphtheriae), as well as in the transfer of genetic
material from one bacterium to another (as in transduction).
Bacteria are prokaryotic cells with majority having an outer covering called the
cell wall that is composed mainly of peptidoglycan. Unlike viruses, they possess both
DNA and RNA. Unlike eukaryotic organisms, bacteria possess a nucleoid instead
of a true nucleus, smaller ribosomes, and lack mitochondria. Based on their physical
characteristics, bacteria may be broadly categorized into (1) gram negative bacteria with
cell wall (e.g., Escherichia coli); (2) gram positive bacteria with cell wall (e.g., Staphylococcus
aureus); (3) acid fast bacteria with lipid rich cell wall (e.g., Mycobacterium tuberculosis);
and, (4) bacteria without cell wall (e.g., Mycoplasma)
 Prokaryotic and Eukaryotic Cells 2
Fungi are eukaryotic cells with an outer surface composed mainly of chitin. Their cell
membrane is made up mostly of ergosterol. Like bacteria, fungi possess both DNA and
RNA. Unlike bacteria, they possess a true nucleus that is enclosed by a nuclear membrane
and mitochondria that function for ATP production. Fungal ribosomes are also larger than
bacterial ribosomes (80 Svedberg units). Table 2.2 summarizes the major differences between
fungi and bacteria.
Protozoa are the representatives for parasites. Like bacteria and fungi, these are also
eukaryotic cells that have an outer surface called a pellicle. These are unicellular organisms that
usually divide through binary fission, similar to bacteria. Majority exist in two morphologic
forms—cysts and trophozoites. The infective stage is the cyst while the pathogenic stage is the
trophozoite. Protozoa possess both DNA and RNA as well as other cellular features seen in
typical eukaryotic cells.

Table 2.2 Comparison between fungi and bacteria

Features Bacteria Fungi
Cell type Prokaryotic; unicellular Eukaryotic; unicellular or
multicellular
Role in ecosystem Can be both producers and Mainly decomposers
decomposers
Optimal pH Neutral pH (6.5–7.0) Slightly acidic (4.0–6.0)
Cell structures No true nucleus and membrane Possess true nucleus and
bound organelles membrane bound organelles
Main component Peptidoglycan, except in Chitin
of cell wall archaebacteria
Sterols in cell Absent except in Mycoplasma Present
membrane
Mode of nutrition Heterotrophic, chemoautotrophic, Heterotrophic; majority aerobic
photoautotrophic, aerobic, and facultative anaerobic
anaerobic, facultative anaerobic
Reproduction Binary fission Sexual and asexual spores
Adapted from https://www.majordifferences.com/2017/07/10differences between bacteria and.html

Algae are eukaryotic organisms whose outer surface consists primarily of cellulose. They are
described as plant like organisms because most of them have chlorophyll and are thus capable
of photosynthesis. Unlike plants, they do not possess true roots, stems, and leaves. Table 2.3
summarizes the major differences between algae and plants. Algae vary in size from the single
celled phytoplanktons to the large seaweeds found in the ocean floor.
Algae do not produce significant disease in humans. Most algae are beneficial in that they
are important sources of food, iodine, and other minerals. They may also be used as fertilizers,
emulsifiers for puddings, and stabilizers for ice cream and salad dressings.
24 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Table 2.3 Comparison between algae and plants

Features Algae Plants
Taxonomic classification Kingdom Protista Kingdom Plantae
Cellular structure Unicellular, multicellular or Multicellular
colony forming
Photosynthetic Yes Yes
Energy source Carbon dioxide Carbon dioxide
Storage form of energy Starch Starch
Vascular system* Absent Present
Habitat Mostly water Mostly rooted to the ground
Composed of roots, stems, No Yes
and leaves
Method of reproduction Both asexual and sexual Sexual (complex)
*Allow for dispersion of nutrients throughout the entire plant

Diatoms are unicellular algae that inhabit both fresh and saltwater. Their cell
wall contains silicone dioxide that may be utilized in filtration systems, insulation, and
as abrasives. Dinoflagellates are also unicellular algae that are important members of the
phytoplankton group. They contribute greatly to the oxygen in the atmosphere and serve
as important links in the food chain. On the other hand, they are also responsible for what
is known as “red tide.” These small organisms produce a powerful neurotoxin which, when
ingested in significant amounts, is responsible for the potentially fatal disease called paralytic
shellfish poisoning.

Cyanobacteria Diatom Dinoflagellate Green algae Coccolithophore

Figure 2.3 Various structures of phytoplanktons that are usually found floating on wate
 Prokaryotic and Eukaryotic Cells 25

CHAPTER SUMMARY

• Living cells can be classified as either prokaryotic or eukaryotic.

• Prokaryotic cells, as exemplified by bacteria, are usually unicellular, do not possess a true
nucleus and membrane bound organelles, and multiply by means of binary fission.

• Eukaryotic cells vary from unicellular (e.g., protozoa) to multicellular (e.g., fungi). They
possess a true nucleus surrounded by a nuclear membrane as well as membrane bound
organelles.

• Viruses are not classified as cells since they only possess an outer covering called capsid
and a nucleic acid (either DNA or RNA). As such, they are dependent on the host cell
machinery for their replication and are thus considered as obligate intracellular parasites.

• Medically important organisms are those which produce significant disease in humans.
These may take the form of viruses, bacteria, fungi, protozoa, and algae.

› Viruses
and may
are acellular, obligate intracellular parasites possessing only DNA or RNA
be classified based on: (1) type of nucleic acid they possess; (2) shape of the
capsid (icosahedral, helical, polyhedral, or complex); (3) number of capsomeres; (4) size
of the capsid; (5) presence or absence of an envelope; (6) type of host they infect
(humans, plants, or animals); (7) type of disease they produce; (8) target cell or tropism
(e.g., T helper cells for HIV); and (9) immunologic or antigenic properties.

› Bacteria are prokaryotic organisms that possess both DNA and RNA. Most possess a
of
cell wall composed predominantly peptidoglycan.

› Fungi are eukaryotic organisms with a cell wall composed mainly of chitin and cell
membrane that contains ergosterol.
› Protozoa are mostly unicellular parasites that are eukaryotic. Most divide by binary
fission similar to bacteria.

› Algae are eukaryotic, aquatic, plant like organisms. Similar to plants, they are
photosynthetic but unlike plants, they do not have true roots, stems, or leaves
This page is intentionally left blank
 Prokaryotic and Eukaryotic Cells 27

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Matching Type.

A. Cell Type
Column A Column B

1. Reproduce by meiosis and mitosis a. Prokaryotic cell

2. With 70S ribosomes b. Eukaryotic cell
3. Do not have membrane bound organelles
4. DNA associated with histones
5. Has true nucleus surrounded by nuclear membrane

B. Organism Group
Column A Column B

6. Obligate intracellular parasite a. Bacteria

7. Outer covering made up mostly of chitin b. Viruses
8. Possess only one type of nucleic acid c. Fungi
9. Capable of photosynthesis d. Protozoa
10. Unicellular parasite e. Algae
11. Include bacteriophages
12. Reproduction is through sexual and asexual spores
13. Outer surface consists primarily of cellulose
14. Possess chlorophyll
15. Use carbon dioxide as energy source
This page is intentionally left blank
 CHAPTER
Bacterial
3 Morphology

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. distinguish among the various general shapes of bacteria, citing examples for each; and
2. compare the external and internal structures of gram positive, gram negative and
acid fast bacteria.

Bacteria, which are prokaryotic, have simpler structures compared to eukaryotic organisms.
In terms of morphology, bacteria may be classified into three basic shapes: coccus (pl. cocci),
bacillus (pl. bacilli), and spiral shaped or curved. Cocci can be described as spherical or round
shaped organisms (e.g., Staphylococcus, Streptococcus). They may be arranged singly, in pairs
(diplococci), in chains (streptococci), in clusters (staphylococci), in groups of four (tetrad),
or in groups of eight (octad). Rod shaped organisms are called bacilli (e.g., Escherichia
coli, Salmonella). Some may be very short, resembling elongated cocci called coccobacilli
(e.g., Haemophilus influenzae). Curved and spiral shaped organisms may show variations in
their morphology. Vibrio cholerae, the organism causing cholera, is described as comma shaped.
The causative agent of syphilis, Treponema pallidum, is spiral in shape while the causative agent
of diphtheria, Corynebacterium diphtheriae, is club shaped
30 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Fundamental Shapes of Bacteria

Flagella

Pseudomonas Salmonella typhi

Pneumococci Streptococci

Treponema
Spores

Mycobacterium Clostridium
tuberculosis tetani
Staphylococci Leptospira

Spheres (Cocci) Rods (Bacilli) Spirals (Spirochetes)

Figure 3.1 Fundamental shapes of bacteria

Envelope Structures
Prokaryotic cells are surrounded by a complex envelope that may vary in composition.
The envelope serves to protect the bacteria from harsh environmental conditions.

Glycocalyx
This is the outermost covering of some bacteria. It is a gelatinous substance that is located
external to the cell wall, composed of polysaccharide or polypeptide, or both. It is called capsule
if it is strongly attached to the cell wall and slime layer if it is loosely attached. The presence of
the capsule is indicative of the virulence of an organism, aiding the organism in the evasion
of phagocytosis. It can stimulate an antibody response from the immune system. The capsule
serves to protect the organism from dehydration.

Cell Wall
The bacterial cell wall is sometimes called the murein sacculus. Its principal component is
peptidoglycan, which is also called murein or mucopeptide. It is multi layered in gram positive
bacteria and single layered in gram negative bacteria. The cell wall provides rigid support and
gives shape to the bacteria. It protects the bacteria from osmotic damage and plays an important
role in cell division
 Bacterial Morphology 31

Special components of gram positive cell walls

1. Teichoic acids – comprise major surface antigens of gram positive organisms and can
elicit antibody response. In some gram positive organisms such as Staphylococcus aureus,
teichoic acids function for the attachment of the organism to the host cell. These also
provide tensile strength to gram positive bacterial cell walls.
2. Polysaccharides – polysaccharide molecules include neutral sugars such as mannose,
arabinose, rhamnose, and glucosamine. It also includes some acidic sugars such as
glucuronic acid and mannuronic acid.

Teichoic acid
Wall associated protein

Lipoteichoic
acid

Peptidoglycan

Cytoplasmic
membrane

Figure 3.2 Diagrammatic representation of a typical gram positive bacterial cell wall

Special components of gram negative cell walls

1. Outer membrane – a bi layered structure where the inner leaflet is composed of a
lipopolysaccharide (LPS). It has special protein channels that allow the passage of
small or low molecular weight hydrophilic substances such as sugars and amino acids.
LPS has a complex glycolipid called lipid A, responsible for its endotoxin activity. It is
located in the outer leaflet of the outer membrane. The inner core is a polysaccharide
made up of repeat units. This repeat unit is also called the O antigen, which is unique for
every species of bacteria.
2. Lipoprotein – functions to anchor the outer membrane to the peptidoglycan layer and
stabilizes the outer membrane.
3. Periplasmic space – a fluid filled space between the outer membrane and the
inner plasma membrane. It contains enzymes for the breakdown of large non
transportable molecules into transportable ones and enzymes that serve to detoxify and
inactivate antibiotics
 32 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Lipoteichoic acid
Teichoic acid Porin
O specific side chains
Lipopolysaccharide
Outer membrane
Peptidoglycan
Broun's lipoprotein
Periplasmic space Peptidoglycan
Periplasmic space
Plasma membrane Plasma membrane
and integral proteins and integral proteins
Gram (+) cell wall Gram (–) cell wall
Figure 3.3 A comparison between gram positive and gram negative cell walls showing the
differences in their constituents

Acid fast cell wall

Unlike gram positive and gram negative bacteria, acid fast organisms such as
Mycobacterium tuberculosis possess an outer layer that is lipid rich. The cell wall of acid fast
organisms is composed of large amounts of waxes that are known as mycolic acids. The inner
layer of the cell wall is also made up of peptidoglycan but because the outermost layer is lipid
rich, cell walls of acid fast organisms are hydrophobic. This is the reason why they cannot be
stained using the reagents used in gram staining. The hydrophobic nature of their cell wall
protects them from harsh chemicals such as strong acids and detergents.

LAM
Lipoteichoic acid Glycolipid
LPS Mycolic acid
Lipoprotein Porin

PeptidoglycanPeptidoglycan Peptidoglycan Galactan

Mannophosphoinositide
Gram positive Bacteria Gram negative Bacteria Mycobacteria
Figure 3.4 Schematic representation comparing gram positive, gram negative, and acid fast
cell wal
 Bacterial Morphology 33

Projecting Structures
Flagella
These are thread like structures made up entirely of molecules of the protein sub unit
flagellin. They project from the capsule and are organs for motility. Flagella are classified into
four types, namely: (a) monotrichous (single polar flagellum); (b) lophotrichous (a tuft of flagella
at one end of the bacterium); (c) amphitrichous (flagella at both ends of the bacterium); and
(d) peritrichous (flagella all around the bacterium). Bacteria without flagella are called atrichous.

a b

c d

Figure 3.5 Typical arrangement of bacterial flagella. a Peritrichous, b monotrichous and polar,
c lophotrichous and polar, and d amphitrichous and polar.

Pili or Fimbriae
These are rigid surface appendages found on many gram negative bacteria. They are fine
and short in comparison with flagella. Their structural protein sub units are called pilins.
Pili may also function for motility. They function for adherence to cell surface (common pili)
or attachment to another bacterium during a form of bacterial gene exchange called
conjugation (sex pili).

Axial Filaments
Axial filaments are also called endoflagella and are found in spirochetes (e.g., Treponema
pallidum causing syphilis). These are composed of bundles of fibrils, the structures of which
are similar to flagella. They arise from the ends of the bacterial cell and spiral around the cell.
The filaments rotate producing movement of the outer sheath of the spirochetes propelling
them forward
34 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Cytoplasmic Membrane
Also called cell membrane or plasma membrane, the cytoplasmic membrane is located beneath
the cell wall. It is sometimes called the cell sac because it encloses the cytoplasm of the cell.
The cytoplasmic membrane is a selectively permeable membrane that allows for transport of
selected solutes. In aerobic organisms, it is the site of the electron transport chain and serves as
the site of ATP production. It therefore serves the function of the mitochondria, which are not
found in prokaryotic cells. The cytoplasmic membrane also contains the enzymes needed for
the biosynthesis of DNA, cell wall components, and membrane lipids.

Internal Structures
Nucleoid
Bacteria have no true nucleus that is surrounded by a nuclear membrane. Its genetic
material is packaged in a structure called the nucleoid. Bacteria possess a single, circular, double
stranded DNA.

Mesosomes
The mesosome functions for cell division. It is also involved in the secretion of substances
produced by bacteria.

Ribosomes
The ribosomes function for protein synthesis. Unlike eukaryotic ribosomes, bacterial
ribosome is smaller (70S).

Granules or Inclusion Bodies

These are found in certain bacteria and serve for storage of food and energy
(e.g., metachromatic granules of Corynebacterium diphtheriae or Much granules of
Mycobacterium tuberculosis)
 Bacterial Morphology 35

Endospores
Endospores are structures produced by many bacteria when they are placed in a hostile
environment. It is composed of dipicolinic acid which confers resistance to heat, drying, chemical
agents, and radiation; making it very difficult to destroy. The process of spore production
is called sporulation, and this occurs when the environmental conditions are detrimental to
the bacteria. When environmental conditions become favorable, the endospores revert to
their vegetative state through a process called germination. Some gram positive, but never
gram negative, bacteria form spores.
Pilus

Capsule
Cytoplasm
Inclusion Ribosomes
Cell wall
Capsule Plasma
membrane
Nucleoid
Cell wall containing DNA
Plasmid
Plasma
membrane

Fimbriae

Flagella

Figure 3.6 Parts of a typical prokaryotic cell

a b c

Figure 3.7 Spores showing a terminal and b central location, c as well as metachromatic
granules of Corynebacterium diphtheria
36 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• There are three basic shapes of bacteria: (a) spherical or cocci; (b) rod shaped or bacilli;
and (c) curved or spiral.

• Awithtypical prokaryotic cell is composed of three major components—an outer envelope

its projecting structures, the cell membrane, and the internal structures.

• The envelope is composed of the following:

» The outermost covering is the glycocalyx, also known as the capsule if it is adherent to
the cell wall and slime layer when it is loosely attached to the cell wall.
» The cell wall or the murein sacculus provides rigid support and shape to the bacteria.
Its main component is peptidoglycan, which is multilayered in gram positive bacteria
and monolayered in gram negative bacteria.
› Gram positive cell wall contains teichoic acids which may function for the
attachment of the bacterium to the host cell, as well as polysaccharide molecules.
› Gram negative cell walls contain lipopolysaccharide made of a lipid A molecule
A is
and polysaccharides. The lipid component responsible for the endotoxic
activity of gram negative bacteria. The lipopolysaccharide is an integral part of the
outer membrane of gram negative bacteria. Gram negative bacteria also have a
periplasmic space where important enzymes are found.
› Acid fast organisms possess a cell wall that is also made up of an inner layer of
peptidoglycan and an outer layer rich in waxes composed of mycolic acid and
other lipids. This is responsible for the hydrophobic nature of its cell wall and
the main reason why acid fast organisms cannot be stained using the reagents
for Gram staining.

• Structures projecting from the bacterial capsule include pili or fimbriae of gram negative
organisms, flagella, and axial filaments of spirochetes.
» There are two types of pili: common pili which functions for attachment and sex pili
which participates in gene exchange among bacteria in a process called conjugation.
» Flagella may be of four patterns: (1) lophotrichous (a tuft of flagella on one end of
the bacterium), (2) amphitrichous (a single flagellum on each end of the bacterium),
(3) peritrichous (flagella surrounding the bacterium), and (4) monotrichous (only one
flagellum at one end of the bacterium).
» Axial filaments are similar in structure to flagella and help propel the spirochetes
forward
 Bacterial Morphology 3

• Bacterial cytoplasmic membrane is the functional analogue of the mitochondria. It is

selectively permeable and is the site of ATP production of aerobic bacteria.

• Bacteria do not have a true nucleus. Its genetic material is packaged in a structure called
nucleoid. Bacterial ribosome is smaller than a typical eukaryotic ribosome.

• Bacteria possess structures that enable them to withstand adverse environmental

conditions. These structures are the endospores which are mainly composed of dipicolinic
acid.

• Other structures found in bacterial cells are the mesosomes, which play a role in cell
division, and inclusion bodies or granules in some bacteria which serve as storage for food.
This page is intentionally left blank
 Bacterial Morphology 39

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Multiple Choice.

1. What bacteria are partly round and partly rod shaped?

a. Cocci c. Spirochetes
b. Bacilli d. Coccobacilli
2. Which among the following bacterial structures acts as a functional analogue of
the mitochondria?
a. Capsule c. Cell membrane
b. Nucleoid d. Outer membrane
3. Which among the following cell wall components is found only in gram positive
bacteria?

a. Lipopolysaccharide c. Mycolic acid

b. Teichoic acid d. Muramic acid
4. Which among the following statements is correct regarding the cell wall of acid
fast bacteria?
a. It can be stained with crystal violet.
b. Its outer layer is hydrophobic due to the presence of lipids.
c. Its outer layer is hydrophilic due to the presence of a multi layered
peptidoglycan.
d. The major component is lipoteichoic acid.
5. A bacterium that has a tuft of flagella on one end is called:
a. Lophotrichous c. Peritrichous
b. Monotrichous d. Amphitrichou
40 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. Which of the following structures is utilized by bacteria in exchanging genetic

material from one bacterium to another?
a. Pilus c. Mesosome
b. Flagella d. Axial filament
7. Which among the following provides rigid support to bacteria and gives shape to
the bacteria?
a. Cell membrane c. Outer membrane
b. Cell wall d. Capsule
8. The bacterial endospore is resistant to heat and drying due to the presence of this
component:
a. Teichoic acid c. Mycolic acid
b. Muramic acid d. Dipicolinic acid
9. Which among the following is a structure utilized by some bacteria for food
storage?

a. Much granules d. A, B, and C

b. Metachromatic granules e. A and B only
c. Endospores
10. Which of the following contain enzymes used by bacteria to break down large
molecules into smaller, easy to transport molecules?
a. Cell wall c. Outer membrane
b. Cell membrane d. Periplasmic spac
 CHAPTER
Bacterial Growth
4 Requirements

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define microbial growth;
2. discuss the various nutritional and physical requirements of bacteria for growth; and
3. illustrate the bacterial growth curve with explanation of the events occurring in each
phase of the bacterial growth curve.

Growth as defined in medical dictionaries involves an orderly and organized increase in

the sum of all components of the organism. The process entails the replication of all cellular
structures, organelles, and components. Microbial growth is concerned with the increase in the
number of cells and not an increase in the size of the organism. A bacterial colony is composed
of thousands of cells; hence, colonies in culture are actually composed of billions of cells. As
in any living organism, bacteria require certain nutrients and physical conditions that will
promote their growth. This chapter discusses the various nutritional and physical requirements
of bacteria for growth.

Nutritional Requirements
Carbon
Carbon makes up the structural backbone or skeleton of all organic molecules. Based
on their carbon source, microorganisms may be classified into autotrophs (lithotrophs) and
heterotrophs (organotrophs)
42 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Autotrophs are microorganisms that utilize inorganic compounds (e.g., carbon dioxide) and
inorganic salts as their sole carbon source. Organotrophs are organisms that make use of organic
substances like sugars or glucose as their carbon source. For both autotrophs and heterotrophs,
their energy may be derived from either light (photolithotrophs and photoorganotrophs) or the
oxidation of inorganic substances (chemolithotrophs and chemoorganotrophs).Most medically
important bacteria are chemoorganotrophs.

Nitrogen, Sulfur, Phosphorus

These are necessary for the synthesis of cellular materials like proteins and nucleic acids.
Nitrogen and sulfur are required for the synthesis of proteins. Nitrogen and phosphorus are
essential for the synthesis of nucleic acids and ATP. Approximately 14% of the dry weight
of a bacterial cell is nitrogen and about 4% is sulfur and phosphorus.

Inorganic Ions
These include magnesium, potassium, calcium, iron, and trace elements (e.g., manganese,
zinc, copper, cobalt). Magnesium stabilizes ribosomes, cell membranes, and nucleic acids.
It also serves as a co factor in the activity of many enzymes. Potassium is required for the
normal functioning and integrity of ribosomes and participates in certain enzymatic activities
of the cell.
Calcium is an important component of gram positive bacterial cell wall and contributes
to the resistance of bacterial endospores against adverse environmental conditions. Iron is
a component of cytochrome, a component of the electron transport chain, and functions as
a co factor for enzymatic activities. Trace elements are components of enzymes and function
as co factors. Some are necessary for the maintenance of protein structure.

Growth Factors
Growth factors are essential to promote the growth and development of the bacterial cell.
These include vitamin B complex and amino acids
 Bacterial Growth Requirements 4

Physical Requirements
Moisture/Water
The bacterial cell is composed mainly of water. It serves as the medium from which bacteria
acquire their nutrients.

Oxygen
Oxygen is used by aerobic bacteria for cellular respiration and serve as the final electron
acceptor. Microorganisms are classified as either aerobes or anaerobes based on their oxygen
requirements.
Microorganisms that utilize molecular oxygen for energy production are referred to as
aerobes. Strict aerobes are organisms that strictly require oxygen for growth. Microbes that
cannot survive in the presence of oxygen are called obligate anaerobes. These organisms do
not have the enzymes that break down free radicals produced in the body (i.e., catalase and
superoxide dismutase).
There are organisms that can grow and survive under both aerobic and anaerobic
conditions. These are called facultative organisms. Most medically important bacteria are
facultative. Some organisms are able to grow at low oxygen tension but their rate of growth is
diminished. These are called microaerophiles. There are some organisms though that may require
the addition of carbon dioxide to enhance their growth. These are called capnophiles.

Temperature
Enhanced enzyme activity requires certain temperatures. Microbes are classified into
three groups based on their temperature requirements, namely: (1) thermophiles, which grow
best at temperatures higher than 40 °C; (2) mesophiles, which require an optimal temperature
of 20 °C–40 °C; and, (3) psychrophiles, which require an optimum temperature of 10 °C–20 °C.
Most medically important bacteria are mesophiles.
44 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

90 °C

80 °C
Thermophile

70 °C

60 °C Pasteurization (62.8 °C)

50 °C

40 °C
Mesophiles

30 °C Human body (37 °C)

Psychrophiles
20 °C

10 °C

0 °C Refrigerator (4 °C)

Figure 4.1 Classification of bacteria into three groups based

on their optimum temperature requirements

pH
Another requirement of bacteria is the extent of acidity or alkalinity of their environment,
which is referred to as the pH. Microorganisms that grow best in pH 8.4–9.0 are called
alkalophiles. Those that grow best in pH 6.5–7.5 are called neutrophiles. Most medically
important bacteria are neutrophiles. The pH of most human tissues are 7.0–7.2. Certain
bacteria require a pH less than 6.0. These bacteria are called acidophiles.

Osmotic Conditions
Most organisms grow best under ideal conditions of osmotic pressure, which is determined
by the salt concentration. The normal microbial cytoplasmic salt concentration is approximately
1%. The optimum condition is if the external environment also has the same salt concentration.
If the extracellular salt concentration is increased (e.g., when food is salted), water will flow out
of the microbial cell and the organism will shrink and die. On the other hand, if the external
environment does not contain salt, water will flow into the bacterial cell causing the organism
to swell and rupture. Organisms that require high salt concentrations for growth are called
halophiles (e.g., diatoms and dinoflagellates) and those that require high osmotic pressure for
optimal growth are called osmophiles.
 Bacterial Growth Requirements 45

Bacterial Growth Curve

The bacterial growth curve illustrates the phases in the growth of the population of
bacteria when they are grown in a culture of fixed volume. It reflects the different stages in the
growth of the organism and is divided into four phases: lag phase, log phase, stationary phase,
and death or decline phase.

microorganism
Stationary
Log, or phase
exponential
growth, Death, or
phase decline, phase

of
Numbers

Survival
Lag phase
phase

Time

Figure 4.2 Bacterial growth curve

Lag Phase
This is the period of adjustment for the bacteria in the new environment. During this
phase, there is no appreciable increase in the number of microorganisms. The organisms will
show increased metabolic activity in order to synthesize DNA as well as secrete enzymes which
might not be present in their new environment but which are needed by the organism. Bacteria
attain their maximum size toward the end of the lag phase. This phase may last for 1 to 4 hours.

Log/Logarithmic/ExponentialPhase
This period is characterized by rapid cell division, resulting in an increase in the number
of bacteria. The organism exhibits high metabolic activity. This is the period when the
generation time or doubling time of the organism (i.e., the time required for the bacterial cells to
double in number) is determined. A generation time of 10 minutes means that the bacteria will
double in number every 10 minutes showing exponential growth. The average duration of this
phase is about 8 hours.
46 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Stationary Phase
This is considered as the period of equilibrium. During this period, the rate of growth slows
down, nutrients start to deplete, and toxic wastes begin to accumulate. As a consequence, some
bacterial cells may die. However, since there are still bacterial cells undergoing cell division, the
number of living cells equals the number of dead cells. Gram positive organisms may become
gram negative organisms in this phase. Sporulation occurs towards the end of this phase, or in
the case of spore forming organisms, during the beginning of this phase.

Death or Decline Phase

This is the period of rapid cell death where the number of dead cells is greater than the
number of living cells. This is due to the continuous depletion of nutrients and accumulation
of waste materials. Sporulation continues to occur during this stage. The duration of this phase
varies from a few hours to a few days
 Bacterial Growth Requirements 47

CHAPTER SUMMARY

• Bacteria require optimum nutrient and physical conditions for their growth.
• Nutritional requirements of bacteria include adequate supply of carbon, nitrogen, sulfur,
phosphorus, inorganic ions, and growth factors.

• Bacteria are classified into two groups based on their carbon source: autotrophs/
lithotrophs and heterotrophs/organotrophs.
» Autotrophs utilize inorganic compounds for their carbon source while organic
compounds such as glucose serve as the carbon source of heterotrophs.

• Bacteria derive energy by two means: from sunlight or from oxidation of inorganic
substances.

• Physical requirements of bacteria include moisture, oxygen, temperature, pH, and osmotic
conditions.
» Bacterial cell is made up mostly of water, which serves as the medium from which
bacteria derive their nutrients.
» Organisms that require oxygen for optimal growth are called aerobes while those that
cannot survive in the presence of oxygen are called anaerobes.
» Facultative organisms are those which can grow in the presence or absence of oxygen.
» Bacteria may be grouped into three based on their temperature requirements: (1) those
that require high temperature (thermophiles); (2) those that require temperature of
20 °C–40 °C (mesophiles); and (3) those that require temperature of 10 °C–20 °C
(psychrophiles).
» Acidophiles are organisms that grow best in pH < 6.0. Neutrophiles grow best
at pH of 7.0–7.2 while alkalophiles are those that grow best at pH of 8.4–9.0.
» Organisms that require salt for growth are called halophiles. Osmophiles are those that
need high osmotic pressure for maximal growth.
• Based on their nutritional and physical requirements, most medically important bacteria
are chemoorganotrophs,facultative, mesophiles, and neutrophiles.

• The bacterial growth curve illustrates the phases of growth of a bacterial population
grown in culture of fixed volume. It is divided into a lag phase, log phase, stationary
phase, and death or decline phase
This page is intentionally left blank
 Bacterial Growth Requirements 49

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. Microorganisms that utilize organic compounds as sole carbon source are called:
a. Phototrophs c. Chemotrophs
b. Heterotrophs d. Autotrophs
2. Which among the following is essential for the synthesis of nucleic acids and
proteins?
a. Iron c. Calcium
b. Nitrogen d. Potassium
3. Organisms that strictly require oxygen for growth are called:
a. Facultative c. Obligate anaerobes
b. Obligate aerobes d. Microaerophiles
4. Most medically important bacteria are:
a. Photoorganotrophs c. Mesophiles
b. Alkalophiles d. Halophiles
5. Bacteria that require an optimum temperature of more than 40 °C are called:
a. Thermophiles c. Psychrophiles
b. Mesophiles d. Neutrophiles
6. Microorganisms that require carbon dioxide for growth are called:
a. Halophiles c. Capnophiles
b. Mesophiles d. Psychrophiles
50 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Matching Type.

Column A Column B
7. Nutrients are depleted and toxic wastes accumulate a. Lag phase
8. Period of adjustment for the bacteria b. Log phase
9. Period of rapid cell division
c. Stationary phase
d. Death or
10. Period when spores begin to form decline phas
 5 Normal Flora
CHAPTER

of the Human Body

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define “normal flora;”
2. differentiate between resident flora and transient flora;
3. explain the role of normal flora in the body; and
4. give examples of organisms that normally inhabit different sites in the body.

Microbial Ecology is the study of the relationships between microorganisms and their
environment. Among these relationships is the relationship of microbes with humans, and such
include the normal flora (or indigenous flora) of the human body. Normal flora consists of the
group of organisms that inhabit the body of a normal healthy individual in the community.
These indigenous flora may be non pathogenic or pathogenic and may at times behave as
opportunistic pathogens.
There are two types of flora, namely: (1) resident flora and (2) transient flora. Resident flora
are organisms that are relatively of fixed types and are regularly found in a given area of the
body at a given age. Transient flora are those that inhabit the skin and mucous membrane
temporarily for hours, days, or weeks and are derived from the environment. Normal flora are
beneficial to the human body because they can inhibit the growth of pathogenic organisms
by priming the immune system of newborns. At the same time, normal flora protects the
body’s organs and systems that are in direct contact with the external environment and are
therefore subject to the attack of invasive organisms. Normal flora do this by either competing
with invasive organisms for nutrients essential for their growth or by producing substances
that can kill them. Normal flora synthesize important vitamins that are essential to humans
52 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Normal intestinal flora secrete vitamin K that is needed for the activity of some clotting factors.
Other beneficial effects of normal flora include the following:
1. Normal flora can prevent pathogenic organisms from attaching to and penetrating the
skin and other tissues by producing mucin which make it difficult for the pathogenic
organisms to attach to the tissues to produce disease.
2. Normal flora in the intestines aid in the digestion of food by producing enzymes such
as cellulase, galactosidase, and glucosidase.
3. Intestinal flora also help in the metabolism of steroids.
The healthy fetus is normally sterile until birth, following the rupture of the bag of
water. Once born, the newborn normal flora is derived from the mother’s genital tract during
delivery, from the skin and respiratory tract of individuals who handled the newborn, and
from the environment.
There are certain body tissues and fluids that are normally sterile. Body fluids that are
sterile include the cerebrospinal fluid (CSF), synovial fluid, and blood. In the blood, there may
be low transient bacteremia brought about by physiologic trauma. The sterile tissues include the
urinary bladder, uterus, fallopian tubes, middle ear, and paranasal sinuses. Presence of bacteria
in these tissues and body fluids may lead to serious infections in these areas. For example,
bacteria in the CSF can gain entry into the central nervous system, leading to a potentially
fatal encephalitis.

Normal Flora on Different Sites of the Body

Skin
The skin is the part of the human body that is in constant contact with the environment,
making it the most exposed to microorganisms. There are certain factors that eliminate
non resident flora from the skin, namely: (1) lysozyme in the skin; (2) acidic pH of the skin
due to sweat; (3) free fatty acids in sebaceous secretions; and (4) the constant sloughing off of
the skin.
The normal flora of the skin consists mainly of bacteria and fungi. The microorganisms
vary depending on the region of the skin. The skin may be divided into three regions: (1) axilla,
perineum, and toe webs; (2) hand, face, and trunk; and (3) upper arms and legs. Skin of the
axilla, perineum, and toe webs is characterized by having higher moisture levels, higher body
temperature, and higher levels of surface lipids. These regions have more microorganisms
compared to the others and are predominantly inhabited by gram negative bacilli. Dry sites
(e.g., hands, forearms, feet, legs) have diverse flora because of their exposure to the environment.
Predominant flora in these areas include Staphylococcus epidermidis and Staphylococcus hominis
 Normal Flora of the Human Body 53

Most microorganisms in the skin are found in its superficial layers (stratum corneum)
and hair follicles. Anaerobes inhabit the deeper structures and layers of the skin, such as hair
follicles, sebaceous glands, and sweat glands. Table 5.1 summarizes the various microorganisms
that inhabit the skin.

Table 5.1 Normal flora found on the skin

Organism Remarks
Staphylococcus epidermidis Major skin inhabitant, comprising approximately 90% of
resident aerobic flora
Staphylococcus aureus Most commonly found in nose and perineum; in the nose,
number varies with age (greater in newborns than in
adults)
Micrococci (Micrococcus luteus) Accounts for 20%–80% of micrococci in the skin
Diphtheroids (Coryneforms) Classified into: lipophilic (common in axilla)
or non lipophilic (more common on glabrous or hairless
skin such as palms of hands)
Anaerobic diphtheroids (Propionibacteriumacnes) – areas
rich in sebaceous glands
Gram negative Bacilli Seen in moist intertriginous areas such as toe webs and
(Enterobacter, Klebsiella, axilla
Escherichia coli, and Proteus spp.)
Nail Flora Similar to that of the skin
Fungi may also be present (Aspergillus, Penicillium,
Cladosporium, Mucor)

Mouth and Respiratory Tract

The tongue and buccal mucosa are inhabited mostly by Streptococcus viridans group, which
includes S. mutans, S. milleri, S. salivarius, and S. sanguis. Although they are part of the normal
flora of the mouth, the viridans streptococci have been implicated in the pathogenesis of dental
caries. The gingival crevices and the tonsillar crypts are primarily inhabited by anaerobic flora.
The normal flora of the pharynx and trachea are similar to those found in the oral cavity.
However, there may be transient carriage in the pharynx of potentially pathogenic organisms.
These include Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis
and Mycoplasma.
54 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

In the upper respiratory tract, initial colonization by pathogenic organisms may be seen.
These include Neisseria meningitidis, Corynebacterium diphtheriae, and Bordetella pertussis.
The lower respiratory tract is usually sterile and organisms that reach this region are usually
destroyed by the defense mechanisms of the body such as the alveolar macrophages.

Conjunctiva
The normal flora in the conjunctivae are very scanty because they are held in check by the
flow of tears that contain lysozyme. The lysozyme may interfere with the cell wall synthesis of
organisms. However, some bacteria may transiently colonize the conjunctiva including Neisseria,
Moraxella, and Corynebacterium. Staphylococci and streptococci may also be present.

Digestive Tract
The esophagus contains transient mouth flora. Minimal bacteria may be found in the
stomach due to the relatively hostile environment in the stomach. Bacteria that may be found
in the stomach are those that may be swallowed with the food or those that are dislodged from
the mouth. The acidity in the environment of the stomach is further increased after meals
because of the release of gastric acid. However, there are certain bacteria that are able to survive
in the acidic environment of the stomach. One of these is Helicobacter pylori, the most common
cause of duodenal ulcer. This organism produces urease that causes alkalinization of gastric acid
thereby enabling it to colonize the stomach.
The number of bacterial flora differs between the small intestine and large intestine. In the
small intestine, scanty flora may be found due to the constant peristaltic movement of the
intestines. Most of the bacteria cultured in the small intestine include streptococci, lactobacilli,
and Bacteroides which are all transient.
The number of bacterial flora in the large intestine is far greater than in the small intestine.
The colon is inhabited predominantly by anaerobes (95%–99%) which includes Bacteroides
fragilis (most common), Bifidobacterium/Lactobacillusbifidum (predominant in breast fed
infants), Eubacterium, Peptostreptococcus, and Clostridium. In bottle fed infants, the predominant
intestinal flora is Lactobacillus acidophilus. About 1%–4% of the flora of the colon are facultative
aerobes, predominantly Escherichia coli and other Enterobacteriaceae.
Intestinal flora play important roles in the body, namely: (1) synthesis of vitamin B complex
and vitamin K; (2) conversion of bile into bile acids; (3) competition with transient flora for
nutrients; (4) prevention of colonization of the intestines by transient flora; and (5) production
of potentially pathogenic end products of metabolism that are toxic to transient flora
 Normal Flora of the Human Body 55

Esophagus
Major bacteria present Organ Major physiological
processes

Secretion of acid (HCl)

Stomach Digestion of
macromolecules
pH 2
Duodenum
Enterococci Continued digestion
Lactobacilli Small Absorption of
Jejunum intestine monosaccharides, amino
acids, fatty acids, water
Enterobacteria pH 4–5
Enterococcus faecalis Ileum
Bacteroides
Bifidobacterium Absorption of bile acids,
Eubacterium vitamin B12
Peptococcus Colon Large pH 7
Peptostreptococcus intestine
Ruminococcus
Clostridia
Lactobacilli
Streptococcus
Staphylococcus
Anus

Figure 5.1 Normal flora of the digestive tract

Genitourinary Tract
The urinary tract is sterile above the distal 1 cm of the urethra. In the anterior
urethra, the predominant flora isolated are S. epidermidis, enterococci, and diphtheroids. In both
males and females, Mycobacterium smegmatis may be found as normal commensals in
their secretions. In addition, Gardnerella vaginalis, bacteroides, and alpha streptococci
may be found in penile urethra. The female urethra is either sterile or contains
Staphylococcus epidermidis.
Vaginal flora varies depending on the age, hormonal levels, and vaginal pH of the host. In
female infants, the predominant vaginal flora is Lactobacillus spp. From 1 month of age until
puberty, there is cessation of glycogen secretion making the vaginal pH higher (around 7.0).
The microorganisms that may inhabit the vagina at this time include Staphylococcus epidermidis,
Streptococci, diphtheroids, and Escherichia coli. At the onset of puberty, there is resumption
of glycogen secretion making the vaginal pH acidic. Predominant flora include Lactobacillus
acidophilus, corynebacteria, peptostreptococci, streptococci, Bacteroides, and staphylococci.
Lactobacillus plays a crucial role in preventing gonococcal infection by producing lactic acid that
adds to the acidity of the vagina. Young girls are more prone to the development of gonococca
56 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

infection compared to adult women because the normal acidic pH of the vagina as well as the
normal vaginal flora are not yet fully developed.
After menopause, the vaginal pH increases once more due to the lessened production
of glycogen. Normal flora that predominate during this period are similar to those found
during pre puberty. Most of these flora are derived from the skin and from the colon. Fungi
such as Torulopsis and Candida may also be found (10%–30%). Conditions that will allow the
overgrowth of these fungi (e.g., intake of antibiotics) can lead to vaginal infections such as
vaginitis.

Bacterial Flora in Normal

Bacterial Flora in Normal
Persons in Hospital
Persons in the Community
or Long term Care Facilities
Trac
• Staphylococcus sp. • Staphylococcus sp.
• Streptococcus pneumoniae •
Streptococcus sp. Anaerobes
UpperRespiratory
– •Enterobacteriaceae
– Viridans Streptococcus – Escherichia coli
• Haemophilus sp. – Klebsiella sp.
• Anaerobes • Candida sp.
• Pseudomonas sp.
• Staphylococcus sp. • Staphylococcus sp.
Skin
• Coryneform bacteria • Enterobacteriaceae
or "Diptheroids" – Escherichia coli
• Propionibacterium sp. – Klebsiella sp.
• Anaerobes • Anaerobes sp.
• Enterococcus sp. • Enterococcus
• Enterobacteriaceae • Enterobacteriaceae
Gastrointestinal

Tract –Escherichia coli –

Escherichia coli
– Klebsiella sp. – Klebsiella sp.
• Streptococcus sp. • Candida sp.
–Streptococcus anginosus
(milleri) group
• Pseudomonas sp.
• Lactobacillus sp.
• Candida sp.
Genital
Tract
• Lactobacillus sp. • Candida sp.
• Streptococcus sp.
– Streptococcus agalactiae

Figure 5.2 Comparison of bacterial flora of persons who are healthy and those that are confined
in hospitals or long term care facilities
 Normal Flora of the Human Body 57

CHAPTER SUMMARY

• Normal or indigenous flora refers to organisms that inhabit the body of a normal healthy
individual.

• Resident flora, also known as normal flora, refers to microorganisms that are regularly
found in a given area at a given age.

• Transient flora are those organisms that inhabit the skin and mucous membrane
temporarily for a few hours, days, or weeks. They do not establish themselves permanently
in the body tissues.

• Normal flora have important roles in the body which can be beneficial or harmful.
» Advantages of normal flora include:
1. Inhibition of growth of pathogenic organisms by priming of the immune system
2. Synthesis of vitamin B12 and vitamin K in the intestines.
3. Synthesis of substances that may inhibit growth of pathogenic organisms
(e.g., enzymes, fatty acids, bacteriocins).
» Disadvantages of normal flora include:
1. Production of disease if the individual becomes immunocompromisedor if they
change their usual anatomic location.
2. Production of disease since most of them are pathogens or opportunistic
pathogens.

• Most of the normal flora in the skin are found in moist, intertriginous areas.
Diphtheroids and epidermidis are the predominant flora of the
Staphylococcus skin.

• The tongue and buccal mucosa are inhabited mostly by viridans group, which
Streptococcus
includes S. mutans, S. milleri, S. salivarius, and S. sanguis. The gingival crevices and the
tonsillar crypts are primarily inhabited by anaerobic flora.

• There may be transient carriage in the pharynx of potentially pathogenic organisms.

These include Haemophilus influenzae, pneumoniae, Neisseria meningitidis, and
Streptococcus
Mycoplasma.

• InThese
the upper respiratory tract, initial colonization by pathogenic organisms may be seen.
include Neisseria meningitidis, Corynebacterium diphtheriae, and Bordetellapertussis.

• Most of thewhich
Bacteroides
bacteria cultured in the small intestine include streptococci, lactobacilli, and
are all transient
58 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

• The colonfragilis
is inhabited predominantly by anaerobes (95%–99%) which includes
Bacteroides (most common), Bifidobacterium/Lactobacillusbifidum (predominant in
breastfed infants), Eubacterium, Peptostreptococcus, and Clostridium.

• Vaginal flora varies depending on the age, hormonal levels, and vaginal pH of the host.
» In female infants, the predominant vaginal flora is Lactobacillus spp.
» From 1 month of age until puberty, microorganisms which may inhabit the vagina
include Staphylococcus epidermidis, Streptococci, diphtheroids, and Escherichia coli.
» At puberty the predominant flora include Lactobacillus acidophilus, corynebacteria,
peprostreptococci, streptococci, Bacteroides, and staphylococci.
» Fungi such as Torulopsis and Candida may also be found (10%–30%)
 Normal Flora of the Human Body 59

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Multiple Choice.

1. Normal flora synthesizes and excretes:

a. Vitamin D and E c. Vitamin K and B12
b. Vitamin D, E, K, and B12 d. Vitamin D, K, and B12
2. The bacterial count in the small intestine:
a. Decreases distally c. Is consistent throughout
b. Increases distally d. Is normally sterile
3. Streptococcus pneumoniae can be found in the:
a. Mouth d. Skin
b. Nostrils e. Small intestine
c. Pharynx
4. Strep viridans is most numerous on/in the:
a. Large intestine d. Skin
b. Mouth e. Small intestin
c. Nasopharynx
5. In commensalism:
a. Both organisms benefit from symbiosis
b. Neither organism is benefited nor harmed
c. One organism benefits at the expense of the other
d. One organism benefits while the other derives neither benefit nor harm
60 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. Enteric gram negative bacilli can be found on/in the:

a. Large intestine d. Small intestine
b. Mouth e. Vagina
c. Skin
7. An outbreak of sepsis caused by Staphylococcus aureus has occurred in the newborn
nursery. You are called upon to investigate. According to your knowledge of the
normal flora, what is the most likely source of the organism?
a. Nose c. Vagina
b. Colon d. Throat
8. Which body site harbors the greatest number of bacteria?
a. Skin c. Respiratory tract
b. Mouth d. Large intestine
9. Which among the following statements is true about normal flora?
a. Collection of organisms found routinely in specific parts of the body
b. Component organisms are the same in all individuals
c. Mostly fungi
d. Most are pathogenic
10. Which among the following is true regarding normal flora?
a. It is absent in the stomach due to the acidic pH.
b. It develops in the body only after the neonatal period.
c. It cannot be eradicated by antimicrobial agents.
d. The flora in the bronchi is similar to that of the trachea
 CHAPTER
Medical and
6 Surgical Asepsis

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. differentiate between sepsis and asepsis;
2. distinguish between medical asepsis and surgical asepsis;
3. enumerate general aseptic procedures followed to maintain a clean environment and
prevent the spread of infectious diseases;
4. explain the various isolation precaution measures;
5. identify aseptic measures utilized in the operating room; and
6. determine general measures that can be used to prevent the development of infection
in the community.

Infection Control is one of the major concerns that healthcare workers in healthcare
facilities and hospitals constantly address. There are certain terminologies associated with
infection control that a healthcare worker must be familiar with. These terminologies are often
related to the chain of infection, how the organisms are transmitted, asepsis, the specific types
of infection, and personal protective equipment (PPE). These include the following:
1. Chain of infection – how an individual acquires the infectious agents and includes
the infectious agent, the source of infection or its reservoir, how the organism is
transmitted, and the organism’s portal of entry into the susceptible host.
2. Mode of transmission – the manner in which the infectious organism is acquired by
the host.
3. Standard precautions – the specific measures used to prevent the spread of infection
among all patients and healthcare workers, including measures to protect them from
contaminated blood and other body fluids
62 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

4. Contamination – denotes contact of a sterile or aseptic item with microorganisms.

Medically aseptic items become contaminated if they get in contact with
disease producing organisms. Sterile items become contaminated if they get in contact
with items that are not sterile.
5. Decontamination – the process where physical or chemical means are used to remove,
inactivate, or destroy pathogens on a surface or item making them safe for handling or
use and incapable of transmitting infectious agents.
6. Disinfection – the process of using physical or chemical means to destroy pathogens,
excluding the spores.
7. Sterilization – the process by which all pathogens are destroyed, including the spores.
The various methods of physical and chemical sterilization will be discussed in the
succeeding chapters.
8. Antiseptic – a chemical solution that inhibits the growth of some microorganisms.
Most antiseptics can be used directly on the skin (e.g., alcohol and iodine).
9. Healthcare associated infection – any infection that is acquired during the time
a patient is admitted in a healthcare facility. The most common healthcare associated
infection is the urinary tract infection (UTI).
10. Iatrogenic infection – infection that is acquired in the course of undergoing diagnostic
tests or therapeutic procedures.
11. Occupational exposure – the acquisition or exposure to an infectious agent of
a healthcare worker during the course of his/her work.
12. Personal protective equipment (PPE) – specialized equipment and attire used by
healthcare workers to protect them from infections. These include gloves, masks,
gowns, and goggles.

Asepsis
Asepsis refers to a condition in which the individual and his/her surrounding environment
are free of any microorganisms. Sepsis, the opposite of asepsis, refers to the clinical condition
where an individual develops a systemic reaction to a bacterial infection that starts from
a localized infection in one part of the body. The goals of asepsis are to protect the patient
from hospital acquired or nosocomial infections and to prevent the spread of pathogenic
microorganisms.
All patients in healthcare facilities are vulnerable to pathogenic organisms. Some of the
factors that play a role in the occurrence of infection among patients include: (1) suppression
of the immune system; (2) prolonged duration of illness; and (3) procedures that patients
undergo in the healthcare facility such as insertion of in dwelling catheters, use of antibiotics
 Medical and Surgical Asepsis 6
and insertion of intravenous lines or endotracheal tubes. The most commonly occurring
pathogenic microorganisms that lead to nosocomial infections are Escherichia coli, Staphylococcus
aureus, Pseudomonas aeruginosa, Candida albicans, and Enterococcus. The primary locations of
infections from these organisms are surgical wounds, the urinary tract, the respiratory tract,
and the bloodstream.
Pathogens may be introduced to the patient through contact with hospital personnel,
the hospital environment, or hospital equipment such as respiratory machines, catheters,
and intravenous lines or needles. Situations that require aseptic measures are surgery and the
insertion of intravenous lines, urinary catheters, and drains. All personnel must constantly
monitor not only their own movements and practices but those of others as well.
Asepsis may be categorized into medical asepsis and surgical asepsis. Medical or clean asepsis
refers to the absence of disease producing microorganisms. It is the infection control process
that aims to reduce the spread of infection. It involves certain procedures aimed to decrease
the number of organisms and prevent their spread in the general clinical setting. Proper hand
hygiene, the administration of all medications except those that are given intravenously, and the
preparation of the patient’s skin before administration of subcutaneous medication are instances
when medical asepsis is applied.
Surgical or sterile asepsis is defined as the absence of all microorganisms. It involves
procedures that aim to eliminate microorganisms from an area in the body where surgical
procedures will be performed as well as the location where the surgical procedure will be carried
out. There are some procedures and treatment modalities that necessitate surgical asepsis and
there are others that would only require medical asepsis. The principles of surgical asepsis
are applied when the skin is not intact and when internal areas of the body are involved in
procedures, whether for diagnostic or treatment purposes. Applications of surgical asepsis
include wound care, during invasive procedures (e.g., endoscopy), administration of intravenous
drugs, and during insertion of urinary catheter and other internally placed tubes.
General aseptic procedures that help to preserve and maintain a clean medical environment
include: (1) frequent handwashing of hospital personnel (doctors, nurses, medical technologists,
and orderlies); (2) prompt and safe disposal of contaminated materials like bandages and
needles; (3) regular checking and emptying of containers for surgical drains; (4) prompt
cleaning of soiled or moist areas; and (5) proper labeling of containers regarding the date and
time of disposal.

Handwashing
The most frequent source of microorganisms leading to outbreaks of infection in health
institutions is the hands of the healthcare workers. This is the reason why proper handwashing
is one of the most basic means of preventing the spread of pathogenic organisms. It is
essential in the healthcare environment for the following reasons: (1) to reduce the flora on
64 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

the healthcare worker’s skin; (2) to protect the healthcare worker in the event that there is a
break in his or her skin; (3) to reduce risk of contact with infectious agents if gloves worn are
punctured; and (4) to reduce the chances of disease transmission. Healthcare workers must be
aware that the healthcare environment is highly susceptible to a number of healthcare acquired
infections. These include infections with methicillin resistant Staphylococcus aureus (MRSA),
vancomycin resistant Enterococcus (VRE), and penicillin resistant Streptococcus pneumoniae.
When should handwashing be done? The United States Center for Disease Control
recommend routine handwashing for at least 15 seconds with a 10 second rinse. For healthcare
workers a longer period of time for handwashing that entails thorough washing of the hands,
lathering at least twice, and careful cleaning of the fingernails is recommended in the following
situations: (1) at the beginning and end of each shift; (2) when the hands are visibly soiled;
(3) after contact with a possible source of microorganisms such as blood or body fluids,
mucous membrane, non intact skin, or contaminated objects; (4) before and after performing
invasive procedures; or (5) before removing gloves if they are visibly soiled and each time after
removing gloves.
Proper handwashing can be done with friction and regular soap and water. Hands must
be thoroughly washed with vigorous scrubbing, paying special attention to the areas around
the nailbeds and between the fingers. These are areas that usually have high bacterial load.
The fingernails should be kept clean and short. Patients, as well as their relatives, must also be
taught the proper way of handwashing. Remember that the best way to prevent the spread of
communicable diseases is health education!
Alcohol based sanitizing antimicrobial solutions or hand cleansers must not be used as
substitute for proper handwashing. However, if running water and soap are not available, one
may use alcohol based hand cleansers to decontaminate the hands. The alcohol based hand
cleansers must be liberally applied to the entire hand after which the hands are rubbed until the
entire hand is completely dried.

Personal Protective Equipment (PPE)

Personal protective equipment (PPE) are specialized equipment and attire used in
healthcare facilities to protect not only the healthcare workers but also the patients and visitors
against infections. These include masks, gowns, and goggles.

Gloves
Among the various PPEs in use, gloves are the most commonly used. Gloves used
during medical procedures are disposable and the most commonly used are of two
types: (1) examination gloves, which may be sterile or non sterile, and (2) surgical gloves
 Medical and Surgical Asepsis 65

which are sterile. They serve as a protective barrier when handling or touching open wounds,
blood, or body fluids. Gloves provide protection from microorganisms and help prevent the
spread of infectious agents from one person to another. Sterile, disposable gloves must be
provided to all personnel in healthcare facilities, particularly those who have direct contact
with patients. The gloves must be disposed of immediately after use. Hands must be washed
thoroughly after using gloves since the wearing of gloves can also promote multiplication of
microorganisms because of the moist environment that it provides.
The World Health Organization (WHO) has come up with guidelines for the proper use
of gloves in healthcare facilities. Some of the recommendations listed in the guidelines include
the following:
1. Gloves are not meant to replace observance of proper hand hygiene. The practice of
hand hygiene must still be observed before and after wearing of gloves.
2. Gloves must be worn if contact with blood or body fluids, mucous membranes,
open wounds, or potentially infectious material is anticipated.
3. Gloves must be removed and disposed of after caring for a patient. Healthcare workers
must not wear the same gloves if caring for more than one patient.
4. Gloves must be removed or changed if moving from a contaminated body site to
another body site in the course of caring for a patient.
5. Re using of gloves after decontamination is not recommended.
Furthermore, WHO recommends the use of gloves in the following situations:
1. Before performing a sterile procedure.
2. When in contact with a patient and his or her surroundings in conditions where
contact precautions are warranted.
3. When contact with blood or body fluids, non intact skin, and mucous membranes is
anticipated.
The removal of gloves is indicated in the following:
1. When hand hygiene is indicated.
2. After contact with a single patient and his or her surroundings is ended or when
contact with a contaminated body site is ended.
3. As soon as the gloves are damaged or there is loss of integrity of the gloves.
4. After contact with blood or body fluids, non intact skin, and mucous membrane
66 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Masks
The mask must cover the mouth and nose. It must be tied in a way that there should be
minimal gaps between the face and the mask. The healthcare worker must also avoid touching
the mask while it is worn. The moment it becomes damp, it should be replaced with a clean
and dry one. Remember that masks are supposed to be single use items. Therefore, it must be
discarded and disposed of as clinical waste the moment the procedure which necessitated its
wearing is completed. It is also recommended that hands are decontaminated by washing with
soap and water or by using alcohol based hand sanitizers after the mask is disposed.

Sterile Gowns
Healthcare workers are recommended to wear gowns or aprons when there is probability
of contact with blood, body secretions excluding sweat, or other body substances. Likewise,
wearing of gowns is recommended if the healthcare worker has close contact with patients,
equipment, or materials that can introduce infectious agents to the healthcare worker’s skin,
uniform, or other clothing. The type of apron or gown to wear depends on the degree of risk
with the infectious agents and the potential for body substances and blood to penetrate through
the clothes or skin of the healthcare worker. The protective wear can either be in the form of an
apron or gown.
If there is a risk for body substances, blood, or body secretions to contaminate the
clothing or skin of the healthcare worker, a fluid resistant apron or gown is recommended.
Clean, non sterile gowns or aprons are generally sufficient to protect the skin and prevent
soiling of clothing during procedures or other in patient activities that may lead to splashing or
spraying of blood and body substances. Fluid resistant gowns or aprons are always worn with
gloves and other personal protective equipment. Healthcare workers must make sure that they
change gowns or aprons in between treating different patients.
Disposable, single use gowns are usually used to protect the healthcare worker during
procedures and other activities related to patient care where there is likelihood of generating
splashing or sprays of blood or body substances. The length of the sleeves will depend on the
specific procedure being performed or how much is the risk of exposure of the healthcare
worker’s arms.
Fluid resistant, single use, long sleeved, full body gowns are usually worn (1) when there is
a risk of contact of the healthcare worker’s skin with a patient who has broken skin, (2) if there
is extensive skin to skin contact between the healthcare worker and the patient, and (3) if the
risk of contact with body substances or fluids cannot be contained such as when the patient has
diarrhea or is vomiting incessantly
 Medical and Surgical Asepsis 6
In cases of surgical procedures and other invasive procedures, care must be taken to prevent
the invasion of microorganisms into the surgical site. Sterility parameters have been developed
to maintain the sterile field. These parameters are as follows:
1. The front of a sterile gown is considered sterile from the chest down to the level of
the sterile field. The reason for this is because most scrubbed personnel work next to a
sterile table and/or bed.
2. The gown sleeves are sterile from two inches above the elbow to the cuff,
circumferentially.
3. The back of the gown is not considered sterile because it cannot be constantly
monitored.
4. The neck, sleeve cuffs, and underarms of the gloves are not considered sterile and are
not considered as effective microbial barriers.
If contamination of the surgical gown occurs at any point during the procedure, the gown
as well as the gloves must be changed. The circulating nurse needs to obtain sterile gloves and
gown for the scrubbed person who needs to change his or her gown. The individual concerned
must step away from the sterile field while the circulating nurse wears sterile gloves and unties
the scrubbed person’s gown at the neck and waist. The scrubbed person in turn grasps the front
of the gown at the shoulders below the neckline, pulls the gown off inside out and rolls it away
from the body. The circulating nurse then turns to face the scrubbed person, grasps the gown
at the shoulders and pulls it off. The gloves are then removed next. The moment the gloves are
removed, the scrubbed person is now ready to re glove and re gown.

Isolation Precautions
Isolation is the process of separating an individual with an infectious disease from the
rest of the healthy population to prevent the spread of the infection to other individuals.
The Center for Disease Control (CDC) in the United States has come up with guidelines
to follow towards this end. These recommendations, which they termed universal precautions,
are geared towards handling of patients with an infection from an unknown pathogen to
decrease the risk of transmission. These precautions apply to all body fluids including blood,
skin, and mucous membranes. These include (1) proper handwashing; (2) the use of personal
protective equipment such as gloves, aprons, gowns, masks or face shields; (3) proper handling
and disposal of secretions and excretions excluding sweat; (4) proper handling and disposal
of soiled linen and equipment; (5) environmental control; (6) prevention of injury from sharp
devices such as needles; and (7) patient placement.
68 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Transmission based Precautions

Transmission based precautions have been developed to further prevent the spread
of infectious agents. These precautions are based on the mode of transmission of the
infectious agents and are classified into (1) contact precautions; (2) droplet precautions; and
(3) airborne precautions.

Contact Precautions
Contact precautions are used to prevent the spread of infections or infectious agents
that are transmitted through touching of patients or items in the room where the
infectious agents may be deposited (called fomites). These include infectious agents such
as methicillin resistant Staphylococcus aureus (MRSA), viruses such as respiratory syncytial
virus, agents that cause diarrhea whether viral or bacterial, and open wounds. All individuals,
whether healthcare personnel or non healthcare personnel, must wear gowns and gloves.

Droplet Precautions
These precautions are used for diseases or infectious agents that are spread in tiny
droplets caused by coughing and sneezing. These are used to prevent contact with secretions
from the respiratory tract. Examples of such disease are influenza, mumps, or pertussis
(whooping cough). These droplets that are spread when the individual coughs or sneezes can
travel a distance of approximately 3 feet (or 90 centimeters). All persons entering the rooms of
these patients are required to wear a surgical mask.

Airborne Precautions
These are measures geared towards preventing the spread of diseases or infectious agents
that are spread through the air from one person to another. These microorganisms are so tiny
that they can float in the air and travel long distances. These include infectious agents that
cause chickenpox, measles, and tuberculosis. Patients who are admitted to the hospital with
the said infections must be placed in a room with negative air pressure where the air is gently
sucked out and not allowed to flow into the hallway thereby preventing contact with the outside
environment. The door must remain closed at all times and all individuals entering the room
must wear a protective mask. This is also called reverse isolation
 Medical and Surgical Asepsis 6

Aseptic Measures in the Operating Room

To prevent post operative infection, asepsis must be strictly observed in the operating
room. Thorough cleaning of the operating room with detergent or detergent germicides, soap
and water must be done. In addition, all equipment that would be directly in contact with the
patient must be properly sterilized. Surgical instruments can be sterilized in the autoclave or
by using chemical agents. Radiation is seldom used because of its toxic effects to body cells.
Personnel must ensure sterility by making sure that sterile packages are dry and intact.
Sterile surgical clothing and operating room gowns and other protective devices
(e.g., surgical gloves, face masks, goggles, eye/face shields) serve as barriers against
microorganisms and must be used to maintain asepsis in the operating room. Gowns used
by the surgical team are considered sterile in front from the chest to the level of the sterile
surgical field and two inches above the elbows to the cuff of the sleeves. This must be put on
with extreme care to avoid contact between its external, sterile surfaces and non sterile objects,
including the skin. The operating room nurse is usually the one who assists the surgeons in
donning the gloves and gowns. The nurse also prepares and arranges the surgical instruments to
minimize the risk of contamination.
Sterile drapes are sterilized linens placed around the field to delineate sterile areas. These
are used to create a sterile field. Only the top surface of the draped area is considered sterile.
The drapes must be used to cover the patient, furniture, and equipment to be included in the
sterile field so that only the incisional site is exposed. Only the scrubbed personnel must handle
the sterile drapes. Once positioned, the sterile drapes should not be removed or rearranged.
Wrapped kits of instruments and/or equipment are opened in such a way that the contents
do not touch non sterile items or surfaces. This is ensured by opening the farthest areas of a
package first. Sterile packages are opened as close as possible to the time of actual use. Leaning
over the contents of packages must be avoided and opened flaps must be prevented from falling
back onto contents. All sterile objects that will touch open wounds or enter body cavities must
be handled only with sterile forceps or with sterile, gloved hands.
To reduce the chances of introducing endogenous flora of the skin to the deeper tissues,
the patient must be prepared prior to surgery. Preparations include shaving of body hair on the
surgical site and thorough cleansing with a disinfectant. The most commonly used and most
effective antiseptic is iodine because it can destroy all forms of microorganisms.
Surgical scrub must be performed by all members of the surgical team and all others
who will perform the surgical procedure or will have access to and contact with the patient,
including equipment and instruments inside the operating room. Surgical scrubbing requires
the use of long acting powerful antimicrobial soap on the hands and forearms and must
be done for a longer period of time (at least 2–5 minutes) than that of typical handwashing.
70 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The interdigital areas of the hands must also be thoroughly washed. The hands must be held
below the elbows during the surgical scrub and above the elbows following the surgical scrub.
Contact with the faucet or other potential contaminants must be avoided. Thorough drying
with a sterile towel is essential, since moist surfaces invite the presence of pathogens. The faucet
can be turned off through use of a foot pedal.
During the operation, only properly scrubbed personnel should be allowed at the vicinity of
the sterile field. The hands and arms of the scrubbed personnel must remain within the sterile
site. Personnel should not turn their backs from the sterile field. Only those areas that can be
seen by the surgeon are considered sterile. Items that are not sterile should not pass over the
sterile field. Talking, laughing, coughing, or sneezing are not allowed across a sterile field.

Preventing Infection in the Community

Controlling the spread of communicable diseases is best achieved at the community level,
before the occurrence of disease. Healthcare personnel must not only be involved in infection
control in the hospital but, more importantly, in the community. It is the duty of healthcare
professionals to educate the public on infectious diseases, particularly their modes of
transmission, because if the people have a clear understanding of the disease process, they would
know what steps to take to prevent its spread.
Infection control in the community includes sanitation techniques, improvement
of health practices, and vaccination. Sanitation techniques include water purification,
improvement of health practices, proper sewage disposal, and other measures that will ensure
a clean environment. Improvement of health practices involves educating the members of the
community on the proper handling, storage, and preparation of food. The members of the
community must be made aware that infectious and parasitic diseases can be obtained from
contaminated and improperly cooked food as well as contaminated water. Lastly, people should
be made aware of the value of immunization. Information about individual vaccines and vaccine
schedules should be made available to the people
 Medical and Surgical Asepsis 71

CHAPTER SUMMARY

• Sepsis is a clinical condition where infectious agents are spread throughout the body
of an individual from a localized site of infection and manifest with symptoms of
organ damage.

• Asepsis is the absence of disease producing organisms and is divided into medical asepsis
and surgical asepsis.
» Medical asepsis is aimed at reducing the number of disease producing organisms to
prevent its spread from healthcare workers to the patients and vice versa.
» Surgical asepsis is aimed at total elimination of disease producing organisms
particularly in areas in the body where surgical procedures will be performed as well as
the location where the surgical procedure will be carried out.

• Handwashing is the most basic and universally accepted measure used to prevent the
spread of infection. Routine handwashing for at least 15 seconds with a 10 second rinse
is recommended.

• Personal protective equipment (PPE) are specialized equipment and attire used in
healthcare facilities to protect not only the healthcare workers but also the patients and
visitors against infections. These include masks, gowns, and goggles. Guidelines have been
set in the proper use of these personal protective equipment.

• Universal precautions are specific measures geared towards handling of patients with
an infection from an unknown pathogen to decrease the risk of transmission. These
precautions apply to all body fluids including blood, skin, and mucous membranes.

• Transmission based precautions have been developed to further prevent the spread
of infectious agents. These precautions are based on the mode of transmission of the
infectious agents and are classified into (1) contact precautions; (2) droplet precautions;
and (3) airborne precautions.

• The operating room is one of the most sterile areas in the hospital. Strict measures must
be followed to ensure sterility not only of the operating room but also of the instruments
and materials to be used in a surgical procedure. All healthcare personnel entering the
operating room must observe strict precautions to maintain its sterility.

• The best way to prevent the spread of infection is at the community level. Proper
health education on the sources of infection as well as the transmission of
disease producing microorganisms is essential. Preventive measures such as vaccination
must also be emphasized
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 Medical and Surgical Asepsis 73

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. The primary reason for aseptic procedures is to:

a. Protect patients
b. Protect patients and healthcare providers
c. Wipe out all bacteria in the office
d. None of the above
2. Which of the following illustrates microorganism transmission via droplet contact?
a. Infected wound drainage that comes in contact with the nurse’s hands
b. A virus is transmitted through sexual intercourse
c. Microorganisms comes in contact with a person’s nasal mucus when
someone coughs nearby
d. Contaminated stethoscope touches a client’s skin
3. Which among the following differentiates medical asepsis from surgical asepsis?
a. Medical asepsis inhibits growth and spread of pathogens while surgical
asepsis destroys microorganisms and spores.
b. Surgical asepsis inhibits growth and spread of pathogens while medical
asepsis destroys microorganisms and spores.
c. They are equally used in any prevention control protocol.
d. Medical asepsis is known as the sterile technique while surgical asepsis is
known as the clean technique.
74 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

4. Which of the following is/are the most common sites for healthcare associated
infections?

a. Urinary and Respiratory Tract c. Scalp and finger nails

b. Blood Stream d. Surgical or Traumatic wounds
5. What should you remember in surgical hand hygiene?
a. Hands above elbows
b. Elbows above hands
c. Elbows should be straight
d. Hands should be on the same line as elbows
6. When a patient is on droplet precaution, what should he or she wear before
he or she is transported?
a. Mask c. Gloves
b. Gown d. Goggles
7. Which of the following is NOT TRUE about surgical asepsis?
a. Includes practices used to render and keep objects and areas free
from microorganisms
b. Such procedures include inserting urinary catheter or IV catheter
c. Also known as sterile technique
d. Involves actions such as handwashing
e. Such techniques are used continuously both within and outside
health agencies

8. Which among the following is a priority nursing action needed when medical
asepsis is used?
a. Handwashing c. Autoclaving of instruments
b. Surgical procedures d. Sterilization of equipmen
 Medical and Surgical Asepsis 7

9. A client has been placed in blood and body fluid isolation. The nurse is instructing
auxiliary personnel in the correct procedures. Which statement by the nursing
assistant indicates the best understanding of the correct protocol for blood and
body fluid isolation?
a. Masks should be worn with all client contact.
b. Gloves should be worn for contact with non intact skin, mucous membranes,
or soiled items.
c. Isolation gowns are not needed.
d. A private room is always indicated.
10. The most effective way for healthcare providers to protect themselves, their family,
and their patients from influenza is to:
a. Wear a surgical mask at all times at work.
b. Stay at home if they have respiratory symptoms.
c. Get an annual flu shot and encourage family, co workers, and patients
to do the same.
d. Not go to work during flu season.
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 CHAPTER Physical and
7 Chemical Methods
of Sterilization

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define important terms related to sterilization and disinfection;
2. compare the different physical and chemical methods of sterilization as to classification,
mechanism of action, and indication for use; and
3. explain the different factors affecting the efficacy of a chemical agent.

Definition of Terms
1. Sterilization – the process of killing or removing all microbial forms, including spores.
2. Disinfection – the process by which most microbial forms on inanimate objects are killed
without necessarily destroying saprophytes and bacterial endospores which leads to
a reduction in the number of organisms to a level that they cannot produce infection.

3. Antisepsis – use of chemical agents on living tissue (e.g., skin) to prevent the spread
of microorganisms either by inhibiting their growth or destroying them.
4. Bactericidal or germicidal agent – agent, physical or chemical, that kills bacteria.
5. Bacteriostatic agent – agent, physical or chemical, capable of inhibiting the growth
of bacteria without necessarily killing them.
6. Sporicidal, fungicidal, viricidal – agents capable of destroying spores, fungi, and viruses,
respectively
78 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Physical Methods of Sterilization

Heating
Heating is the most common physical method of sterilization. The rate of killing is
expressed in thermal death time, i.e., the minimum time required to kill a suspension of an
organism at a predetermined temperature and environment. The mechanisms of action of
heating include: (1) formation of single strand breaks in the bacterial DNA; (2) coagulation
and denaturation of proteins; (3) accumulation of toxic levels of electrolytes; and (4) alteration
of cell membrane structure. Several factors can affect the process of sterilization through
heating. These include:
1. Nature of the heat – moist heat has greater killing action than dry heat.
2. Temperature and time – as temperature increases, the time taken to sterilize decreases.
In other words, there is an inverse relationship between time and temperature.
3. Number of microorganisms – the more microorganisms there are, the higher the
temperature and the longer the duration of the process required to destroy all of them.
4. Nature ofmicroorganisms – spore forming microorganisms are more difficult to destroy
than non spore forming ones.
5. Type of material – the temperature required to sterilize materials depend on the
sensitivity of the material to heat. Heat sensitive materials will require lower
temperature than heat resistant materials.
6. Presence of organic material – the presence of organic materials such as fats, proteins,
and sugars may necessitate higher temperatures.

Types of Heat
1. Moist heat – preferred over dry heat because of its more rapid killing action. Its main
mechanism of action is to cause coagulation and denaturation of proteins. The
various methods of moist heat may be classified according to the temperature used.
These include:
a. Temperature below 100 °C
• Pasteurization
This is the method of destroying disease producing organisms in milk and
milk products as well as other beverages. There are several variations of this
method based on the temperature utilized. One method is called the conventional
method where the milk is heated at 60 °C–65 °C followed by rapid cooling
 Physical and Chemical Methods of Sterilization 7
The flash method involves heating at 72 °C for 15 seconds followed by quick
cooling to 13 °C. A newer pasteurization method developed is what they call
ultra high temperature (UHT) method where heating is done at 140 °C for a
period of 15 seconds and 149 °C for 0.5 seconds.

• Vaccine bath
This is used to destroy contaminating bacteria in vaccine preparations.
The vaccine preparation is heated in a water bath at 60 °C for one hour.
This procedure is not sporicidal. Only the vegetative forms of the bacteria
are destroyed.

• Serum bath
This is used to inactivate bacteria contaminating serum preparations and
is done by heating at 56 °C for several successive days. Similar to vaccine bath,
only the vegetative forms are destroyed since higher temperatures will cause
coagulation of proteins present in the serum.

• Inspissation
This technique is used to solidify and disinfect egg containing and serum
containing media. The culture medium is placed in the slopes of a device called
an inspissator and is heated at 80 °C–85 °C for 30 minutes for three successive
days. The basis for the method is that on the first day, vegetative forms will die
and the spores that will germinate the following day will also die.
b. Temperature of 100 °C
• Boiling
This method involves utilizing water at boiling temperature of 100 °C. It is
not sporicidal and will destroy only the vegetative forms. The killing action can
be enhanced by the addition of 2% sodium bicarbonate. Certain metal articles
and glasswares can be disinfected using this method for 10–20 minutes without
opening the lid of the boiler.

• Fractional sterilization (Tyndallization)

This method is also known as intermittent sterilization and involves exposing
the material to be sterilized to live steam at 100 °C for 30–90 minutes for three
consecutive days, depending on the material to be sterilized. This sterilization
method can be used to sterilize culture media such as TCBS and selenite broth.
The vegetative forms are killed on the first day and the spores that will germinate
will be destroyed on the next successive days. Only vegetative forms of the
bacteria are destroyed with this method.
80 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

c. Temperature above 100 °C

• Autoclave (Steam under pressure)
This is the most efficient method of sterilization because it can destroy all
microbial forms. The temperature for sterilizing is dependent on the pressure
of the steam. When the pressure reaches 15 pounds per square inch (psi), the
temperature inside the vessel reaches 121 °C. Because of the high temperature and
pressure, it would take only 15–20 minutes to sterilize the material. This method
is used to sterilize instruments, surgical bandages, culture media, and other
contaminated materials that can withstand high temperature and high pressure.

Figure 7.1 Modern laboratory autoclave machine

Figure 7.2 Inspissator and water bat

 Physical and Chemical Methods of Sterilization 81

2. Dry heat – the effectiveness of dry heat depends on the penetration of heat through the
material to be sterilized. It is used to sterilize materials in enclosed tubes, oils, jellies,
powders, and glasswares such as test tubes and Petri dishes.
a. Red flame
This method is used to sterilize articles like bacteriological wire loops, straight
wires, tips of forceps, and searing spatulas. The materials are held over the flame of
a Bunsen burner until they become red hot. It is limited only to articles that can be
heated to redness in flame.
b. Open flame (Flaming)
This method also makes use of the Bunsen burner or alcohol lamp. The material
to be sterilized is passed over the flame several times but is not heated to redness.
It is aimed at burning the organism into ashes and is used to sterilize such articles as
mouths of test tubes, scalpels, glass slides, and cover slips. Only vegetative forms are
destroyed. In addition, cracking of the glassware may occur.

a b

Figure 7.3 a Sterilization of inoculation loop using red flame and b a test tube
being sterilized using open flame

c. Incineration
This method is aimed at burning the organism into ashes. The contaminated
material is burned using an incinerator. Articles that must be incinerated
include soiled dressings and beddings, animal carcasses, and pathological material.
This will result in loss of the article and hence must be used only for articles that
have to be disposed. Some materials such as polystyrene emit dense smoke and must
not be incinerated
82 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

d. Hot air oven

The use of the hot air oven was first introduced by Louis Pasteur. Articles
to be sterilized are placed in the oven with a temperature of 160 °C for a period
of one hour. This can be used to sterilize metallic instruments such as forceps,
scalpels, and scissors. It can also be used to sterilize certain glasswares (e.g., petri
dishes, pipettes, flasks) and it is the only method used to sterilize powders and
ointments. The disadvantage of using this method is that because air is a poor
conductor of heat, then hot air will have poor penetration of the materials to be
sterilized. In addition, cotton wool and paper may get slightly charred and glasses
can become smoky.
e. Infrared rays

In this method, the articles to be sterilized are placed in a conveyor belt and
passed through a tunnel that is heated by infrared radiators. The temperature
to which the materials are subjected to is 180 °C for a period of 7.5 minutes. It can
be used to sterilize metallic equipment and glassware.

Dessication
This method is based on the principle of depriving the microorganism of moisture. It is
used mainly for food preservation, such as in the preparation of dried fish and fruits. It may
destroy vegetative forms. Endospores are resistant to drying.

Freezing
Freezing is not a reliable method of sterilization because most pathogenic organisms
are resistant to low temperatures. Its main use in the laboratory is for the preservation
of microorganisms in a process called lyophilization or freeze drying where the organism is
rapidly frozen then dehydrated in high vacuum and stored in a vacuum sealed container.

Filtration
This is a form of mechanical sieving that does not kill microorganisms but merely
separates them from the fluid. A cellulose ester filter with a pore size of 0.22 μm–0.45 μm is
used which can filter all microorganisms except viruses and the three smallest
Rickettsia, and Chlamydia. It is used for liquid solutions that will be destroyed
bacteria—Mycoplasma,

by heat or freezing such as serum, antibiotic solutions, sugar solutions, or urea solution. This
method can be used to remove bacteria from culture media or to prepare suspensions of
viruses and phages
 Physical and Chemical Methods of Sterilization 8

Radiation
1. Ultraviolet Light (UVL)/Non ionizing radiation – the effective UVL wavelength is in
the range of 200 nm–280 nm, with 260 nm as the most effective. This corresponds with
the maximum absorption of bacterial DNA. UVL acts by inducing formation of thymine
thymine dimers resulting in lethal frameshift mutations. Microorganisms such as bacteria,
viruses, and yeasts can be inactivated within seconds. However, UVL is not sporicidal
and is more frequently used for surface disinfection. It is used to disinfect hospital wards,
operating rooms, laboratories, and other rooms in the hospital that need to be sterilized.
The disadvantage of UV ray is that it has low penetrance. It is also limited by the lifespan
of the UV bulb. In addition, there are some bacteria that have DNA repair systems that
can counteract the damage done by UV rays. Care should also be observed by the handler
because UV rays can be harmful to the skin and eyes.
2. Ionizing radiation – ionizing rays have greater penetrance than UV rays. It exerts its
effect by causing formation of free radicals that chemically interact with proteins and
nucleic acids, resulting in cell death. It is not routinely used because of its potential to
harm human tissues. There are two types of ionizing radiation used for sterilization
purposes: electron beams and electromagnetic rays.
a. Electron beams
Electron beams are particulate in nature. A linear accelerator from a heated
cathode is used to generate high speed electrons. It can be used to sterilize syringes,
gloves, dressing packs, food, and some pharmaceuticals. It has lower penetrance and
requires sophisticated instruments.
b. Electromagnetic rays (Gamma rays)
Electromagnetic rays are produced from nuclear disintegration of selected
radioactive isotopes. They have greater penetrance than electron beams but require
longer exposure time. The high energy radiation produced cause damage to the
microorganism’s nucleic acid. It is bactericidal, fungicidal, viricidal, and sporicidal. It
is used commercially to sterilize disposable Petri dishes, plastic syringes, vitamins,
antibiotics, hormones, fabrics and glassware.

Sonic and Ultrasonic Vibrations

Some bacteria can be killed after exposure to certain frequency of sound waves. Exposure
to sound waves at a frequency of approximately 20,000 cycles/second for one hour can kill
some bacteria and viruses. High frequency sound waves act by disrupting cells. They are used to
disinfect and clean instruments and to reduce microbial load.
84 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Osmotic Pressure
This method is based on the principle of osmosis, so that when the concentration of the
fluid surrounding the organism is altered, this will cause the bacterial cell to collapse. This is
used for preservation of fruits in syrup and meats in brine.

Chemical Methods of Sterilization

Chemicals can inhibit the growth of pathogenic organisms, either temporarily or
permanently. Several factors can affect the efficacy of a chemical agent. These include:
1. Concentration and potency of the chemical agent. In general, a higher concentration is
bactericidal whereas a lower concentration may only be bacteriostatic. This is not true
for alcohol. For alcohol, the effective bactericidal concentration is at 50% to 80%.
2. Duration of exposure. The longer the time of exposure to the chemical agent, the
better the killing action.
3. Temperature. A higher temperature speeds up the rate of a chemical reaction and thus
accelerates killing action. However, there are also certain chemical agents that exert
optimal effect at lower temperatures.
4. Nature of the surrounding medium. The pH of the medium and the presence of
extraneous materials like pus or blood decreases the efficiency of the chemical agent.
These materials may inactivate or lower the concentration of the chemical agent or
may bind the chemical agent to its surface.
5. Nature of the organism. This refers to the innate resistance of the microorganism to
disinfectants. Microorganisms vary in their resistance to disinfectants. Bacteria that
produce endospores may be resistant to most chemical agents. Mycobacterial cell wall
is lipid rich that makes it difficult for the chemicals to penetrate it. Gram negative
bacteria have an outer membrane that confers resistance to disinfectants.
6. Number of organisms/Size of inoculum. The larger the number of microorganism
present, the more time needed for a disinfectant to destroy all of them.
A chemical agent, to be effective as a disinfectant or antiseptic, must be chosen carefully
based on the specific purpose, pathogen, and environment. A good chemical agent must possess
the following characteristics:
1. It should be broad spectrum, able to destroy a wide variety of microorganisms.
2. It should be fast acting, able to destroy microbes within a short period of time.
3. It should be active in the presence of organic matter.
4. It should be active in any pH.
5. It should be stable
 Physical and Chemical Methods of Sterilization 8

6. It should be non toxic, non allergenic, non irritative, and non corrosive.
7. It should be soluble in water and easy to apply.
8. It should leave a residual antimicrobial film on the treated surface.
9. It should have high penetrating power.
10. It should not be expensive and must be easily available.
11. It should be safe under storage and shipping for reasonable periods of time.
12. It should not have a bad odor.

Classification of Chemical Disinfectants

Chemical disinfectants may be classified based on the following: (1) consistency (liquid
or gaseous); (2) spectrum of activity (high level, intermediate level, low level); or (3) mechanism
of action.

Mechanism of Action
Damage to the cell membrane
Damage to the cell membrane can cause smaller molecules to leak out of the bacterial cell
and interfere with the active transport and energy metabolism within the cell. Chemicals under
this include the following:
1. Surface active agents – compounds have long chain hydrocarbons that are fat soluble
and charged ions that are water soluble. They concentrate on the surface of
membranes and disrupt membrane resulting in leakage of cell components. These
agents are active against vegetative microbial forms including Mycobacteria as well as
enveloped viruses. They are widely used as disinfectants in homes and hospitals but
their activity is reduced in the presence of hard water and organic matter.
a. Cationic agents
These are detergents where the fat soluble portion is positively charged due
to combination with a quaternary nitrogen atom. These are called quaternary
ammonium compounds and are effective at alkaline pH. Examples are cetrimide
and benzalkonium chloride.
b. Anionic agents
These are negatively charged agents that contain long chain hydrocarbons.
Examples are soaps and bile salts. They remove dirt through the process of
emulsification and are most effective at acidic pH.
86 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. Phenolic compounds – these act by disrupting cell membranes as well as causing

precipitation of proteins and inactivation of enzymes. These are coal tar derivatives
that act as disinfectants at high concentration and as antiseptic at low concentrations.
Phenols are bactericidal and fungicidal with good activity against Mycobacteria but
have poor activity against spores and most viruses.
a. Phenol is no longer used as a disinfectant because it is toxic to human cells. It is
used as a gold standard in the chemical evaluation of new chemical agents using
the phenol coefficient test.
b. Cresols are phenol derivatives more potent and safer than phenol. An example is
Lysol®.

c. Chlorhexidine is used as a skin disinfectant if in isopropanol solution. The aqueous

preparation is used for wound irrigation. Its main use is as antiseptic hand wash.
d. Chloroxylenols are used for topical purposes. They are effective against
gram positive bacteria.
e. Hexachlorophene is a chlorinated diphenyl which has greater activity against
gram positive bacteria similar to chloroxylenols.
f. Triclosan, an organic phenyl ether, has good activity against gram positive bacteria
and a number of gram negative bacteria including Pseudomonas. It has some
activity on fungi and viruses.
3. Alcohols – disorganize the lipid structure of the cell membrane, dehydrate cells,
and cause denaturation and coagulation of cellular proteins. The microbial killing
property of alcohol is seen better in a 70% aqueous solution compared to absolute
alcohol. The disadvantage of using alcohols is that they are skin irritants and are
also flammable.
a. Ethyl alcohol – used as skin antiseptic, it is bactericidal and removes lipids from
skin surfaces.
b. Isopropyl alcohol – it has greater bactericidal activity than ethyl alcohol and
is less volatile. It can be used to disinfect surfaces. Inhalation of its fumes can
cause narcosis.
c. Benzyl alcohol – it is used mainly as a preservative.
d. Methyl alcohol – it is fungicidal and sporicidal used in disinfecting inoculation
hoods.

Denaturation of cellular proteins

Substances that cause denaturation or loss of the normal structure of proteins pave the
way for the eventual destruction of the bacterial cell. Denaturing agents include: (1) acids and
alkalis, (2) alcohol and acetone, and (3) phenol and cresol
 Physical and Chemical Methods of Sterilization 8
Modification of the functional groups of proteins and nucleic acids
1. Heavy metals – cause damage to the enzyme activity of bacteria. They also cause
precipitation of proteins and oxidation of sulfhydryl groups. Heavy metals are mostly
bacteriostatic than bactericidal.
a. Mercurials (e.g., mercurochrome and merthiolate) are biocidal and are used as
antiseptics. These are active against viruses at dilution of 1:500 to 1:1000.
b. Silver compounds (e.g., silver nitrate) are bactericidal. 1% silver nitrate solution is
used clinically as treatment for ophthalmia neonatorum (Crede’s prophylaxis).
Silver sulfadiazine is used topically in the treatment of burn wounds.
2. Halogens – bactericidal oxidizing agents that cause oxidation of essential sulfhydryl groups
of enzymes causing inactivation of the enzymes.
a. Iodine (tincture of iodine, iodophores) is considered the best antiseptic because it is
sporicidal, bactericidal, fungicidal, viricidal, and amoebicidal. It can be combined
with neutral carrier polymers to produce iodophores (e.g., povidone iodine).
A 10% solution of povidone iodine is used for pre operative and post operative
skin disinfection.
b. Chlorine is mainly used in the treatment of water (chlorine gas). Hypochlorites
are used for sanitizing dairy and food processing equipment. It is also a
common household disinfectant. At higher concentrations, it is used to disinfect
swimming pools.
c. Hydrogen peroxide is a weak antiseptic and used only for cleaning wounds and in the
disinfection of surgical devices and soft plastic contact lenses.
3. Alkylating agents
a. Aldehydes damage nucleic acids by alkylation of amino , carboxyl , or hydroxyl groups.
It kills all microorganisms including spores.
• Formaldehyde (formalin) is used for surface disinfection. It can be used to
sterilize bedding and furniture. It is also used to kill Mycobacterium tuberculosis
in sputum and fungi in athlete’s foot.

• Glutaraldehyde is sporicidal and used as a cold sterilant in sterilizing medical

equipment such as respiratory therapy machines and other equipment that can
be damaged by heat. It is more potent than aldehyde. It requires alkaline pH for
its action and exposure time of at least 3 hours to be effective.
b. Ethylene oxide is also sporicidal and is used in the gaseous sterilization of
heat sensitive materials or equipment like heart lung machine, respiratory and
dental equipment, and polyethylene tubes in anesthesia machines. It is more
potent than glutaraldehyde but slower acting. It is highly flammable and is usually
combined with 10% CO2 . It causes eye irritation and is mutagenic and carcinogenic.
88 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• Sterilization is the process of removing all microbial forms, including spores.

• Disinfection is the process of removing most of the microbial forms. It is meant to reduce
the microbial load to prevent the development of infection.

• Factors that affect the effectivity of physical methods of sterilization include:

1. Nature of the heat
2. Temperature and time
3. Number of microorganisms
4. Nature of microorganisms
5. Type of material
6. Presence of organic material
• Heating is the most common physical method of sterilization. It acts by the following
mechanisms:
1. formation of single strand breaks in bacterial DNA
2. coagulation and denaturation of proteins
3. accumulation of toxic levels of electrolytes
4. alteration of cell membrane structure
• An effective chemical disinfectant must have a broad spectrum of activity, be fast acting,
inexpensive, easy to apply, odorless, and non toxic to human tissues.

• Chemical agents used for disinfection and sterilization are classified based on their main
mechanisms of action.
» Agents that cause damage to the cell membrane include surface active agents, phenolic
compounds, and alcohols.
» Acids and alkalis, alcohol and acetone, phenols and cresols all cause denaturation of
proteins.
» Modification of functional protein groups is the mechanism of action of heavy metals,
halogens, and alkylating agents
 Physical and Chemical Methods of Sterilization 89

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. What is the process where all microbial forms in non living objects, including the
spores are destroyed:
a. Sterilization c. Tyndallization
b. Disinfection d. Lyophilization
2. An agent capable of inhibiting the growth of bacteria but does not kill them
is called:

a. Bactericidal c. Bacteremia
b. Bacteriostatic d. None of the above
3. This method is used to effectively sterilize instruments, surgical bandages, culture
media, and other contaminated materials. It can destroy all microbial forms
including spores:
a. Autoclaving c. Pasteurization
b. Boiling d. Tyndallization
4. The method of pasteurization called Ultra High Temperature (UHT) involves
which of the following?
a. Heating at 60 °C–65 °C followed by rapid cooling
b. Heating at 72 °C for 15 seconds followed by quick cooling to 13 °C
c. Heating is done at 140 °C for a period of 15 seconds and 149 °C
for 0.5 seconds
d. Heating at 121 °C for 15–20 minutes at 15 psi
90 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

5. This form of radiation causes disruption of H bonds in bacterial DNA, causing

formation of thymine dimers and frameshift mutations:
a. Ultraviolet light (UVL) c. Gamma rays
b. Ionizing radiation d. X ray
6. Which of the following is correct regarding ethylene oxide?
a. It is used in gaseous sterilization.
b. It is more potent than glutaraldehyde.
c. It is sporicidal.
d. All of the above
7. This chemical agent is used as a gold standard in the evaluation of new
chemical agents:
a. Phenol c. Cresols
b. Alcohol d. Acetone
8. Which among the following chemical agents act by modifying functional groups
of proteins and nucleic acids?
a. Detergents c. Formaldehyde
b. Chlorhexidine d. Isopropyl Alcohol
9. Which among the following statements is true regarding physical methods
of sterilization?
a. Dry heat is more effective than moist heat.
b. Heat sensitive materials will require lower temperature than
heat resistant ones.
c. Spores are destroyed using boiling at 100 °C.
d. In autoclave, when the temperature is 100 °C, the pressure is 15 psi.
10. Which among the following statements is true regarding chemical methods
of disinfection?
a. Chemical agents must not leave a residual antimicrobial film
on the treated surface.
b. A lower temperature is needed to speed up the rate of chemical reactions.
c. Alcohol is bactericidal at 100% concentration.
d. A good chemical agent must be odorless and easy to prepare
 CHAPTER
Antimicrobial
8 Agents

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define what an antimicrobial agent is;
2. explain the characteristics of an ideal antimicrobial agent;
3. classify antimicrobial agents based on their mechanism of action, giving examples
for each class;
4. define “antimicrobial resistance;” and
5. discuss the mechanisms by which microorganisms develop antimicrobial resistance.

Antibiotics or antimicrobials are substances produced from microorganisms or

synthetically that are capable of inhibiting or destroying microorganisms even at low
concentrations. Natural sources include fungi and bacteria. The antibiotic penicillin, for
example, was derived from the fungus Penicillium. Polymixin and bacitracin were developed
from the bacterium Bacillus sp. while Actinomyces was the source for the drugs tetracycline,
chloramphenicol, and streptomycin.
Antibiotics are mainly used in the treatment of infectious diseases. An ideal antimicrobial
agent must possess the following characteristics:
1. It should be able to kill the microbial agent or inhibit its growth.
2. It must have a broad spectrum of activity.
3. It should not cause any damage or adverse effect to the patient.
4. It should remain stable when stored in either a solid or a liquid form.
5. It should be able to remain in specific body tissues long enough for it to be effective
92 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. It should be able to kill the organism or inhibit its growth before it has had a chance to
mutate and develop resistance.
7. It must exhibit selective toxicity. It must be toxic to the microbial cell but not to the
host’s cells.
Antibiotics may be classified in several ways. Based on spectrum of activity, they may
be classified as broad spectrum or narrow spectrum antibiotics. Broad spectrum antibiotics
are those with a wide coverage of activity against a wide spectrum of microorganisms while
narrow spectrum antibiotics are those with a limited coverage of activity, effective only against a
limited number of microorganisms.
Antibiotics may also be classified based on their antimicrobial activity. An antibiotic is said
to be bactericidal if it is capable of killing the microorganism. An antibiotic is bacteriostatic if it
can only inhibit the growth of the organism. In the choice of antibiotics, bactericidal agents are
more preferred than bacteriostatic drugs.
Another way of classifying antibiotics is based on their absorbability from the site of
administration. A locally acting antibiotic is one that limits its action at the site where
it is administered. Examples are topical agents such as topical ointments or eye drops.
A systemically acting antibiotic is one that affects several body systems. Examples are
antibiotics that are administered intramuscularly or intravenously.

Classification of Antibiotics According

to Mechanism of Action
Agents that Interfere with the Synthesis
of Bacterial Cell Wall
These agents act by inhibiting the different stages of peptidoglycan synthesis or by
destroying an already formed peptidoglycan by activating autolytic enzymes. The most
commonly used are the β lactam antibiotics as exemplified by penicillins and cephalosporins.
Also called penicillin binding proteins (PBPs), these act by inhibiting the conversion
of immature peptidoglycan to mature peptidoglycan by directly inhibiting bacterial
transpeptidases. The peptidoglycan produced is weakly cross linked making the organism
susceptible to cell lysis and death.
Another class of cell wall synthesis inhibitor is the glycopeptides (e.g., Vancomycin).
Members of this class inhibit the transglycosylase and transpeptidase enzymes that are essential
for the completion of the synthesis of the peptidoglycan component of the bacterial cell wall
 Antimicrobial Agents 9

Agents that Alter the Function or Permeability

of the Cell Membrane
The microbial cell membrane is essential to the survival of the organism because not
only does it serve as a barrier by its selective permeability but more importantly it is the site
of bacterial ATP production. Agents that target the cell membrane can be classified into
cationic, anionic, and neutral agents. The most well known are polymyxin B and colistemethate
(polymyxin E) which are cationic agents. These agents initially act by disrupting the outer
membrane structure enabling them to enter the cell and inhibit metabolic processes in the
bacterial cell. Among the damaging effects of polymyxin B are (1) disturbance of the surface
charge and lipid composition of the cell membrane, (2) disruption of the potassium gradient on
the cell membrane, and (3) depolarization of the cell membrane.
Antifungal drugs such as polyenes (nystatin, amphotericin B) alter the permeability of the
cell membrane. Azoles (clotrimazole, ketoconazole, miconazole, fluconazole), another group of
anti fungal drugs, interfere with the synthesis of ergosterol, a major component of the fungal
cell membrane.

Agents that Inhibit Protein Synthesis

These agents bind with the ribosomes, either the 30S or the 50S ribosomal sub units
or both. Binding with the ribosome results in failure to initiate the synthesis of proteins,
interference with protein elongation or misreading resulting in deformed proteins. Inhibitors
of the 30S ribosomal subunit interfere primarily with the initiation process. The representative
drugs are the aminoglycosides and tetracycline. Aminoglycosides cause formation of non
functional complexes and misreading. Spectinomycin is an antimicrobial agent related to the
aminoglycosides that binds to a protein in the 30S of ribosomes different from the target
of aminoglycosides. Similarly, tetracycline also targets bacterial 30S ribosomal subunit.
Spectinomycin and tetracycline are only bacteriostatic but inhibit a wide variety of bacteria
including Chlamydia and Mycoplasma.
Agents that bind to the 50S ribosomal sub unit are inhibitors of the elongation process
of protein synthesis. There are three classes of drugs under this—chloramphenicol,macrolides,
and lincinoids. Chloramphenicol acts by binding to a peptidyl transferase enzyme thereby
inhibiting peptide bond formation. It is a bacteriostatic agent that is effective against a number
of gram positive and gram negative organisms. Macrolides also act on peptidyl transferase
enzyme by interfering with its reaction or translocation. The most popular macrolide is
erythromycin which can effectively inhibit certain gram positive and gram negative bacteria
including Haemophilus, Mycoplasma, Chlamydia, and Legionella. Newer classes of macrolides are
azithromycin and clarithromycin which have broader spectrums of activity than erythromycin.
94 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The most important among the lincinoids or lincosamines is clindamycin. It has a similar target
as macrolides and like macrolides, it is only bacteriostatic. Agents that bind with both 30S and
50S ribosomal sub units include gentamycin and kanamycin.

Agents that Act on the Nucleic Acid

1. Agents that inhibit DNA topoisomerases – topoisomerase enzymes (types I and II)
are essential to DNA synthesis and are critical enzymes involved in protein translation
and cell replication. DNA gyrase, a type II topoisomerase is found only in prokaryotic
organisms and is essential for their survival. Among the different antimicrobial agents,
quinolones have been found to be most effective against DNA gyrase. First generation
quinolones include nalidixic acid and oxolinic acid. Newer generation of quinolones
include the fluoroquinolones, which have wider spectrum of activity than the quinolones.
2. Agents that inhibit RNA synthesis – agents that act by interfering with the β subunit of an
RNA polymerase that is needed for RNA synthesis. Rifampicin is a first line drug used
for the treatment of tuberculosis that specifically inhibits bacterial RNA synthesis.

Agents that Inhibit Microbial Metabolic Pathways

These agents interfere with metabolic pathways crucial for the survival of the
microorganism. Trimethoprim and sulfonamides are antibiotics that interfere with folic acid
metabolism. They act as competitive inhibitors of tetrahydrofolic acid which is important in the
synthesis of DNA, RNA, and bacterial cell wall proteins. Bacteria cannot utilize preformed folic
acid from the environment and thus must synthesize their own. Sulfonamides act specifically by
inhibiting formation of dihydrofolic acid. Trimethoprim inhibits formation of tetrahydrofolic
acid by inhibiting the enzyme dihydrofolate reductase.

Mechanisms of Drug Resistance

Drug resistance is a growing concern in the field of infection control. An organism is said
to have developed resistance to an antibiotic if it is not affected anymore by that particular
antibiotic. Development of resistance may either be innate (intrinsic) or acquired. Intrinsic
resistance is a stable genetic property that is encoded in the chromosome of the organism and
shared by all strains of the species. Acquired resistance is resistance arising from the ability
of an organism to resist an antimicrobial drug to which the species, as a whole, is naturally
susceptible. It is not normally encoded in the chromosome of the organism but developed in
the course of time due to constant exposure to the antimicrobial agent involved. It can be due to
chromosomal mutation or the result of genetic exchange between organisms
 Antimicrobial Agents 9
There are several factors that contribute to the development of antimicrobial resistance of
microorganisms. The most common is the overuse of broad spectrum antibiotics due to over
prescription. Other factors include incorrect diagnosis, unnecessary prescription of antibiotics,
indiscriminate or improper use of antibiotics by the patient, and the use of antibiotics as
additives to livestock feeds to improve the growth of the animals.
Resistance acquired through genetic exchange can occur through any of three
transduction, and conjugation. Transformation is the simplest and the ways—transformation,

earliest form of genetic exchange studied. In transformation, naked or free microbial DNA
inserts itself into the DNA of the same species. Transduction is the transfer of genetic
material by a bacteriophage. Conjugation is the transfer of genetic material through the
sex pilus. In conjugation, what is transferred to another bacterium is an extrachromosomal
DNA called plasmid. The resistance gene is carried by the plasmid.

Mechanisms of Drug Resistance

Drug modification or inactivation
Certain resistance genes may affect the activity of an antibiotic in two ways. A resistance
gene may code for enzymes that can alter its chemical structure leading to the inactivation of
the antibiotic, or the products of the resistance genes may cause hydrolysis of the antibiotic
thereby destroying the antibiotic. For example, certain bacteria produce beta lactamases which
can hydrolyze the beta lactam bonds in the chemical structure of the antimicrobial agent.
This is the most common mechanism of beta lactam resistance and is the mechanism involved
in the resistance of certain microorganisms to penicillin and cephalosporin.

Prevention of cellular uptake or efflux

Gram negative bacteria have developed the ability to change the lipid composition of
their outer membrane thereby preventing the antibiotic from reaching its cellular target. This
prevents their accumulation in the bacterial cell. In addition, there are gram positive and
gram negative bacteria that have developed an efflux pump that can prevent the antibiotic
to accumulate within the bacterial cell. This is true in the case of bacterial resistance to
tetracyclines and fluoroquinolones. Efflux pumps are effective against a wide range of
antimicrobials in multiple classes.

Modification of target sites

Antimicrobials have specific targets in the bacterial cell. Any change in the structure
of these target structures will lead to the inability of the antibiotic to exert its action on
the target bacteria. Certain bacteria have developed the ability to alter the normal target
96 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

binding sites of antibiotics thereby effectively inhibiting the drug to act on the infectious
agent. For example, the target site of penicillin on the bacterial cell is a structure called
penicillin binding protein (PBP). The organism Streptococcus pneumoniae has developed
resistance to penicillin by causing alteration in the structure of its penicillin binding protein.
In the case of Staphylococcus aureus, in addition to producing beta lactamase, the genetic changes
in the organism include the formation of a new PBP that is of low affinity to penicillin.
Other targets of antibiotics that have undergone alteration are indicated in Table 8.1.

Table 8.1 Examples of antimicrobial target sites that have undergone modifications
Target site modified Antibiotic involve
Peptide sub units of peptidoglycan Glycopeptides
Ribosome sub units Macrolides, tetracyclines, aminoglycosides
Metabolic enzymes Sulfa drugs, sulfones, trimethroprim
Lipopolysaccharide structure Polymyxins
DNA gyrase Fluoroquinolones
RNA polymerase Rifampin

Overproductionor bypass of target enzyme

One of the mechanisms developed by bacteria is targeting specific enzymes that are
essential to the metabolism of the organism. This is true in the case of antimicrobials that
function as anti metabolites. One way by which this is achieved is by over production of the
target enzyme of the bacteria. By overproducing the target enzyme of the antibiotic, there will
still be enough amount of the enzyme that is free from the antibiotic allowing the organism
to still carry out the essential enzymatic reaction. Some bacteria have developed alternative or
bypass mechanisms that can serve as alternative for the target enzyme. Both these mechanisms
are involved in bacterial resistance to sulfonamides.

Target mimicry
Target mimicry is a new mechanism of antimicrobial resistance that has been discovered.
It involves bacteria producing proteins that are similar in structure to the target sites of the
antibiotics. Due to the similarity in structure of the new proteins and the target proteins, the
antimicrobial binds the new proteins and not the target protein. For instance, the organism
Mycobacterium tuberculosis produces a protein that can be mistaken for the structure of DNA.
The protein selectively binds fluoroquinolones preventing its binding to the organism’s DNA
making the organism resistant to the drug.
 Antimicrobial Agents 97

Efflux pump Blocked penetration

• fluoroquinolones • β lactams
• aminoglycosides • tetracyclines
• tetracyclines • fluoroquinolones
• β lactams
• macrolides

Inactivation of enzymes Target modification

• β lactams • fluoroquinolones
• aminoglycosides • rifamycins
• macrolides • vancomycin
• rifamycins • β lactams
• macrolides
• aminoglycosides
Figure 8.1 Summary of mechanisms of antibiotic resistance by bacteri
98 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• Antimicrobials are substances that may be acquired from natural sources or are
synthetically produced in laboratories. These agents are designed to either kill the target
bacteria (bactericidal) or inhibit its growth (bacteriostatic).

• An ideal antimicrobial agent must have a broad spectrum of activity, be stable when
stored in either solid or liquid form, remain in target tissues for a specified amount of
time, destroy the target organism before it has a chance to produce disease, and not cause
damage or harmful effects on the host.

• Antimicrobials are classified in several ways:

1. Based on the spectrum of activity (broad spectrum or narrow spectrum)

2. Based on their antimicrobial activity (bactericidal or bacteriostatic)
3. Based on their absorbability from the site of administration (systemic or local)
4. Based on their mechanism of action
• Based on the mechanism of action, antibiotics may be classified into:
1. Agents that inhibit the synthesis of the bacterial cell wall
2. Agents that alter the function or permeability of the cell membrane
3. Agents that inhibit protein synthesis
4. Agents that act on the nucleic acid
5. Agents that interfere with bacterial metabolic pathways
• The indiscriminate use of antibiotics, together with inaccurate diagnosis and poor patient
compliance have contributed to the emergence of drug resistance of microorganisms.

• Resistance may be innate or acquired.

» Innate resistance is encoded in the chromosome of the organism.
» Acquired resistance is not normally coded in the chromosome of the organism but
may be the result of chromosomal mutation or genetic exchange between organisms.
» Acquisition of resistance genes through genetic exchange can occur in three
transduction, and conjugation. ways—transformation,

• There are several mechanisms developed by bacteria that enable them to develop
resistance to selected antimicrobials. These include (1) drug modification or interaction,
(2) prevention of cellular uptake or efflux, (3) modification of target sites, (4) production
or bypass of target enzyme, and (5) target mimicry
 Antimicrobial Agents 99

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. Which of the following mechanisms describes the action of beta lactamases?

a. Efflux pump c. Drug inactivation
b. Target mimicry d. Target overproduction
2. Which of the following resistance mechanisms is commonly effective against a wide
range of antimicrobials in multiple classes?
a. Efflux pump c. Target modification
b. Target mimicry d. Target overproduction
3. Which among the following methods of gene exchange involves the transfer
of naked or free DNA?
a. Transformation c. Conjugation
b. Transduction d. Binary fission
4. Which among the following methods of gene exchange involves the transfer
of plasmid DNA from a donor bacterium to a recipient bacterium?
a. Transposition c. Transduction
b. Transformation d. Conjugation
5. Which among the following is not a characteristic of a good antimicrobial agent?
a. Broad spectrum
b. Stable when stored
c. Does not remain for a long time in body tissues
d. Demonstrate selective toxicity
100 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Matching Type.

Column A Column B
6. Trimethoprim a. Inhibition of cell wall synthesis
b. Alteration of cell membrane
7. Ampicillin
8. Metronidazole
c. Inhibition of protein synthesis
d. Inhibition of DNA synthesis
9. Amphotericin B e. Inhibition of RNA synthesis
10. Rifampicin f. Inhibition of folic acid synthesi
 CHAPTER
Host Response
9 to Infection

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. differentiate “antigen” from “immunogen;”
2. discuss the properties of antigen that would make them immunogenic;
3. describe the different lines of defense of the body;
4. determine the role of the B cells and T cells in providing defense against infectious
agents;
5. differentiate innate immunity and adaptive immunity;
6. identify the differences between humoral and cell mediated immune responses;
7. examine the functions of the different immunoglobulins;
8. differentiate primary immune response and secondary immune response;
9. distinguish from each other the mechanisms involved in the various types of
hypersensitivity reactions, citing examples for each type; and
10. explain the role of vaccines in the prevention of the development of infectious diseases.

Definition of Terms
Immunology – the study of the immune system and the immune response.
Immunogen – any substance capable of inducing an immune response, whether humoral or
cell mediated or both.
Antigen – a substance recognized by the immune system, whether by the B cell or the T cell,
that serves as the target of the immune response but may not necessarily lead to an immune
response.
Epitope – the structure in the antigen that is recognized by the B cell or the T cell.
Hapten – a substance that is of low molecular weight that can only induce an immune response
if bound to another substance that is already immunogenic (carrier molecule)
102 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Properties of Antigens
There are several properties that an antigen must possess to make it immunogenic. These
include (1) foreignness and genetic composition, (2) chemical composition and complexity,
(3) molecular size and stability, and (4) mode of entry of the antigen. Antigens are genetically
foreign to the host or recognized by the body as non self. For example, if a pig’s heart is
transplanted into a human being, the chances of the human body developing an immune
reaction leading to the rejection of the transplanted heart will be very high because it is
genetically different from humans. In the same manner, most humans are exposed to similar
environmental components (e.g., dust) but not all will have similar reactions. One person may
manifest a hypersensitivity reaction to a substance that will provoke no reaction in another.
This is because each individual has a different genetic composition from another.
The chemical composition and complexity of an antigen may also affect its
immunogenicity. Most organic substances can be antigenic except for pure lipids and nucleic
acids. Of the different chemical groupings, proteins are the most immunogenic. This is because
proteins are larger molecules than others that have more complex structures. Likewise, between
a pure protein and a glycoprotein, a glycoprotein will be more antigenic because its structure is
more complex.
The molecular size of an antigen is another property that can affect its immunogenicity.
As a rule, molecules with molecular weights below 10,000 daltons are weakly immunogenic
or not immunogenic at all. Those with molecular weights greater than 10,000 daltons are very
potent immunogens. However, one needs to consider the stability of the molecule. There are
some substances that have high molecular weights that break up into smaller molecules once
they enter the body, in which case they lose their immunogenicity.
Finally, immunogenicity also depends on how the antigen is administered. The dose of the
antigen as well as the mode of administration should be taken into consideration. For instance,
one might need a small amount of antigen to induce an immune response if the antigen
introduced is a protein as compared to a larger amount if the antigen were a polysaccharide.
In the same manner, antigens may not elicit a reaction intramuscularly but may provoke a good
response when given subcutaneously.

The Immune System

The immune system is composed of molecular and cellular components that are derived
from the central (primary) and peripheral (secondary) lymphoid organs. The central lymphoid
organs are the primary sites for differentiation and maturation of the important cells that
play an important role in adaptive immunity which are the T lymphocytes (or T cells) and
the B lymphocytes (or B cells). These consist of the bone marrow and the thymus. The bone
marrow is the site from where blood cells originate. The precursor cells for lymphocytes ar
 Host Response to Infection 103

found in the adult bone marrow and this is where they differentiate into B cells and T cells.
Once differentiated, B cells remain in the bone marrow and undergo maturation in the bone
marrow. On the other hand, the T cells will go out of the bone marrow as immature and
incompetent forms then go to the thymus where they mature and become competent. After
maturation, the mature B cells and T cells proceed to the peripheral lymphoid organs to await
any antigen that may enter the body.
The peripheral lymphoid organs consist of the lymph nodes, spleen, and the mucosa
associated lymphoid tissues (MALT), which include the tonsils, adenoids, Peyer’s patches in the
ileum, and the appendix. These organs are the site of reactivity of lymphoid cells. These are
where antigens are trapped and subsequently encounter the T and B cells. Antigens are brought
to these peripheral lymphoid organs where the cells needed for their destruction are located.
Both mature T cells and B cells are found in the peripheral lymphoid organs.

Primary Lymphoid Organs Secondary Lymphoid Organs

Thymus
Spleen

Bone Marrow
Lymph nodes

(Tonsils and Adenoids Bronchus

Mesenteric Peyer’s patch)

Figure 9.1 The primary (central) and secondary (peripheral) lymphoid organs

Cells of the Immune System

The various responses of the human immune system are mediated by specific cells
and the substances they produce. These cells include the white blood cells which include
(1) granulocytes (e.g., neutrophil) which are 50%–80% of white blood cells; (2) lymphocytes,
approximately 20%–45% of total white blood cells; and (3) monocytes and macrophages,
3%–8% of white blood cells. Neutrophils play a major role in acute inflammation as well as in
bacterial infections. Lymphocytes and macrophages are mainly involved in chronic inflammation.
Lymphocytes are the predominant inflammatory cells in viral infections. Macrophages are
also predominant in chronic inflammation. In addition, cells that belong to the mononuclear
phagocyte system play crucial roles as antigen presenting cells
104 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

As the name implies, antigen presenting cells are cells that are involved in the processing
and presentation of antigens to the T cells. These include the macrophages, B cells, dendritic
cells, Langerhans cells in the skin, Kupffer cells in the liver, and glial cells in the central nervous
system. B cells, macrophages, and dendritic cells are the professional antigen presenting cells,
the most important of which are the dendritic cells. Some of these cells are found in association
with lymphoid follicles in the lymph nodes and are thus called follicular dendritic cells.
Langerhans cells in the skin also bring antigens to the paracortical zone of the lymph node
where they are called interdigitating dendritic cells. Dendritic cells are considered as the true
link between innate and adaptive immunity.
Other white blood cells that are part of the innate arm of the immune system include
eosinophils, basophils, and platelets. Eosinophils possess eosinophilic granules that play a role
in type I hypersensitivity reaction or allergy. In addition, eosinophils also secrete a substance
that is called major basic protein that is toxic to parasites, especially helminths or worms.
Like eosinophils, basophils also play a role in allergies. The granules of both eosinophils and
basophils contain histamine which when released is responsible for the changes seen during
the initial phase of an allergic reaction. Finally, platelets are membrane bound cell fragments
that are derived from large cells called megakaryocytes. Platelets are mainly involved in blood
coagulation, however, they secrete substances that play a role in inflammation.
Natural killer cells are large granular lymphocytes that are also called NK cells or Null cells.
They were originally classified as cytotoxic T cells because they had the same manner of killing
target antigens. However, studies conducted on their structures showed that all T cells had a
T Cell Receptor (TCR) on their surface that was not present in NK cells, hence NK cells are
not classified anymore as T cells. NK cells are classified as large granular lymphocytes and are
part of the body’s innate immune system.

Bone marrow Thymus Tissues

Fetal liver
T cell

NK Cells NK Cells
B Cell Blood
Lymphocytes
recirculating lymphocytes
Stem
cell Mast cell

Monocytes Monocytes Tissue macrophages

Megakaryocyte Platelets
Granulocyte Granulocyte

Lymphocytes
Bloo

Macrophages
Interdigitating cells antigen presenting cells
Dendritic cells

Secondary lymphoid tissues

 Host Response to Infection 105

Multipotential hematopoietic stem cell

(Hemocytoblast)

Common myeloid progenitor Common lymphoid progenitor

Erythrocyte Mast cell Myeloblast Natural killer cell Small lymphocyte

(Large granular lymphocyte)
Megakaryocyte

Basophil Neutrophil Eosinophil Monocyte T lymphocyte B lymphocyte

Thrombocytes

Plasma cell

Macrophage Dendritic cell

Figure 9.2 Origins of the cells of the immune system

Granulocytes Agranulocytes

Neutrophil Eosinophil Basophil Lymphocyte (small) Monocyte

Multilobed nucleus, Bilobed nucleus, Bilobed nucleus, Large spherical Kidney shaped
pale red and red cytoplasmic purplish black nucleus, thin rim of nucleus, abundant
blue cytoplasmic granules cytoplasmic granules pale blue cytoplasm pale blue cytoplasm
granules

Figure 9.3 White blood cells that play important roles in the body’s immune response
106 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The most important cells of the immune system that play pivotal roles in adaptive
immunity are the T lymphocytes and the B lymphocytes. As mentioned, both cells originate
from the bone marrow. B cells mature in the fetal liver and in the adult bone marrow, which
is the equivalent of the bursa of Fabricius in birds. In the peripheral lymphoid organs, they are
located mostly in the germinal centers of the lymph nodes and in the spleen. In the presence of
the appropriate antigen, the B cells differentiate into antibody producing plasma cells as well
as memory B cells. They are involved in the body’s humoral immunity. At the same time, the
B cells also function as a professional antigen presenting cell.
T cells are located mainly in the paracortical and interfollicular areas of the lymph nodes
and spleen. They are involved in the body’s cell mediated immunity. The T cells further
differentiate into CD4+ T cells and CD8+ T cells (cytotoxic or cytolytic). The CD4+ T cells
consist of the helper T cells and the regulatory T cells (CD4+CD25+ T cells). The helper
T cells do not have the direct capacity to destroy an antigen. Instead, it activates the cytotoxic
T cells and stimulates differentiation of B cells into antibody producing plasma cells. The
regulatory T cells play an important role in the maintenance of self tolerance or the ability
of the immune system to recognize self from non self. The T cells, most especially the CD4+
T cells are the predominant lymphocytes in the circulation and constitute part of the body’s
immune surveillance. Some T cells also differentiate into memory T cells.

Innate Immunity
Innate immunity is also known as natural immunity. This immunity is already active from
the time of birth, prior to exposure to an antigen. Innate immunity is non specific. It includes
host barriers that prevent entry of microorganisms such as the skin and mucous membranes
(first line of defense), and processes such as phagocytosis and inflammation (second line of
defense) which prevents the multiplication of organisms that gain entry to the body preventing
them from multiplying before they have a chance to produce disease. It is activated within
minutes following exposure to the antigen. However, it does not improve after exposure to the
antigen and does not possess memory and thus provides only short term protection.
The innate arm of the immune system performs two major functions: killing invading
microorganisms and activating adaptive immune responses. As mentioned, it consists of
the body’s first and second lines of defense. The first lines of defense serve to prevent entry
of the organism to the body and limit microbial survival. Physical and chemical barriers
prevent attachment and entry of the organisms. These include the skin, fatty acids in
sebaceous secretions, and sweat. The low pH of the fatty acids and sweat inhibit the growth of
microorganisms. In addition, the normal flora of the skin and other parts of the body form a
biological barrier that inhibits the colonization and multiplication of pathogenic organisms by
competing with the pathogenic organisms for nutrients and by priming the immune system
 Host Response to Infection 10

Microorganisms that penetrate the first line of defense are prevented from multiplying
inside the body by the body’s second line of defense. If the primary barriers are breached,
inflammation is activated as well as the natural killer cells. The microorganisms are recognized
by innate immune cells and soluble mediators because of their molecular patterns called
pathogen associated molecular patterns (PAMP). Inflammatory cells possess pattern recognition
receptors which allow them to act on these pathogenic organisms. Soluble host proteins
specifically those that are part of the complement system also possess such pattern recognition
receptors (e.g., mannose binding lectin). Recognition of these patterns in turn activate the
inflammatory cells and the complement system. Activation of inflammatory cells lead to
phagocytosis of the antigen while activation of the complement system results in the production
of membrane attack complex which help degrade the antigen.

Table 9.1 Important components of innate immunity

Factors that limit entry of microorganisms Mode of action
Keratin layer of intact skin Act as a mechanical barrier
Lysozyme in tears and other secretions Degrades bacterial cell wall
Respiratory cilia Directs organisms trapped in mucus out of the
Low pH of stomach and vagina; fatty acids in respiratory passages
skin Inhibits growth of microorganisms
Surface phagocytes Ingest and destroy microbes
Normal flora Prevent colonization by pathogens
Factors that limit growth of the Mode of action
microorganisms within the body
Natural killer cells Kills virus infected cells
Neutrophils Ingest and destroy microbes
Macrophages and dendritic cells Ingest and destroy microbes; present antigens
Interferons to T cells
Complement system Inhibit viral replication; produce anti viral state

Fever Membrane attack complex creates holes in

bacterial cell membrane; components activate
Inflammatory response inflammation
Inhibits bacterial growth
Limits spread of microbes by destroying them
Adapted from Levinson, W.: Review of Medical Microbiology and Immunology [9th ed.], p. 396, McGraw Hill, 2006

Microbes able to escape the second line of defense are acted upon by the final line of
defense which is the immune response. This involves the B cells and the T cells, cells that are
involved in adaptive immunity.
108 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Adaptive Immunity
Adaptive immunity is specific. It is activated after exposure to a particular antigen. Unlike
innate immunity, it is an acquired response to an antigen that is initiated by recognition of
specific epitopes of the foreign invaders. It involves production of antibodies by the B cells and
activation of the cytotoxic T cells. The response is delayed compared to innate immunity since
it takes about 7–10 days before sufficient levels of antibodies are produced by the body. At the
same time, antigens need to be processed first before they can be acted upon by the cytotoxic
T cells. However, unlike innate immunity, the protection given by adaptive immunity is longer
and, in most cases, lasts throughout the lifetime of the individual.
An important distinction between innate and adaptive immunity is the fact that adaptive
immunity possesses memory. Once the B or T cells are activated, some of the B and T cells are
converted to memory cells. The presence of these memory cells ensures a higher response once
there is re exposure to the same antigen, making the response amplifiable.

Table 9.2 Comparison between innate immunity and adaptive immunity

Property Innate Immunity Adaptive Immunit
Activity at birth Yes No
Response time Immediate Delayed
Specificity for microorganisms Relatively low (PAMPs) High (specific antigens)
Cells Phagocytic cells, NK cells, B lymphocytes and
epithelial cells T lymphocytes

Memory No Yes
Response amplifiable No Yes
Components
Physical and chemical Skin, mucosa; antimicrobial Secreted antibodies
barriers substances (e.g., defensins)
Blood proteins Complement Antibodies
Line of defense First and second Third (immune response)

Immune Response
First exposure with an antigen leads to the activation of a specific set of helper T cells
called the Th1 cells. Activation of Th1 cells leads to activation of the inflammatory response
and delayed type hypersensitivity as well as stimulation of B cells to produce IgM and IgG. The
antibodies become detectable in the serum after about 7–10 days but can be longer depending
on the nature of the antigen and the dose of the antigen. The serum level of antibodies
continues to rise for several weeks and then declines and may drop to very low levels.
 Host Response to Infection 109

Secondary immune response occurs after re exposure to the same antigen. A second
encounter with the same antigen or a closely related one occurring months or years after the
primary response will activate another set of helper T cell called the Th2 cells. This will lead to
further production of antibodies (except IgM). This response leads to a rapid antibody response
of a much higher intensity than the primary response. This is explained by the persistence of
antigen specific memory cells. The predominant antibody involved is IgG and the levels tend to
persist much longer than the primary response. However, if there is a need for other antibodies,
some of the IgG in the circulation can undergo modifications in their structure to become
converted to another antibody (e.g., IgA or IgE). This process is called class switching.

Primary Secondary
antibody response antibody response

First infection Repeat infection

Plasma cells
IgG IgG

IgM

Short lived plasma

cells in lymphoid
organs Low level Memory
antibod antibody B cell
Low lived
Activated production
plasma cells
B cells in bone marrow
of
Amount
Low lived
plasma cells Memory
in bone marrow B cell
Naive B cell

0 7 >30 0 3 10 >30
Days after
antigen exposure

Figure 9.4 Primary and secondary immune responses

Humoral Immunity
Innate and adaptive immunity can be humoral or cell mediated. Innate humoral immunity
involves cytokines and the complement system. Adaptive humoral immunity involves the action
of antibodies. Antibody mediated immunity is directed primarily against (1) extracellular
pathogens, (2) toxin induced diseases, (3) certain viral infections, and (4) infections caused by
encapsulated pathogens (e.g., pneumococci and Haemophilus influenzae).
110 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Antibodies
Antibodies are globulin proteins (immunoglobulins) that react specifically with the
antigens that stimulate their production. The most important functions of antibodies
are (1) to neutralize toxins and viruses; (2) to opsonize microbes so that they will be readily
recognized and more easily phagocytosed; (3) to activate complement system; and (4) to
prevent the attachment of microbes to mucosal surfaces.

Antibody Structure
A typical immunoglobulin is shaped like a letter “Y” and consists of polypeptide chains
linked by disulfide bonds. An immunoglobulin is made up of two identical heavy chains
(50–70 kD) and two identical light chains (23 kD). The heavy chains consist of polypeptide
chains of 440–550 amino acid residues in length. Each immunoglobulin class has its own
structurally distinct heavy chain—gamma (γ) for IgG, mu (μ) for IgM, alpha (α) for IgA, delta
(δ) for IgD, and epsilon (ε) for IgE. The light chains are approximately 220 amino acid residues
long and are either kappa (κ) chains or lambda (λ) chains.
Each chain is composed of a variable region and a constant region. The variable region
contains the hypervariable region that represents the antigen binding site of the antibody.
The antigen binding site is therefore composed of the variable regions of both the heavy and
light chains. Interchain disulfide bonds hold together two heavy chains. Intrachain disulfide
bonds are found within each of the polypeptide chains.
The region at which the arms of the antibody molecule form a letter Y is a flexible region
called the hinge region. Digestion of this region with either papain or pepsin will yield two
identical antigen binding fragments (called Fab) and one crystallizable fragment (called Fc)
which binds to effector cells
 Host Response to Infection 111

Light chain
hypervariable
regions
Light chain

VL
Heavy chain

Antigen
Fab
V CL
binding
Heavy chain
CH hypervariable
regions
Interchain
disulfide Papain cleavage site
bonds CH 2 Hinge region
Biological Papain cleavage sites
activity Fc Intrachain
disulfide Complement binding region
mediation CH 3
bonds Carbohydrate
VL and VH: variable regions
CL and CH : constant regions

Figure 9.5 Basic structure of an antibody showing the Fab and Fc fragments as well as the heavy
chains and light chains with their respective variable and constant regions. The chains are held
together by interchain disulfide bonds.

Classes of Immunoglobulins
There are five classes of immunoglobulins found in all species and all individuals. Each
immunoglobulin class is defined by its component heavy chain.
1. IgG – a monomer and is the predominant antibody in the secondary immune
response (anamnestic response) and is a major defense against bacteria and viruses.
It comprises approximately 73% of the immunoglobulins in the serum. It consists
of four subclasses or isotypes: IgG1 (most common), IgG2 , IgG3 , and IgG4 . It is the
only antibody to cross the placenta (except IgG4). It is therefore the most abundant
antibody in newborns. Together with IgM, it can fix or activate complement system
(except IgG4 ). It also functions as an opsonin, thus enhancing phagocytosis. It is the
main immunoglobulin in chronic infections.
112 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. IgM – the largest among the immunoglobulins and is a pentamer. It has a J chain
(joining chain) that holds the IgM pentamer together. It is the main immunoglobulin
produced early in the primary response and is the predominant antibody in acute
infections. Together with IgG, it can activate the complement system. It is the more
efficient activator of complement owing to its large size. It is also present on the
surface of B cells where it acts as an antigen receptor.
3. IgA – called the secretory immunoglobulin and is the main immunoglobulin in
secretions such as colostrum, saliva, and tears, as well as respiratory, gastrointestinal,
and genitourinary tract secretions. It exists as a monomer in serum and as a dimer
in secretions where the two monomeric units are held together by a J chain. It is an
important component of mucosal immunity.
4. IgE – also called the reaginic antibody. It is medically important
for two
reasons: (1) it mediates immediate or anaphylactic hypersensitivity reaction, and
(2) it provides defense against parasites such as helminths or worms. It binds to the
surface of mast cells and basophils where it serves as antigen receptor for the allergen.
It exists in monomeric form.
5. IgD – a monomer that has no known antibody function. It is found on the surface of
many B cells and serves as the surface marker for B cells but may also function as an
antigen receptor. It is present in small amounts in serum (approx. 1%).

Table 9.3 Comparison of the major classes of immunoglobulins

Secretory
component
IgM IgG IgA IgE IgD
Heavy Chain μ (mu) γ (gamma) α (alpha) ε (epsilon) δ (delta)
MW (Da) 900k 150k 385k 200k 180k
% of total
antibody 6% 80% 13% 0.002% 1%
in serum
Fixes
Yes Yes No No No
complement
Primary response, Main blood Secreted Antibody of B cell
fixes complement. antibody, into mucus, allergy and Receptor
Function
Monomer serves neutralizes toxins, tears, saliva anti parasitic
as B cell receptor opsonization activity
Adapted from: Prosci. (n.d.) Antibody Structures and Properties. Retrieved from https://www.prosci inc.com/
resources/antibody development guide/antibody structure and propertie
 Host Response to Infection 11

Cell mediated Immunity

Cell mediated immunity has four basic functions, namely: (1) provide resistance and aid
in recovery from infections due to intracellular organisms (e.g., viruses); (2) important defense
against fungi, parasites, and bacteria; (3) involved in transplant and graft rejection; and (4) main
defense against tumor cells.
The components of the cell mediated immune system include several cell types. These
are the macrophages, natural killer cells, helper T cells, and cytotoxic T cells. Macrophages,
together with B cells and dendritic cells present antigens to T cells. In addition, macrophages
are phagocytic cells that ingest and destroy microbes.
Helper T cells (CD4+ T cells) are of several sub types. The most important are Th1 ,
Th2 , and Th17 . In general, the helper T cells function to stimulate differentiation of B cells
to antibody producing plasma cells as well as to activate the cytotoxic T cells. Th1 cells are
activated on first encounter with an antigen and is responsible for triggering inflammation,
delayed type hypersensitivity and synthesis of IgM and IgG. Th2 response is seen on
re encounter with the same antigen leading to further synthesis of antibodies as well as class
switching. Th17 cells are chemotactic for neutrophils and play a role when further inflammation
is required.
Cytotoxic T cells or the CD8+ T cells destroy antigens primarily through the perforin
granzyme mechanism. Once the antigen is presented to the CD8+ T cells, these release perforin
which will cause formation of pores on the wall or membrane of the antigen. Afterwards,
granzyme is released by the CD8+ T cells which will cause destruction of the antigen while at
the same time activate caspases leading to activation of apoptosis or programmed cell death.
CD8+ T cells are the body’s main defense against intracellular pathogens. It is also involved in
transplant and graft rejection as well as destruction of tumor cells.

Complement System
The complement system consists of a group of soluble proteins (C1 – C9) which are
proteases that cleave and activate one another in a sequential manner. They are secreted as
inactive enzymes which are enzymatically activated by other complement proteins. This
pathway is mediated by a single molecule of IgM or two molecules of IgG (IgG1 , IgG2 ,
or IgG3). There are three main effects of activation of the complement system, namely:
(1) lysis of cells; (2) generation of inflammatory mediators; and (3) opsonization leading
to enhanced phagocytosis. It involves four basic steps: (1) initiation, (2) formation of C3
convertase, (3) formation of C5 convertase, and (4) formation of membrane attack complex
(MAC). C3 convertase is required to cleave C3 to form C5 convertase, which is essential to
cleave C5 to form the terminal product which is MAC. There are three pathways that act
synergistically with each other—alternative or properdin pathway, classical pathway, and
mannose binding lectin or MBL pathway.
114 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The alternative or properdin pathway is activated by bacterial products such as endotoxin

or complexes of immunoglobulins. It is part of innate immunity and is the first to be
activated on initial exposure to the antigen. It bypasses C1, C2, and C4. Upon exposure to
the antigen, the C3 component undergoes spontaneous hydrolysis and binds to activated
factor B forming C3bBb (C3 convertase). The C3 convertase cleaves C3 to form C3bBb3b
(C5 convertase) which cleaves C5 to form C5b6789 (MAC). The C3 convertase is stabilized
by properdin P. C3b produced by the other pathways can be utilized by the pathway in what is
known as an amplification loop.
The classical pathway of complement system is activated by antigen antibody complexes.
These complexes bind to C1qrs (recognition unit) which will lead to spontaneous activation
and cleavage of complement proteins C2 and C4. The resulting C4b and C2b combine to form
C4b2b (C3 convertase) which will cleave C3 to form C4b2b3b (C5 convertase). This pathway
functions in both innate and adaptive immunity. It is the last to be activated since it requires
some time for specific antibodies to be produced.
The mannose binding lectin or MBL pathway is activated by specific patterns of sugars
found on the bacterial cell wall. Lectin, the first to be discovered, is a pattern recognition
receptor that recognizes the pattern of mannose containing sugars. Other sugar patterns that
do not contain mannose are recognized by another set of pattern recognition receptors called
ficolins. Upon binding of lectin with the sugar on the bacterial cell wall, there is simultaneous
activation of C4 and C2 as in the classical pathway. The sequence of activation of the
complement proteins in both classical and MBL pathways are the same except that the MBL
pathway does not utilize complement protein C1 and the C3 convertase produced is C4b2a.
C5 convertase in this pathway is therefore composed of C4b2a3b.
It should be noted that all three pathways lead to the production of C3b, called the central
molecule of the complement system. C3b functions as an opsonin. At the same time, B cells
have a receptor for C3b and binding of C3b to the receptor on the B cells will stimulate
differentiation of B cells into antibody producing plasma cells, promoting further production of
antibodies. In addition to C3b, C1q in the classical pathway also functions as an opsonin. Other
important products of complement activation include the following:
1. C3a and C5a – chemotactic for neutrophils; chemical mediators in inflammation
causing vascular leakage or increased vascular permeability
2. C3a, C4a, and C5a – function as anaphylatoxins, causing degranulation of mast cells
and release of histamine
3. Membrane attack complex (MAC) – cause lysis of the bacterial cel
 Host Response to Infection 11

HypersensitivityReactions
Hypersensitivity reactions are exaggerated and inappropriate immune responses that lead
to tissue injury resulting in harm to the host. It occurs when an already sensitized person is
re exposed to the same foreign antigen. The injury may be brought about by the various
substances and chemical mediators activated during inflammation as well as the activation of
the complement system. Hypersensitivity reactions may be categorized into four—types I, II,
III, and IV. Types I, II, and III are mediated by antibodies while type IV is mediated by T cells.

Type I: Immediate (Anaphylactic) Hypersensitivity

This is what is commonly known as an allergic reaction and is mediated by IgE. It occurs in
response to environmental or administered antigens. Common allergens include pollens, animal
fur, foods, and various drugs. The process begins when an allergen is presented by dendritic
cells to naïve CD4+ T cells which differentiate into Th2 cells. The Th2 cells in turn release
interleukin 4 (IL 4) which stimulate class switching to IgE. The allergen binds to the IgE and
the complex formed binds to and sensitizes mast cells. Subsequent exposure to the allergen
leads to cross linking among the IgE allergen complexes stimulating degranulation of mast
cells and subsequent release of histamine.
Type I hypersensitivity can be divided into two phases: immediate phase and late phase.
The immediate phase represents the vascular events of inflammation which include vasodilation
and increased vascular permeability. The major chemical mediator during this phase is
histamine. The reaction occurs within minutes of re exposure to the allergen and will eventually
subside. About 2–24 hours after the initial phase, there is a recurrence of the symptoms of the
patient. The late phase represents the cellular events of inflammation where the tissues will
show infiltration by inflammatory cells which include neutrophils and eosinophils. Eosinophils
recruit release enzymes that further cause damage to the mucosa. Main chemical mediators
during this phase include slow reacting substances of anaphylaxis or SRS A consisting mainly
of leukotrienes C4, D4, and E4 (LTC4, LTD4, and LTE4, respectively). Prostaglandins also
play a role during this phase.
116 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Antigen

IgE Signals for

IgE Fc receptor cytokine gene
activation

Signals for Signals for

degranulation Nucleus
activation of
phospholipase A2

Degranulation

SECRETED CYTOKINES

GRANULE CONTENTS MEMBRANE

Late phase reaction
• Histamine PHOSPHOLIPIDS
Leukocyte infiltration
• Proteases
• Chemotactic factors Epithelial damage
(ECF, NCF) Bronchospasm
Arachidonic acid PAF

Prostaglandin Leukotrienes
D2 B4 , C4 , D4

Immediate reaction
Vasodilation
Vascular leakage
Smooth muscle spasm

Figure 9.6 Pathogenesis of type I hypersensitivity reaction showing the important chemical
mediators involved in both the initial and late phases of the reaction

The clinical manifestations are typical in a given individual. These are classified into local
anaphylaxis and systemic anaphylaxis. Examples of local anaphylaxis include food allergy,
urticaria (hives), eczema, allergic rhinitis or hay fever, and asthma. Systemic anaphylaxis is a
severe allergic response where patients manifest symptoms of circulatory collapse such as
hypotension, severe bronchoconstriction,and laryngeal edema. This is a serious reaction that
is potentially fatal and can be induced by foods such as peanuts and seafoods, bee venom
 Host Response to Infection 117

and certain drugs (e.g., aspirin and penicillin). Individuals with type I hypersensitivity are said
to be atopic and will have IgE levels higher than the general population.
Diagnosis of type I hypersensitivity involves accurate history taking. The condition
is usually familial and good history taking techniques will elicit information of other family
members suffering from the same condition. Other modalities are available aimed at identifying
the specific antigen to which an individual is allergic to. These include the skin prick test and
the scratch test. In the skin prick test, known allergens are administered subcutaneously like
doing a skin test. After one hour, the resulting wheal and flare reaction is measured where a
size of > 10 mm is considered positive. In the scratch test, superficial scratches spaced equally
are created on the ventral aspect of the forearm after which varying solutions of known food
allergens are applied. The size of the wheal and flare reaction is again measured with > 10 mm
considered as positive.

a b

Sheep Wool
Feather Cat Dog Horse
Histamine

Alternaria
Grass Daisy (Mould)
Plane Birch
Pollen Pollen
Negative Pollen Pollen
Control

Figure 9.7 a Skin prick test and b scratch test performed for allergy testing

Type II: Antibody mediated Hypersensitivity

Type II hypersensitivity, formerly known as cytotoxic or cytolytic hypersensitivity, involves
three sub types, two of which involve destruction of cells. However, a third sub type does not
involve cytotoxicity but instead involve cellular dysfunction which is the reason why the name
was changed. This hypersensitivity reaction is mediated by IgG or IgM. The classification of
the sub types will be based on the mechanism involved.
The first sub type involves the processes of opsonization and phagocytosis. Opsonization is
the process where an antigen is coated by molecules that facilitate recognition by phagocytic
cells resulting in enhanced phagocytosis. An antigen stimulates production of antibodies,
usually IgG, and the complex formed stimulates the classical pathway of complement
system. This leads to the generation of C1q and C3b which both function as opsonin
thereby enhancing phagocytosis. Note that IgG is also an opsonin. Opsonization of cells by
IgG antibody can also cause activation of natural killer cells which can destroy the targe
118 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

cells without phagocytosis. This is what is known as antibody dependent cellular cytotoxicity
or ADCC. Examples of conditions under this mechanism are transfusion reactions, blood
incompatibilities (ABO and Rh incompatibilities), autoimmune hemolytic anemias, and certain
drug reactions that will lead to hemolysis of red blood cells.

Opsonization and phagocytosis

Phagocytosed
Opsonized cell Fc receptor cell

C3b
Phagocyte
C3b receptor
Complement activation Phagocytosis

Figure 9.8 Pathogenesis of type II hypersensitivity reaction illustrating the mechanism

of opsonization and phagocytosis

The second sub type involves the process of complement and Fc receptor mediated
inflammation. This is initiated when antibodies (IgG or IgM) deposit in fixed tissues such
as basement membrane or extracellular matrix. This will again lead to activation of classical
pathway of complement system leading to the generation of C3a and C5a which are both
chemotactic for neutrophils thus promoting inflammation. Acute rheumatic fever is an example
of a condition under this mechanism.

Complement and Fc receptor mediated inflammation

Neutrophil
Fc receptor
Complement enzymes,
by products reactive oxygen
(C5a, C3a) intermediates
Complement activation Inflammation and tissue injury
Figure 9.9 Pathogenesis of type II hypersensitivity reaction illustrating the mechanism
of complement and Fc receptor mediated inflammation

The third subset under type II hypersensitivity reaction involves formation of

autoantibodies directed against specific cellular receptors (called antibody mediated
cellular dysfunction). In this mechanism, there is no destruction or lysis of target cells.
The autoantibodies produced may act as competitive inhibitors or may mimic the action o
 Host Response to Infection 119

the normal ligand for the receptor. There are two conditions that illustrate this mechanism.
The first example is myasthenia gravis. It is a neuromuscular disease that is characterized by
progressive muscle paralysis. The pathogenesis involves formation of antibodies directed against
acetylcholine receptors. In normal conditions, acetylcholine is released by vesicles found at the
terminal portion of the nerves. The acetylcholine binds to the receptor found on muscle cells.
Binding of the acetylcholine with its receptor will initiate muscle contraction. In myasthenia
gravis, autoantibodies against acetylcholine receptors bind to the receptors preventing the
binding of acetylcholine to the receptors. In this manner, the autoantibodies act as competitive
inhibitors for acetylcholine. Because acetylcholine cannot bind to the receptor, there is no
stimulation of muscle contraction. The muscles that are involved are initially those that
are frequently used such as the eyelid muscles. The earliest manifestation of the condition is
inability to open the eyelids causing drooping of the eyelids (ptosis).
A similar process is seen in Graves’ disease. In this condition, antibodies against receptors for
thyroid stimulating hormone (TSH) are produced. TSH normally stimulates the thyroid gland
to produce thyroid hormone in cases when the hormone level is reduced. The autoantibodies
produced bind to the TSH receptors found in the thyroid gland and mimic the action of TSH.
The net result is continuous stimulation of the thyroid gland to produce hormones so the
patient manifests symptoms of hyperthyroidism.

Antibody mediated cellular dysfunction

Nerve ending Acetylcholine Antibody TSH receptor

(ACh) against
Antibody TSH receptor Thyroid
to ACh epithelial
receptor ACh receptor cell

Muscle Thyroid hormones

Antibody inhibits binding of
neurotransmitter to receptor Antibody stimulates receptor without hormone
Figure 9.10 Pathogenesis of type II hypersensitivity reaction illustrating the mechanism of
antibody mediated cellular dysfunction seen in myasthenia gravis and Graves’ disease

Figure 9.11 a Ptosis of the right eye in a patient with myasthenia gravis and b exophthalmos in a
patient with Graves’ diseas
120 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Type III: Immune Complex mediated Hypersensitivity

Immune complex mediated hypersensitivity reaction is initiated by the formation of
immune complexes in the circulation. The immune complexes form deposit in various tissues,
usually in the kidneys and on the endothelium of blood vessels. Like type II hypersensitivity
reaction, classical pathway of complement system is activated leading to recruitment of
inflammatory cells and inflammation at the site of deposition. As a result, not only is the
target antigen destroyed but the underlying tissues where the immune complexes are deposited
as well, leading to damage to the tissues. In blood vessels, this leads to a condition known as
fibrinoid necrosis. Infectious processes that may involve this type of hypersensitivity reaction
include involvement of the kidneys in malaria (“black water fever”), dengue, and hepatitis B
infection (polyarteritis nodosa).
Type III hypersensitivity is of two forms—local immune complex disease or systemic
immune complex disease. Local immune complex disease is exemplified by Arthus reaction.
This is seen as a complication of immunization especially with vaccines that are given with
multiple doses (e.g., DPT). These vaccines are usually given at prescribed intervals. If the
vaccine is given before the next schedule is due and is injected at the same site as the previous
dose, immune complexes form and precipitate in the walls of blood vessels leading to fibrinoid
necrosis. The result is the formation of a localized area of tissue necrosis at the injection site.

Membrane of
blood vessel 1 Immune complexes are
deposited in the wall
Ag of the blood vessel
Neutrophils

2 Presence of immune 3 Enzymes released

complexes activates from neutrophils
complement cause damage
and attracts to endothelial
inflammatory cells cells of basement
Endothelial such as neutrophils membrane
cell

Figure 9.12 Pathogenesis of type III hypersensitivity reaction illustrating the formation of immune
complexes and subsequent deposition in tissue
 Host Response to Infection 121

Figure 9.13 Arthus reaction seen complicating

administration of TDaP vaccine

The second form of type III hypersensitivity is systemic immune complex disease,
exemplified by acute serum sickness. This is triggered by the administration of large amounts of
foreign serum (e.g., anti tetanus serum) or after receiving antibodies from another person or
species. Some drugs like penicillin may also induce this reaction. The manifestations are seen
around one week after receipt of the foreign serum, drug, or antibodies and include fever,
urticaria, and joint pains. Enlargement of lymph nodes (lymphadenopathy) and the spleen
(splenomegaly) are also noted.

Type IV: T Cell mediated Hypersensitivity

This type of hypersensitivity reaction was formerly known as delayed type of
hypersensitivity. It involves T lymphocytes (either CD4+ or CD8+ T lymphocytes), not
antibodies. The tissue destruction is due to either inflammation brought about by cytokines
produced by the CD4+ T cells (CD4+ T cell mediated inflammation) or direct killing of target
cells by the cytotoxic T cells (CD8+ T cell mediated cytotoxicity).
Reaction involving the helper T cells (CD4+ T cells) is now what is specifically referred to
as delayed type hypersensitivity. It can be induced by intracellular pathogens like Mycobacterium
tuberculosis as well as certain viruses and fungi, including their skin tests. For instance, in the
tuberculin skin test for tuberculosis, the antigen (PPD) is administered into the skin of a
previously immunized individual or someone who was exposed to a known case of tuberculosis.
The detectable skin reaction (called induration) is seen within 24–48 hours (sometimes up
to 72 hours) after administration. A positive tuberculin test is seen as induration measuring
> 10 mm and does not necessarily mean that the tested person has tuberculosis but may mean
positive exposure to an active case. Other examples are contact dermatitis and rheumatoid
arthritis. The helper T cells involved in this form are the Th1 cells which activate macrophages,
and the Th17 cells which recruit neutrophils leading to the inflammatory reaction seen
122 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Delayed type hypersensitivity and immune inflammation

Cytokines
CD4+ (IFN–γ, TNF)
Macrophage activation,
T cell inflammation
(TH 1)

Cytokines Inflammation
CD4+ (IL 17, IL 22)
T cell Tissue injury
APC
(TH 17)
presenting
antigen
T cell mediated cytolysis

CD8+
CTLs
Cell killing
and tissue injury

Figure 9.14 Pathogenesis of type IV hypersensitivity reaction illustrating the mechanism

of delayed type hypersensitivity involving helper T cells
Adapted from: Kumar, Abbas, and Aster: Robbins and Cotran Pathologic Basis of Disease 9th ed., Reed Elsevier
India Private Limited, p. 209, 2014.

In CD8+ T cell mediated cytotoxicity, the cytotoxic T cells destroy cells bearing specific
antigens on its surface leading to tissue destruction. This mechanism has been implicated in
type I diabetes mellitus and plays an important role in the destruction of virus infected cells as
well as graft rejection and destruction of tumor cells. The principal mechanism of destruction
involves perforins and granzymes that are secreted by the cytotoxic T cells. Once the target cells
are recognized by the cytotoxic T cells, they release perforin which perforates the wall of the
antigen. They also facilitate the release of granzymes which in turn causes activation of caspases
thereby leading to apoptosis of the target cell
 Host Response to Infection 123

Delayed type hypersensitivity Inflammation

CD4+ Cytokines
T cell

CD8+
T cell

APC presenting Tissue injury

tissue antigen
Normal
tissue

T cell mediated cytolysis

CD8+
CTLs

Cell killing
and tissue injury

Figure 9.15 Pathogenesis of type IV hypersensitivity reaction illustrating the mechanism

of T cell mediated cytolysis
Adapted from: Kumar, Abbas, and Aster: Robbins and Cotran Pathologic Basis of Disease 9th ed., Reed Elsevier
India Private Limited, p. 209, 2014.

Vaccines
A great number of infectious diseases can be prevented by administering vaccines that
induce either active or passive immunity. It contains a weakened (also called attenuated)
or inactivated form of the organism. It may contain the entire organism or a specific portion
of the microbe (also known as sub unit). It may also be derived from toxins produced by the
microorganism (toxoids). Whatever the component, vaccines are designed to stimulate the
body’s immune system to produce the antibodies specific to the organism or its components
so that these are recognized as foreign and will be destroyed immediately upon entry of the
organism into the body. The immunization of a population stops the spread of an infectious
agent by reducing the number of susceptible hosts (herd immunity). Immunization programs
have achieved the following goals:
1. Protection of population groups from the development of common infectious diseases
such as pertussis, diphtheria, tetanus, and rabies
2. Control of the spread of measles, mumps, and rubella
3. Elimination of smallpox in the worl
124 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Types of Immunization
Passive immunization involves the administration of purified antibody in preparations
called immune globulins or antibody containing serum. It is given for rapid, temporary
protection (usually 3–4 months) or treatment of a person (e.g., in the treatment of rabies).
The protection given is short lived. It is used with the following goals in mind: (1) to prevent
disease after a known exposure; (2) to reduce the symptoms of an ongoing disease; (3) to protect
immunosuppressed patients; or (4) to block the action of bacterial toxins and prevent the
diseases that they cause. Immune serum globulin preparations are derived from infected
humans or animals and are available as prophylaxis for several bacterial and viral diseases.
Examples are human rabies immune globulin (HRIG) and immune globulins against hepatitis
A or B, measles, chickenpox, and diphtheria.
Active immunization involves the injection of vaccines prepared from organisms or their
products. This stimulates the body’s immune system to produce the specific antibodies against
the component organism of the vaccine. The response takes days to weeks to develop but the
protection given is long term or even lifelong. Active immunization is preferred over passive
immunization. Examples include TDaP, MMR, and BCG.

Type of Vaccines
There are four major groups of vaccines—toxoid, live attenuated, subunit or killed/
inactivated. Live attenuated vaccines are prepared using organisms with limited ability to cause
disease. These are especially useful for protection against infections caused by enveloped
viruses. Immunization with a live attenuated vaccine resembles the natural infection leading
to development of humoral, cell mediated, and memory responses. Immunity acquired is
usually long lived and, depending on the route of administration, can mimic the normal
immune response to the infecting agent. The first vaccine was developed by Edward Jenner
for smallpox. Albert and Sabin developed the first live oral polio vaccine. Other examples are
the Bacille Calmette Guarin (BCG) vaccine for tuberculosis and vaccines against measles,
mumps, rubella (German measles), and chickenpox. There are two problems with the use of live
vaccines. First, the organism may still revert to its original virulent form once it enters the body.
Second, the vaccine may be dangerous to immunocompromisedpatients and pregnant women.
Toxoid vaccines were developed based on the principle that certain diseases are caused
by exotoxins produced by the causative agents. Examples are tetanus, botulism, pertussis,
diphtheria, and cholera. The toxoids were produced from the exotoxins. Because the source is
exotoxin, they are not as immunogenic and large or multiple doses are needed which may lead
to tolerance to the antigen so that the addition of an adjuvant is necessary to elicit a higher and
longer lasting immune response
 Host Response to Infection 12

Toxoid vaccines are advantageous because: (1) they are safe without possibility of reverting
to a virulent form; (2) the component antigens are non replicating; and (3) they are more stable
compared to live vaccines. The disadvantages include the need for adjuvant and multiple doses,
and the possibility of developing a type III Arthus reaction. This is because of the presence of
excess antibodies forming complexes with the toxoid molecules and activation of the classical
pathway of complement.
Killed vaccines in general refer to vaccines derived from bacterial sources while inactivated
vaccines are derived from viruses. The first killed vaccine to be produced was the typhoid vaccine
during the latter part of the 19th century. Examples of inactivated vaccines that are popularly
used are the polio vaccine and hepatitis A vaccine. Immune response to the killed/inactivated
vaccine is similar to the response to the toxoid vaccine but with a wider range of target antigens.
Several disadvantages are associated with killed/inactivated vaccines. Same as toxoids, multiple
doses are needed to elicit a strong immune response. In addition, because of the adjuvant used,
local reactions may be seen at the site of injection. Also, the immunity induced may only be
humoral and not cell mediated. Lastly, they do not elicit a local IgA response.
The last vaccine type is the subunit vaccine. This type of vaccine is produced the same way
as the killed/inactivated vaccine but instead of using the entire organism as the antigen to
stimulate antibody production, only a specific antigen or structure on the organism is used. Its
effectivity relies on accurate selection of the sub unit to be used. Examples are the hepatitis B
vaccine where the surface antigen of the virus was used in its development. Other examples are
the vaccines against Haemophilus influenzae and Streptococcus pneumoniae where the capsules of
both organisms were used.
The benefits of using subunit vaccines are similar to toxoid vaccine. An additional
advantage is that with subunit vaccines, one can identify or distinguish the infected individuals
from the vaccinated individuals. For example, in hepatitis B, immunized individuals will only
have the antibody to the surface antigen (anti HBs) and will be the only ones detected in their
blood while infected patients will have additional antibodies present against the core antigen
(anti HBc) and the envelope (anti HBe) of the hepatitis B virus. The disadvantages of using
of subunit vaccines are comparable to toxoid vaccines. Such vaccines also requires multiple
doses and addition of an adjuvant as well as local reactions at the site of infection similar to an
Arthus reaction.

Problems with vaccine use

Vaccines have been instrumental in the prevention of a great number of infectious
diseases. However, the use of vaccines also entails certain problems. As mentioned earlier, live
vaccines can occasionally revert to virulent forms and may be life threatening when given to
immunosuppressed individuals or pregnant women. Adverse side effects have been reported
in association with certain vaccines. These include hypersensitivity and allergic reactions to
126 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

the antigen in the vaccine, to non microbial material in the vaccine, and to contaminants in
the vaccine. Fever is a common complaint of parents after having their children vaccinated.
In some instances, depending on the susceptibility of the child, the fever may lead to benign
febrile seizures.
There are also instances of vaccine failures. Certain organisms such as viruses may have
more than one serotype that may be difficult for a vaccine to control. For example, Rhinovirus,
the most common cause of the common colds has more than 100 serotypes. The existence of
several strains of the influenza virus led to reduced and limited effectivity of the flu vaccine.
Lastly, vaccines do not 100% guarantee that the disease will not develop. A child who receives
a vaccine against chickenpox may still develop the disease, especially if there is an outbreak in
the community. The advantage of giving the vaccine is that it can prevent the development
of complications
 Host Response to Infection 127

CHAPTER SUMMARY

• The immune system is composed of cells and soluble proteins that are designed to defend
the body against any invading organism.

• Cells involved in the immune system are derived from the bone marrow. These are the
white blood cells, the most important of which are the lymphocytes, the major cells
involved in adaptive immunity.

• Antigen presenting cells are derived from the mononuclear phagocyte lineage. They
function to process and present antigens to the T cells. The professional antigen
presenting cells are the macrophage, B cells, and dendritic cells. The most important
among the three are dendritic cells.

• The bone marrow and the thymus are the central or primary lymphoid organs where
the immune cells undergo differentiation and maturation. For example, B lymphocytes
differentiate and mature in the bone marrow. T lymphocytes differentiate in the bone
marrow and undergo maturation in the thymus.

• Mature B cells and T cells enter the circulation to go to the secondary or peripheral
lymphoid organs. These include the lymph nodes, spleen, and the mucosa associated
lymphoid tissue—sites where antigens encounter the immune cells.

• Innate immunity immunity that is already present and active at birth. It is non specific,
is
acts immediately upon encounter with the antigen but gives short term protection.
It does not possess memory. It includes the body’s first and second lines of defense.
» The first line of defense functions to prevent the entry of the organism into the body.
These include the skin and mucous membranes as well as the normal flora found in
different parts of the body.
» The second line of defense aims to destroy the invading organism before it has
a chance to multiply and cause disease. Included are the natural killer cells,
inflammation, and the body’s normal resident flora.
• Adaptive immunity is activated by certain antigens which makes the response more
specific. It has a more delayed reaction because it takes time for antibodies to be
produced and for cytotoxic T cells to be activated. The protection obtained is long term
and, in most instances, lifelong. The most important property is memory which allows
recognition of the antigen on re exposure making the immune response amplifiable.
It constitutes the body’s third line of defense.

• Innate and adaptive immunity may both be humoral or cell mediated

128 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

» Innate, humoral immunity involves the action of specific proteins and molecules that
act to destroy antigens. This includes cytokines and the complement system.
» Innate, cell mediated immunity involves the action of natural killer cells and
phagocytic cells.
» Synthesis of specific antibodies by activated B cells is what constitutes adaptive
humoral immunity.
› IgG is the major immunoglobulin in the circulation and is predominant in the
secondary immune response. It is the only immunoglobulin that truly functions as
an opsonin and the only immunoglobulin that can cross the placenta. It is involved
in chronic inflammation and is able to fix complement.

› IgM is predominant in acute inflammation and the primary immune response. It

the largest and more effective fixer of complement. It also functions as an antigen
is

receptor on the surface of B cells.

› IgA is also known as the secretory immunoglobulin and acts to prevent adhesion
of microbes to mucosal surfaces. It provides protection to the gastrointestinal tract,
digestive tract, and genitourinary tract.

› IgE is the reaginic antibody and is the predominant immunoglobulin in type I

hypersensitivity and parasitic infections.

› IgD functions as a surface marker for B cells and has no biologic activity.
• Adaptive humoral immunity is the body’s defense against extracellular organisms.
Antibodies produced can help neutralize viruses. These antibodies also serve as the body’s
defense against encapsulated organisms as well as toxin producing microbes.

• Adaptive
T cells.
cell mediated immunity involves both the helper T cells and the cytotoxic

» Helper T cells are CD4+ and have several important subsets.

› Th1 acts on first exposure to an antigen and is responsible for stimulating
inflammation, delayed type hypersensitivity and synthesis of IgG and IgM.
› Th2 is activated on re exposure to the same antigen and is involved in stimulating
class switching to form other antibodies.

› function to recruit neutrophils leading to inflammation.

Th17

» CD4+ T cells act to stimulate differentiation of B cells to antibody producing plasma

cells and activate cytotoxic T cells.
» CD4+CD25+ T cells are the regulatory cells which forms part of the body’s immune
surveillance. It recognizes self from non self and is therefore important in the
maintenance of immunologic tolerance (ability of the body to recognize self from
non self). These cells regulate the action of the immune cells preventing exaggerated
response of the immune cells
 Host Response to Infection 12

» Cytotoxic T cells are CD8+ and are the body’s main defense against intracellular
organisms such as viruses and tumor cells. They are involved in graft and transplant
rejection. The major mechanism by which they destroy antigens is through the
perforin granzyme mechanism.
• Complement system is composed of various proteins that are activated or cleaved in
sequential manner. It consists of three pathways.
a

» The alternative or properdin pathway is the first to be activated on exposure to an

antigen. Its main activator is bacterial products such as endotoxins.
» The mannose binding lectin pathway is activated upon recognition of specific patterns
of sugar found on the cell wall of bacteria. There are two pattern recognition receptors
involved in the pathway—lectin which recognizes mannose containing sugars and
ficolin which recognizes other patterns of sugar. It is the second to be activated on first
exposure to an antigen.
» The last to be activated upon antigen exposure is the classical pathway. It is triggered
by antigen antibody complexes.
» All three pathways lead to formation of C3b which is required in the formation of
C5 convertase, the enzyme needed to form the terminal product of complement which
is membrane attack complex.
• Hypersensitivity
and injury.
reactions are exaggerated immune responses that lead to tissue damage

» Type I hypersensitivity is mediated by IgE and is commonly known as allergy.

The effector cells are mast cells and the predominant inflammatory cells are the
eosinophils.
» Type II hypersensitivity is called antibody mediated hypersensitivity. It is triggered
by antigen antibody complexes that causes activation of the classical pathway of
complement system.
» Type III hypersensitivity is immune complex mediated hypersensitivity. It is also
triggered by antigen antibody complexes. Like type II, the antibody involved is IgG or
IgM and similar to type II also involves activation of classical pathway of complement.
The main difference is the site of formation of immune complexes and the resulting
deposition of these complexes in tissues and blood vessels leading to fibrinoid necrosis.
It has two forms—acute serum sickness (systemic form) and the Arthus reaction
(localized form).
» Type IV hypersensitivity reaction is the T cell mediated hypersensitivity reaction
which involves either CD4+ T cells (delayed type hypersensitivity) and CD8+ T cells
(cytotoxic T cell mediated).
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 Host Response to Infection 131

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. The following are secondary lymphoid organs, EXCEPT:

a. Appendix c. Lymph node
b. Bone marrow d. Spleen
2. Destruction of organisms by phagocytosis is an example of which type of immune
reaction?

a. Adaptive, cell mediated c. Innate, cell mediated

b. Adaptive, humoral d. Innate, humoral
3. The following are characteristics of adaptive immunity, EXCEPT:
a. Specific c. Possess immunologic memory
b. Short term protection d. Amplifiable response
4. Which cell is involved in antibody production?
a. B lymphocyte c. CD8+ T lymphocyte
b. CD4+ T lymphocyte d. Natural killer cell
5. What is true regarding the secondary immune response?
a. Antibody response is lower than the primary immune response.
b. It is seen on first encounter with the antigen.
c. The predominant immunoglobulin present is IgG.
d. All of the above
132 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. The following constitutes the body’s first and second lines of defense, EXCEPT:
a. Tears c. Phagocytosis
b. Saliva d. Antibodies
7. Which of the following is a characteristic of live, attenuated vaccines?
a. Needs more doses
b. Can revert to virulent form
c. Can be safely given to pregnant women
d. Composed of organism that is rendered virulent
8. Which hypersensitivity reaction has been implicated in diabetes type I?
a. Type I c. Type III
b. Type II d. Type IV
9. Which immunoglobulin is also an opsonin?
a. IgA c. IgG
b. IgE d. IgM
10. Hepatitis A vaccine is an example of which type of vaccine?
a. Killed vaccine c. Live, attenuated vaccine
b. Inactivated vaccine d. Subunit vaccin
 CHAPTER

10 Bacteria and Disease

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define common terms involved in the production of disease by bacteria;
2. explain the components of Koch’s postulates;
3. discuss thoroughly the various factors that play a role in the chain of infection;
4. compare the various mechanisms by which bacteria produce disease, citing examples for
each mechanism;
5. describe the various ways by which infectious diseases are classified; and
6. compare the events involved in the various stages of an infectious disease.

Definition of Terms
Disease – result of an undesirable relationship between the host and the pathogen, marked by
interruption in the normal functioning of a body part or parts.
Infection – invasion of the body by pathogenic microorganisms. The term is not synonymous
with disease.
Symbiosis – prolonged and close interaction between organisms of different species.
Mutualism – a form of symbiosis in which both organisms benefit from the relationship.
Commensalism – a form of symbiosis in which one organism benefits from another organism
without causing harm to it.
Parasitism – a form of symbiosis where one organism benefits from another organism and at
the same time causes harm to the other.
Pathogenicity – ability of an organism to produce disease. An organism that can produce
disease in humans is said to be pathogenic
134 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Virulence – describes the degree of pathogenicity of an organism or the degree to which an

organism can produce disease.
Contamination – presence of unwanted materials (chemical, biological, or radiological)
where they should not be or at concentrations above the normal. The presence of these
substances may not necessarily lead to harm.
Pollution – presence of contaminants that can cause adverse biological effects to humans and
communities. All pollutants are contaminants but not all contaminants are pollutants.
Bacteremia – presence of bacteria in the blood.
Septicemia – presence of actively multiplying bacteria in the blood, usually from a source of
infection. The condition is called sepsis.
Pyemia – presence of pus producing bacteria in the bloodstream.
Viremia – presence of viruses in the blood.
Toxemia – presence of toxins in the blood.

Koch’s Postulates
Robert Koch was a German physician who made significant contributions to the field of
microbiology. One of his greatest and most well known contribution was proving that certain
microorganisms caused specific diseases. Together with some of his colleagues, he developed
a scientific experimental procedure to prove this relationship. This experimental procedure was
published in 1884 and came to be known as Koch’s postulates. These postulates are as follows:
1. The suspected organism must be absent in healthy individuals but present in those with
the disease.
2. The suspected organism must be isolated from the infected host and grown in pure
culture.
3. The organisms grown from pure culture must produce the same disease as that of the
infected source when inoculated to a susceptible animal.
4. The same organism must be isolated from pure culture from the experimentally infected
host.
Once all the above conditions are fulfilled, it can now be concluded that the organism
isolated is indeed the cause of the disease under study.
The validity of Koch’s postulates lies in the ability of the pathogen to grow in the laboratory
using artificial culture media. However, there are certain organisms that cannot be grown in
artificial culture media. Viruses are obligate intracellular parasites that need to be grown in
living cells. Likewise, Mycobacterium leprae, the causative agent of leprosy needs to be grown on
foot pads of mice and armadillo
 Bacteria and Disease 135

Red blood cell

Causative
agent absent
Injection with
cultured agent

Healthy
organism Suspected agent

Causative Causative
Diseased agent present agent present
organism
1
The suspected 2 The causative 3 The cultured 4 The same
causative agent must be agent must causative
agent must be isolated from cause agent must
absent from the diseased the same then be
all healthy organism and disease when reisolated from
organisms grown in pure inoculated the inoculated,
but present in culture. into a healthy diseased
all diseased susceptible organism.
organisms. organism.

Figure 10.1 Illustration of Koch’s postulates as applied to laboratory animals

Another limitation of Koch’s postulates is that not all people who acquire an infection
develop overt disease. Most of the time, infections are sub clinical. Also, the reaction of humans
to specific pathogens may differ given a specific microorganism. This is because human beings
have different genetic compositions that may modulate their responses to the same organism.
One individual might develop minor illness from a particular pathogen but the same pathogen
may produce fatal infection in another host.
An issue involving Koch’s postulates is the requirement that the cultured organism must
be inoculated into a susceptible animal. However, there are certain organisms that are species
specific. There are organisms that produce disease only in animals in the same manner that
there are infectious agents that produce disease only in humans. Therefore, organisms that
produce disease only in humans cannot be tested using laboratory animals and vice versa.
One also needs to consider the ethical issues involved in such testing procedures. Finally, there
are certain pathogens that become altered when grown in artificial media. Some become less
pathogenic while others may lose their pathogenicity, in which case Koch’s postulates cannot
be applied
136 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Factors that Influence the Occurrence

of Infection: The Chain of Infection
The development of an infectious disease is a consequence of the interaction among three
components—the etiologic agent, the host, and the environment. Transmission starts when the
pathogenic organism leaves its host or a reservoir through a portal ofexit. A susceptible organism
acquires the infection through a given mode oftransmission, entering the body of the susceptible
host through a portal ofentry. Once inside the body, the organism starts to multiply and produce
disease. This is called the chain of infection.

Reservoir
Reservoirs serve as the continual source of disease producing microorganisms. It is the
site where an infectious agent normally resides and multiplies. It provides the organisms with
conditions that enable them to survive and multiply and provide opportunity for transmission
to a susceptible host. Reservoirs include animals and humans as well as the environment.

Animal Reservoirs
Certain infectious diseases can be transmitted from an animal to humans. These are called
zoonotic infections. In most instances, humans serve only as an incidental host and dead end host
and thus the disease will not be transmitted to another human. Examples of zoonotic infections
include anthrax, plague, and rabies.

Human Reservoirs
A number of pathogenic organisms have humans as their reservoir. These organisms may
be directly transmitted from one individual to another. Examples are respiratory pathogens
and sexually transmitted infections. The human reservoir may not necessarily manifest with
the disease. There are certain infected humans who may harbor the organism but only develop
sub clinical disease. There are those who developed the disease, got well but still harbor the
organism thereby transmitting them to others. These are what are known as carriers and
comes in several forms. Those who are infected but do not manifest symptoms are known as
asymptomatic or healthy carriers. Carriers who transmit the causative agent during the incubation
period of the illness are called incubatory carriers. Chronic carriers are those who harbor the
organism for months or longer after the patient developed the initial infection. Individuals
who developed the disease, recovered but remain capable of transmitting the causative agent
are known as convalescent carriers. Carriers are individuals who are not aware that they are
transmitting the infectious agent which makes them public health hazards
 Bacteria and Disease 137

Next Sick Person Germs

(Susceptible Host) (Agent)
• Babies • Bacteria
• Children • Viruses
• People with a weakened • Parasites
immune system
• Unimmunized people Where Germs Live
• Anyone (Reservoir)
• People
How Germs Get In • Animal/Pets
(Portal of Entry) Chain of Infection (dogs, cats,
reptiles)
• Mouth • Wild animals
• Cuts in the skin • Food
• Eyes • Soil
• Water
Germs Get Around How Germs Get Out
(Mode of Transmission) (Portal of Exit)
• Contact (hands, toys, sand) • Mouth (vomit, saliva)
• Droplets (when you speak, • Cuts in the skin (blood)
sneeze, or cough) • Stool

Figure 10.2 An illustration of how an infectious agent is transmitted beginning with the source of
infection until it enters a susceptible host to cause disease

Environmental Reservoirs
Water, soil, and plants can harbor infectious organisms. For instance, the fungus Histoplasma
capsulatum is associated with soil. Water serves as a reservoir for Entamoeba histolytica,
a protozoan parasite that cause amoebiasis. Aquatic vegetation such as watercress and
“kangkong” harbor Fasciola hepatica larvae which causes damage to the liver.

Portal of Exit
The portal of exit is the route by which an infectious agent exits its host. It is usually the
site where the infectious agent is commonly located or localized. For example, the blood fluke
Schistosoma haematobium which preferentially infects the urinary bladder exits the host via urine.
Infectious agents causing respiratory tract infection will leave the host via droplets or aerosols
from the respiratory tract. Sexually transmitted infectious agents exit via vaginal or urethral
secretions. There are also organisms that exit the host through blood sucking arthropods such
as Plasmodium spp., the causative agent of malaria
138 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Mode of Transmission
Infectious agents may be transmitted from the source to a susceptible host in several ways.
These can be broadly categorized as direct or indirect contact.

Direct Contact
Most infectious agents are transmitted through direct contact. Contact with environmental
sources harboring infectious agents are also considered direct contact. For example, the blood
fluke Schistosoma spp. can be transmitted when one wades in fresh water containing snails
that harbor the larvae of the parasite. The larvae in turn enter the human host through skin
penetration. The most important methods though of direct contact are the person to person
contact and droplet spread.
1. Person to person contact – involves transmission through skin to skin contact, kissing,
or sexual transmission. Warts can be transmitted through direct contact with the
lesion on the skin of infected persons. Infectious mononucleosis caused by Epstein
Barr Virus is transmitted through saliva, hence the name “Kissing Disease.” Syphilis,
gonorrhea, and other sexually transmitted infections are spread through vaginal and
urethral secretions of infected persons.
2. Droplet spread – patients with respiratory tract infection such as the common colds or
influenza can transmit the causative agents during coughing and sneezing. Droplets
are differentiated from aerosols by its larger size (> 5 microns in size). It is considered
as direct contact because the droplets are sprayed over a few feet before they fall to the
ground. Close proximity with the source is necessary for droplets to be transmitted.

Indirect Contact
1. Airborne transmission – infectious agents may be transferred from an infected person to
a susceptible host through dust or aerosols. Aerosols are droplets with nuclei less than
5 microns in size. Due to their small size, they may remain suspended in air for a longer
time and may cover farther distance than droplets. There are also organisms that can be
carried with dust. For example, the fungus Cryptococcus neoformans can be transmitted
through aerosolized pigeon or fowl droppings and inhaled by a susceptible host. Measles,
a common childhood illness, can be transmitted through aerosols.

2. Vehicle transmission – refers to transmission of organisms through media such as food,

water, milk, or biologic substances such as blood and body secretions. Fomites or
inanimate objects such as beddings and clothing may also serve as vehicles. Vehicles
carry an infectious agent passively or may provide an environment that promotes
growth and multiplication of an infectious agent. The most common vehicles are foo
 Bacteria and Disease 13

and water. Gastrointestinal infections such as cholera and typhoid are transmitted
through contaminated water. In food borne transmission, the causative agent is
transmitted through ingestion of raw or improperly cooked, poorly refrigerated food
that is contaminated by the causative agent. The food ingested may be contaminated by
feces of the infected patient (fecal oral transmission). Examples are food poisoning and
gastroenteritis.
3. Vector transmission – vectors are usually insects that can transmit an infectious agent.
These spread the infectious agent by two general methods: mechanical and biological.
Mechanical transmission refers to the passive transport of the organism on the insect’s
feet or other body parts. For example, cockroaches and flies can transfer the organisms
from the feces of infected persons to food, which is later swallowed by the host.
Biological transmission is the active transport of the organism. Here, the organism enters
the insect vector after the insect vector bites an infected person. The organism then
multiplies within the insect vector and is transmitted by the insect vector to another
person through bites. For example, malaria is transmitted to a susceptible host through
the bite of the female Anopheles mosquito. Dengue virus, chikungunya virus, and zika virus
are also transmitted through bites of mosquito vectors. Bite of the rat flea is the mode of
transmission of Yersinia pestis, the causative agent of the plague.

Portal of Entry
How the infectious agent enters a susceptible host is referred to as the portal of entry.
It provides access to tissues where the infectious agent can multiply. More commonly, the
portal of exit of an infectious agent is also the portal of entry into another host. For example,
organisms that leave the respiratory tract will also enter another host through the respiratory
tract via inhalation. Organisms that are transmitted through food and water enter the host
through the mouth but exit through the feces. In infection with the blood fluke Schistosoma
haematobium, the organism leaves the body of the host through urine but enters through skin
penetration by the infective larva. Hepatitis B virus and HIV enter the susceptible host through
blood and blood products.

Host
The final link in the chain of infection is the susceptible host. The host’s susceptibility is
affected by several factors such as constitutional or genetic factors and immune status of the
host. Susceptibility to infection may be increased or decreased in certain individuals with
specific genetic make up. For example, patients born with the gene that codes for the sickle cell
trait, an abnormality in morphology of red blood cells, are less prone to develop malaria than
those who were not born with the trait.
140 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The immune status of the host is probably the most important factor that can affect
development of a disease process. Humans have natural barriers that prevent entry of potential
pathogenic organisms. Besides these, they are also equipped with a highly functional immune
system that can mount adequate defenses to fight and destroy any invading pathogen.
Once there is a breakdown in all these defenses, microorganisms can easily gain entrance into
the body, multiply, and produce disease. Factors that may impact the human immune system
include poor nutritional status, chronic intake of alcoholic beverages, or any condition that
dampens the immune response.

How Organisms Produce Disease

Mechanical: Invasiveness
Organisms can produce disease by directly damaging tissues or body surfaces. This
involves invasion of the epithelial surface and penetration into deeper tissues. Invasiveness
encompasses three important steps—colonization, ability to evade host immune defenses, and
production of extracellular substances that can promote invasion. Colonization involves the
ability of the invading organism to enter the susceptible host and establish itself in the portal
of entry. This can be facilitated by substances produced by the organism that facilitate adhesion
of the organism to specific target cells. These substances are collectively called adhesins.
Gram negative bacteria possess pili or fimbriae that promote adherence of the organism to
susceptible cells. By promoting adhesion, the organism can easily invade the surfaces and enter
the body of the host.
Once the organism enters the body, the immune system of the host immediately mounts an
immune response that will lead to the destruction of the invading pathogen. However, there are
certain factors that allow the organism to evade these immune defenses. For instance, bacteria
that possess a capsule enables the organism to evade phagocytosis. Staphylococcus aureus secretes
the enzyme coagulase that promotes formation of a coagulum within which the organism may
hide to escape detection by the immune surveillance cells. Mycobacterium tuberculosis can survive
and multiply inside macrophages by inhibiting phagosome lysosome fusion.
Finally, some microorganisms produce substances or have developed mechanisms that
can promote invasion. Neisseria gonorrheae can enter and multiply within host cells and after
multiplication is extruded from the host cell allowing it to infect other host cells. The process
of extrusion from the host cell causes direct destruction of the host cells. Some bacteria
produce enzymes that aids them in invading target cells. Collagenase is an enzyme produced
by Clostridium perfringens that causes breakdown of collagen, a major component of connective
tissue of muscles thereby contributing to the development of the disease called gas gangrene
 Bacteria and Disease 14

Chemical: Toxin Production

Toxins are poisonous substances and are often the primary factors that contribute to disease
production. There are two major types of toxins—exotoxins and endotoxins. Endotoxins are
integral components of the outer membrane of gram negative bacteria such as Salmonella,
Shigella, and Escherichia coli. The specific component responsible for the endotoxin activity of
these bacteria is the lipopolysaccharide (LPS), which is further composed of a lipid A moiety
and a polysaccharide moiety. The lipid A component is associated with its toxic activity
while the polysaccharide component is antigenic. Endotoxins exert their effects when the
gram negative bacteria die and their cell walls undergo lysis, thereby releasing the endotoxin.
All endotoxins produce similar signs and symptoms, although not to the same degree.
Exotoxins are intracellular products of some bacteria as part of their growth and
metabolism and are released into the surrounding medium. These are mainly proteins and
many of them are enzymes. Most of them are produced by gram positive bacteria but may
also be produced by some gram negative bacteria. Exotoxins are soluble in body fluids and
are thus easily diffused into the blood and rapidly transported throughout the body. There are
three principal types of exotoxins: (1) cytotoxins which kill host cells or affect their function;
(2) neurotoxins which interfere with normal nerve impulse transmission; and (3) enterotoxins
which affect the cells lining the gastrointestinal tract. Diseases produced by exotoxin producing
bacteria are often due to the effects of the exotoxin and not of the bacteria themselves.
Exotoxins are therefore disease specific. Important examples are the diphtheria toxin,
botulinum toxin, and the tetanus toxin.

Table 10.1 Comparison between exotoxin and endotoxin

Property Exotoxin Endotoxin
Bacterial source Mostly gram positive bacteria; Gram negative bacteria only
some gram negative bacteria
Relation to microorganism Metabolic product of growing Present in LPS of outer
cell; secreted outside cell membrane of cell wall;
released after lysis of cell wall
Toxicity Extremely toxic; sometimes fatal Weakly toxic
Chemistry Protein or short peptides Lipid A of LPS of outer
membrane
Pharmacology Specific for a particular cell General such as fever, malaise,
(effect on the body) structure or function in the host and shock; all produce the
(mainly affects cell functions, same effects
nerves, and GIT)
Antigenicity Extremely antigenic Less antigenic
142 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Property Exotoxin Endotoxi

Enzymatic activity Yes No
Heat stability Heat labile (except Heat stable
staphylococcal enterotoxin)
Fever production No Yes
Specificity High degree Low degree
Relation to antibodies Can be converted to toxoids; Cannot be converted to
neutralized by anti toxins toxoids; not neutralized by
anti toxins
Denaturation on boiling Yes No
Location of genes Present on plasmids or Bacterial chromosome
bacteriophages
Representative diseases Gas gangrene, botulism,
diphtheria, tetanus, scarlet fever
Typhoid fever, urinary tract
infections, meningococcal
meningitis, and
meningococcemia

Immunologic
Some organisms produce disease not as a consequence of mechanical invasion or toxin
production but as a consequence of the immune response of the host to the microorganism
or its product. In hepatitis caused by the hepatitis viruses, the damage to the liver is not a
direct effect of the virus but of the response of the immune system to the virus. Antibodies
are produced against the virus and cytotoxic T cells are activated leading to the destruction of
hepatocytes. In childhood measles and German measles, the rashes seen are due to the specific
immune response of the body to the measles virus.

Classification of Infectious Diseases

Based on how they behave within a host
and within a given population
A disease that is spread from one host to another, either directly or indirectly, is called a
communicable disease. Examples are measles, tuberculosis, and typhoid fever. If the disease
is easily and rapidly spread from one person to another, then it is classified as a contagious
disease (e.g., measles and chickenpox). If the infection results in the death of the patient over a
short period of time, it is called a fulminant infection. An example is meningococcemia where
a patient may die hours after confinement in the hospital.
 Bacteria and Disease 14

A non communicable disease is one that is not spread from one person to another.
It is usually caused by organisms that normally inhabit the body and produce disease only
occasionally or by organisms that produce disease only when introduced into the body such
as Clostridium tetani, the agent that causes tetanus. It produces disease only when it enters the
body through breaks in the skin.

Based on the source of the microorganism

An infection is said to be exogenous if the source of the infectious agent is from outside
the body. For example, cholera is an exogenous infection because the causative agent enters the
body through ingestion of contaminated water. Hospital acquired infections or nosocomial
infections can also be considered as exogenous infections where the offending organism was
obtained from the hospital environment during the period of confinement of the patient in the
hospital. On the other hand, an endogenous infection is one where the source of the causative
organism is from inside the body. Escherichia coli is part of the normal flora of the colon that can
enter the urinary tract via the urethra, especially in women, due to its close proximity to the anal
orifice. This can lead to the development of urinary tract infection which can ascend to involve
the kidney and can lead to sepsis in immunocompromisedpatients.

Based on the occurrence of a disease

A disease that occurs occasionally is called a sporadic disease. If the disease is constantly
present in a population at low levels, then it is called an endemic disease. Malaria is said to be
endemic in Palawan while schistosomiasis is considered endemic in Leyte. If a great number of
people in a given locality develop an infectious disease in a relatively short period of time, it is
called an epidemic. Influenza is an example of a disease that can cause epidemics. If a disease
has a worldwide occurrence or involves at least three regions in the world, it is said to be a
pandemic. Influenza, especially due to influenza A, SARS, and AIDS are examples of diseases
that can cause pandemics.

Based on the severity or duration of a disease

An acute disease is one that develops rapidly but lasts for only a short period of time. An
example is the common cold. If the disease develops more slowly and occurs for long periods of
time, it is called a chronic disease. A good example of this is tuberculosis. Hepatitis B infection
is an example of a disease that can have either an acute form or a chronic form. A latent disease
is one in which the causative organism remains inactive for a time but can become active again
and produce symptoms of the disease. Viruses that belong to the family Herpesviridae are
characterized by latency. An example is shingles, a reactivation of a latent chickenpox infection
which occurs years after the initial infection.
144 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Based on the extent of host involvement

Infections can be classified according to the extent to which the body of the host is affected.
A localized infection is one in which the invading organisms are limited to a relatively
small area of the body. Boils and abscesses are examples of localized infections. A systemic
or generalized infection is one where the causative organisms or their products are spread
throughout the body through blood or lymph. In some cases, the causative agents of a localized
infection may enter a blood or lymphatic vessel, spread to specific parts of the body and become
confined to specific areas. This is called a focal infection. Focal infections can arise from
infections in areas such as the teeth, tonsils, or sinuses.
A primary infection is an acute infection that causes the initial illness, while a secondary
infection is one which is caused by opportunistic pathogens after the primary infection has
weakened the body’s defenses. Secondary infections are common in patients whose immune
system have been compromised by another disease process (e.g., AIDS) and can prove to be
more devastating than the primary infection. A subclinical or inapparent infection is one that
does not cause noticeable illness. Hepatitis due to hepatitis A virus can, for instance, occur in
certain individuals who do not necessarily develop the typical signs and symptoms associated
with the disease.

Stages of an Infectious Disease

Once a microorganism invades a susceptible host, disease follows. The development of the
disease follows a sequence of events that tends to follow a similar pattern whether the disease is
chronic or acute. These periods are the following:
1. Incubation period – the time interval between entry of the offending agent and the
appearance of the initial signs and symptoms of the disease. In most cases, this period
is variable and is usually stated in the form of a range (e.g., 6–12 days). The length of
this period can be affected by the virulence of the organisms as well as the number of
infecting microorganisms. It also depends on the resistance of the host. An organism
that is considered virulent can produce disease within a short incubation period. As
for the number of infecting microorganisms, in general, the greater the number of
microorganisms that invade the body, the shorter the incubation period. However,
if the organism is highly virulent, it may take only a small number of organisms to
produce disease, hence a shorter incubation period.
2. Prodromal period – a relatively short period, is characterized by early, mild symptoms
of disease which are generally non specific. In measles infection, the prodromal
period is characterized by non specific constitutional symptoms such as fever, cough,
colds, general aches, and malaise—symptoms which can also be seen in other disease
processes and are thus not specific to measles
 Bacteria and Disease 145

Incubation Prodromal Illness Decline Convalescence

or symptom
period period (most severe signs (declining signs (no signs or
microorganisms

(no signs or (vague and symptoms) and symptoms) symptoms)

symptoms) general
symptoms)
or
signs

of of
Number intensity

Time
Figure 10.3 Stages of an infectious disease

3. Period of illness – corresponds to the period of maximal invasion. It is during this period
that the disease is most acute. During this period, the patient manifests signs and
symptoms distinctive of the disease. For example, the period of illness in measles is
marked by the appearance of the typical rashes seen in measles. Examination of the
patient’s complete blood count (CBC) will generally show elevation of the white blood
cells although in some infections there may be a reduction in the total WBC count.
As a rule, most bacterial infections will usually show increased neutrophil count while
most viral infections will have a high lymphocyte count.
Several outcomes can arise during this period. The infection may remain acute
where the body’s defense mechanisms successfully destroy the invading microorganism
leading to resolution of the infection and recovery of the patient. When the patient
does not successfully overcome the disease producing agents, he or she may develop
severe disease that can lead to a fulminant infection. The infection may also progress
from an acute form into a chronic form (e.g., hepatitis B infection). Finally, the
infection can progress to a carrier state where the patient is asymptomatic but
continues to transmit the infecting microorganism.
4. Period of decline – corresponds to what is known as the period of defervescence.
During this period, the signs and symptoms of the patient start to subside. Body
temperature may return to normal and the feeling of weakness may diminish.
However, it is also during this period that the patient becomes vulnerable to secondary
infections.
5. Period of convalescence – this period is marked by recovery of the patient from the
disease. The patient regains strength and the body returns to its pre diseased,
normal condition.
146 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• Robert Koch was the first to scientifically prove that a specific infectious disease is
caused by a specific infectious agent. Together with some colleagues, they experimentally
provided evidence for this in what is now known as Koch’s postulates.

• The chain of infection involves the following elements—reservoir, portal of exit, mode of
transmission, and host.
» The reservoir is the site where the organism resides and multiplies. It provides an
environment conducive to the growth and replication of the organism. Reservoirs
provide continual source of the infectious agent and may be humans, animals, or the
environment.
» Portal of exit refers to where the organism exits from its reservoir. For example,
organisms that have the gastrointestinal tract as their reservoir will exit through
the feces.
» Mode of transmission refers to how the organism is spread. It is generally classified
into direct contact transmission and indirect contact transmission.
› The major routes of direct contact transmission are through person to person
contact and through droplet transmission.
› Indirect transmission includes airborne transmission, vector transmission, and
vehicle transmission.
» The final link in the chain of infection is a susceptible host. Development of infection
in the host is affected by several factors such as genetic constitution of the host, the
nature of the organism, and the immune status of the host. The most important
among these factors is the defensive powers of the host.

• Bacteria produce disease in three ways—mechanical (invasion), chemical (toxin

production), and immunological.
» Invasion involves adhesion to mucosal surfaces and penetration into deeper tissues.
It is facilitated by structures found on the external surface of certain bacteria (e.g., pili
in gram negative bacteria), as well as extracellular substances secreted by the bacteria
to promote adhesion (adhesins).
» Bacteria produce toxins that may cause damage to the cells or affect the normal
physiologic function of the susceptible host. These are generally classified into
exotoxins and endotoxins.

› Endotoxins are components of the outer membrane of gram negative bacteria.

a
These are mainly lipopolysaccharides composed of lipid component and A
polysaccharides. The endotoxin activity is attributed to the lipid A component
 Bacteria and Disease 14

› Exotoxins are substances which are secreted by bacteria. These are mainly produced
by gram positive bacteria but may also be produced by some gram negative
bacteria.

» In some infections caused by microorganisms, the damage to the host tissues is not a
direct effect of the infecting agent but is a consequence of the body’s immune response
to the organism. An example is damage to the liver seen in patients with hepatitis due
to infection with the hepatitis viruses.
• Infectious diseases are classified based on the following: (1) how they behave within
a host and within a given population; (2) the source of the microorganism; (3) the
occurrence of a disease; (4) the severity or duration of a disease; and (5) the extent of
host involvement.

• An infectious disease may be divided into five stages: (1) incubation period, (2) prodromal
period, (3) period of illness, (4) period of decline, and (5) period of convalescence.
» Incubation period corresponds to the time from initial entry of the infectious agent
until the time the patient first manifests signs and symptoms.
» Prodromal period corresponds to the initial manifestations of the patient. These
manifestations are usually non specific constitutional symptoms such as fever, body
malaise, cough, and colds.
» The period where there is maximal invasion by the infecting agent is the period of
illness. It is during this period where signs and symptoms characteristic of the disease
are seen.
» The decline phase is the period when the manifestations of the patient begin to
diminish. It is also known as the period of defervescence.
» The period of convalescence is also known as the recovery period where the patient
already becomes asymptomatic and the body returns to its normal, pre diseased state.
This page is intentionally left blank
 Bacteria and Disease 149

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. Which of the following is the proper order on the stages of an infectious disease
process?

a. Convalescence, Incubation, Illness, Prodromal, Decline

b. Prodromal, Convalescence, Incubation, Decline, Illness
c. Incubation, Prodromal, Illness, Decline, Convalescence
d. Illness, Incubation, Prodromal, Convalescence, Decline
2. The site where pathogens grow is called:
a. Reservoir c. Portal of Entry
b. Portal of exit d. Host
3. An individual who is more likely than others to acquire an infection is a:
a. Vector c. Fomite
b. Reservoir d. Susceptible host
4. A 6 year old child was brought to the Emergency Room because of high grade
fever and petechial rashes. Dengue was suspected. Dengue exemplifies which type
of transmission?
a. Person to person contact c. Vector transmission
b. Vehicle transmission d. Airborne transmission
5. Blood can be classified under which type of transmission?
a. Direct contact c. Droplet transmission
b. Vehicle transmission d. Vector transmission
150 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. Which among the following clinical conditions can be considered as an outbreak

or epidemic?
a. Two or more people with diarrhea and vomiting
b. Two people living together with diarrhea and vomiting
c. Two or more people which exceeds the expected number experiencing
the same illness in the same place and at the same time
d. Two or more people with a respiratory infection
7. Which of the following IS NOT considered a portal of entry for bacteria?
a. Eyes c. Mouth
b. Nose d. Intact skin
8. A 75 year old male patient was admitted for elective cataract extraction. While in
the hospital, he developed pneumonia. This is classified as what type of infection?
a. Community acquired infection c. Epidemic
b. Nosocomial infection d. Sporadic
9. A patient was brought to the hospital because of symptoms of tetanus. The disease
is due to the bacterium Clostridium tetani and the manifestations are the effects of
tetanospamin, a neutrotoxin produced by the causative agent. Which mechanism
of disease production is involved?
a. Mechanical c. Immunological
b. Chemical d. A and B
10. A 5 year old child was exposed to a neighbor who has measles. After 5 days, she
started to manifest cough, colds, and conjunctivitis. The time from exposure until
the development of the signs and symptoms presented by the child corresponds
to which stage of the disease process?
a. Incubation period c. Period of illness
b. Prodromal period d. Period of defervescenc
 CHAPTER
Introduction
11 to Parasitology

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. define symbiosis;
2. enumerate the different types of symbiotic relationships and give examples for each;
3. classify the various forms of parasites and hosts;
4. enumerate the various sources of parasitic infections and cite examples for each;
5. give the modes of transmission for parasites and cite examples for each;
6. discuss the various mechanisms by which parasites produce disease in humans;
7. describe the proper way of collecting and handling specimens for laboratory diagnosis;
8. compare the various procedures used for laboratory diagnosis of parasitic infections; and
9. illustrate by means of a diagram the taxonomic classification of parasites.

A relationship where unlike organisms exist together is called symbiosis. There are
three types of symbiotic relationships. Commensalism is a form of symbiotic relationship
in which two species live together and one species benefits from the other without harming
or benefitting the other. The relationship between the human body and most of the normal
flora in the body exemplifies this type of relationship. Mutualism, on the other hand, is a
symbiotic relationship in which two organisms mutually benefit from each other. The normal
intestinal flora for instance produces vitamin K which is needed for the activity of some of the
body’s clotting factors. These flora benefit from humans by obtaining nutrients from the body
which they need for their metabolism while humans also benefit from them because of the
vitamin K that they produce. Finally, parasitism is the form of symbiotic relationship where
one party or symbiont (i.e., the parasite) benefits to the detriment of the other (the host). In
almost all cases of parasitic relationships, the parasite deprives the host of essential nutrients
and produce disease in the host
152 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

There are two important elements in parasitism—the parasite and the host. Parasites are
organisms that usually depend on the host for survival and are classified in several ways, namely:
1. Based on habitat
a. Ectoparasites – parasites that live outside the host’s body (e.g., fleas, lice). Invasion
of the body by ectoparasites is called infestation.
b. Endoparasites – parasites that live inside the body of the host (e.g., helminthes
or worms). Invasion of the body by endoparasites is called infection and is the
result of entry and multiplication of the parasite within the host.
2. Based on ability to live independently of the host
a. Facultative parasites – parasites that can live independently of the host (i.e., free
living). These parasites do not have to live inside a host to complete their
life cycle.
b. Obligate parasites – parasites that must live inside a host (e.g., Plasmodium,
Leishmania, hookworms). Majority of the parasites that infect humans are
obligate parasites.
3. Based on mode of living
a. Permanent parasites – parasites that remain in a host from early life to maturity
(e.g., Plasmodium)
b. Intermittent parasites – parasites that simply visit the host during feeding time
(e.g., non pathogenic parasites)
c. Incidental parasites – parasites that occur in an unusual host (e.g., dog tapeworm
in humans)
d. Transitory parasites – parasites whose larva develops in a host while the adult is
free living (e.g., Echinococcus granulosus or dog tapeworm).
e. Erratic parasites – parasites that are seen in an unusual organ, different from that
which it ordinarily parasitizes (e.g., Ascaris lumbricoides in the lungs or kidneys).
Hosts are essential to the existence of parasites. Hosts are organisms that harbor the
parasite and provide nourishment to the parasite. There are four types of hosts. Definitive
hosts are hosts that harbor the adult stage of the parasite (e.g., humans for the intestinal round
worm Ascaris), or where the sexual stage or sexual phase of the life cycle of the parasite occurs
(e.g., mosquito for the malaria parasite Plasmodium). Intermediate hosts are those that harbor
the larval stage of the parasite (e.g., cow for the cysticercus larva of the beef tapeworm Taenia
saginata), or where the asexual stage of the life cycle of the parasite occurs (e.g., humans for the
malaria parasite Plasmodium). Reservoir hosts are vertebrate hosts that harbor the parasite and
may act as additional source of infection in man. Migratory birds serve as the reservoir host for
the parasite Capillaria philippinensis which people normally get from contaminated fresh water.
Finally, paratenic hosts are those that serve as a means of transport for the parasite (e.g., insect
vectors) so that the infective stage of a certain parasite may reach its final host
 Introduction to Parasitology 153

Sources of Exposure to Infection or Infestation

Exposure to parasites may occur through one or more of the following sources:
(1) contaminated soil or water; (2) food containing the parasite’s infective stage; (3) a blood
sucking insect; (4) a domestic or wild animal harboring the parasite; (5) another person and
his or her clothing, bedding, or the immediate environment he or she has contaminated;
or (6) one’s self (auto infection).
The most common source of exposure to infection is soil contaminated or polluted
with human feces. This is true for most of the parasitic worms or helminths such as
Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, and human hookworms. Water
may be the source of the viable cysts of the parasitic amoebae and intestinal flagellates, the
larvae of the blood flukes, and the eggs of the pork tapeworm Taenia solium. Freshwater fish
serve as the source for the fish tapeworm Diphyllobothrium latum, as well as other intestinal and
liver flukes. Raw pork is the source of Trichinella spiralis and Taenia solium, while improperly
cooked or raw beef is the source of Taenia saginata.

Table 11.1 Summary of terms associated with parasite host relationships

Type of Parasite
Obligatory parasite Parasite that cannot survive outside the host.
Facultative parasite Parasite capable of existing independently of the host.
Endoparasite Parasite that is established inside the host.
Ectoparasite Parasite that is established in or on the exterior surface of host.
Type of Host
Accidental or Host other than the normal one that is harboring a parasite.
incidental host
Definitive host Host in which the adult/sexual phase of parasite development occurs.
Intermediate host Host in which the larval/asexual phase of parasite development occurs.
Reservoir host Host harboring parasites that are parasitic for humans and from which
humans may become infected.
Transport/Paratenic Host responsible for transferring a parasite from one location to
host another.
Parasite Host
Relationships
Symbiosis Living together; the association of two living organisms, each of
different species.
Commensalism Association of two different species of organisms that is beneficial to
one and neutral to the other.
Mutualism Association of two different species of organisms that is beneficial to
both.
Parasitism Association of two different species of organisms in which one benefits
at the other’s expense
154 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Blood sucking insects may serve as source for certain parasites—the female Anopheles
mosquito for the malaria parasite Plasmodium; sand fly for leishmaniasis; tsetse fly and reduviid
bug for trypanosomes; and the Culex and Mansonia mosquitoes for filariasis. Dogs, on the other
hand, are the direct source of infection with the hydatid cyst of the dog tapeworm Echinococcus
granulosus. Other animals that may serve as sources for parasites include pigs, cows, and birds.
Other human beings are directly responsible for a considerable amount of infection with
the pathogenic amoeba Entamoeba histolytica, the pinworm Enterobius vermicularis, and the
dwarf tapeworm Hymenolepis nana. Auto infection accounts for some of the infections and
some re infections with Hymenolepis nana, Enterobius vermicularis, and Strongyloides stercoralis.

Modes of Transmission
Ingestion of contaminated food and water (fecal oral transmission) is the most common
mode of transmission of most intestinal parasites. Those that are transmitted by ingestion of
contaminated water include the intestinal protozoa (cyst stage), and the embryonated egg stage
of the intestinal roundworms (e.g., Ascaris lumbricoides, Trichuris trichiura). Trichinella spiralis,
Taenia solium, Taenia saginata, Diphyllobothrium latum, intestinal flukes and the lung flukes are
transmitted by eating food containing the larval stage of the parasites.
Some parasites actively enter the body through penetration of the skin from the
soil (e.g., hookworms and Strongyloides) or from contaminated water (e.g., blood fluke).
Other modes of transmission include: (1) bite of blood sucking insect vectors (e.g., malaria,
leishmaniasis, trypanosomiasis, and filariasis); (2) inhalation of eggs (pinworm or Enterobius
vermicularis); (3) transplacental or congenital infection (Toxoplasma gondii and occasionally
Plasmodium); (4) transmammary (mother’s milk) infection (Strongyloides, Ancylostoma);
and (5) through sexual intercourse (Trichomonas vaginalis).

Portals of Exit of Parasites

The most common portal of exit of parasites is through the anus. Eggs of medically
important roundworms are excreted together with human feces and contaminate soil
and water. Urine may serve as the portal of exit for Trichomonas vaginalis, Strongyloides
stercoralis, and Schistosoma haematobium while the lung fluke Paragonimus westermani and
the intestinal roundworm Ascaris lumbricoides (larval stage) may be excreted with sputum.
Trichomonas vaginalis, which is transmitted through sexual intercourse, may be isolated from
vaginal discharge
 Introduction to Parasitology 15

Mechanisms of Disease Production by Parasites

The term pathogenesis refers to the dynamics of any disease process. Some parasites
may cause inapparent infection, causing no symptoms, and producing no detectable harm.
The infection can remain inapparent continuously for long periods or between short periods
of relapse (e.g., malaria). For most helminthic infections, signs and symptoms usually manifest
only if a large number of worms are present.
Parasites damage the host through one or more of the following mechanisms: (1) trauma or
physical damage; (2) lytic necrosis; (3) stimulation of host tissue reaction; (4) toxic and allergic
phenomena; and (5) opening of pathways for entry of other pathogens into the tissues.

Traumatic Damage
In this mechanism of damage, the manifestations may be due to the direct physical damage
caused by the parasite in the organ it parasitizes or at the point of entry of the parasite. Entry
of the infective larvae of hookworms or blood flukes into the skin may produce relatively slight
physical damage. Small lesions may result from the bite of mosquitoes (e.g., malaria) and other
insects (e.g., tsetse fly in African sleeping sickness). Migration of the larval stage of certain
roundworms (Ascaris and hookworms) may lead to ruptured capillaries in the lungs. Large
number of worms may produce acute intestinal obstruction.

Lytic Necrosis
Enzymes and other substances produced by many parasites that are necessary for them to
digest food available in the immediate environment may cause harm to the host tissues. A good
example is that of the parasitic protozoan Entamoeba histolytica which releases enzymes that
lyse tissues for their nutritional needs. These enzymes also enable the parasite to penetrate the
tissues of the colon, producing ulcerations in the colon, and extra intestinal viscera.

Stimulation of Host Tissue Reaction

Majority of animal parasites provoke host tissue reactions. These reactions may be in the
form of cellular proliferation and infiltration at the site of the parasite entry or may involve
systemic increase in certain types of cells, especially those circulating in the blood. One may
see an increase in circulating eosinophils. This is true for most infections caused by helminths.
In some cases, stimulation of red blood cell production may occur, especially in infections that
lead to mechanical loss or destruction of the red blood cells, as in hookworm infection or in
malaria. Certain parasitic infections may also lead to stimulation of neoplastic (cancer) growth
in the organ infected. For example, infection with the blood fluke Schistosoma japonicum may
eventually lead to development of cancer of the liver. Cancer of the biliary ducts may be seen in
infection with the liver fluke Clonorchis sinensis.
156 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Toxic and Allergic Phenomena (Immunopathology)

Proteins or other metabolites produced by the parasites may lead to hypersensitivity or
allergic reactions due to stimulation of antibody production. An example of this is infection
with pinworm Enterobius vermicularis where an allergic reaction occurs in the anus as a response
to the female worm and its eggs leading to its most prominent manifestation of pruritus ani.
Table 11.2 summarizes the various immunopathologic reactions of humans to parasites.
a
of

Table 11.2 Immunopathologic reactions of humans to various parasites

oa
th

do

not

Reaction Mechanism Result Example Cili

Type 1: Parasite antigen + IgE attach Anaphylactic shock; Helminths;

whip

allergy or to mast cells → histamine bronchospasm; local African hic

anaphylactic release inflammation trypanosomiasis

Type 2: Antibody + parasite antigen Lysis of cell bearing parasite Trypanosome
antibody on cell surface → complement antigens cruzi
mediated activation or ADCC
Type 3: Antibody + extracellular Inflammation & tissue Malaria,
immune antigen complex damage; complex deposition schistosomiasis,
complex in organs and tissues trypanosomiasis
Type 4: Sensitized T cell reaction Inflammation, mononuclear Leishmaniasis,
cell with antigen, release of accumulation, macrophage schistosomiasis,
mediated lymphokines, triggered activation; tissue damage trypanosomiasis
cytotoxicity

Opening of Pathways for entry of Other Pathogens

into the Tissues
The presence of the parasites and the damage they produce to the tissues may favor the
entry and proliferation of other organisms, especially bacteria. For instance, infection with
pinworm leads to intense itchiness in the anus. This causes the infected individuals, usually
children to scratch their anus and in the process, may lead to development of superficial
erosions in the anus. These erosions may serve as the point of entry for bacteria present in the
anus or those contained in the feces causing secondary bacterial infection in the eroded areas.

General Life Cycle of Parasites

The life cycle of parasites may vary from the very simple to the most complex. There
are several important components in the life cycle of parasites—source of infection, mode of
transmission, the infective stage (morphologic form that infects humans), the pathogenic stage
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(phylum Apicomplexa), and ciliates (phylum Ciliophora). The parasitic helminths or worms are
subdivided into two phyla: Nemathelminthes (roundworms) and Platyhelminthes (flatworms).
The flatworms are composed of two classes: Trematoda (flukes) and Cestoda (tapeworms).
158 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Most parasitic protozoa reproduce by binary fission except the sporozoa which undergo
both sexual and asexual reproduction. Flagellates are equipped with one or more whip like
flagella that enable them to move. Amoebae move by means of pseudopodia. Ciliates possess
rows or patches of cilia that serve as their organs of locomotion. Finally, sporozoa do not
possess any organ for motility. Figures 11.2 and 11.3 below show the summary of the
classification of medically important parasites.

Subkingdom
Protozoa

Phylum Phylum Phylum

Sarcomastigophora Ciliophora Apicomplexa

Subphylum Subphylum
Sarcodina Mastigophora

Class Class Class Class

Lobosea Zoomastigophora Kinetofragminophorea Sporozoa
(Amoebas) (Flagellates) (Ciliates)

Figure 11.2 Classification of protozoa

Adapted from Zeibig, Clinical Parasitology: A Practical Approach 2nd ed., 2013, p. 10

Subkingdom
Metazoa

Phylum Phylum
Nemathelminthes Platyhelminthes

Class Class Class Class

Nematoda Filariae Cestoda Trematoda
(Roundworms) (Tissue Roundworms) (Tapeworms) (Flukes

Figure 11.3 Classification of helminths

Adapted from Zeibig, Clinical Parasitology: A Practical Approach 2nd ed., 2013, p. 10
 Introduction to Parasitology 159

Table 11.3 Comparison of the biologic, morphologic, and physiologic properties of protozoa
and helminths
Parasite Biologic, Morphologic, and Physiologic Characteristics
Protozoa
Amoeba Unicellular; cyst and Binary fission Pseudopods Facultative Assimilation by
trophozoite forms anaerobe pinocytosis or
phagocytosis
Flagellates Unicellular; cyst and Binary fission Flagella Facultative Simple diffusion
trophozoite forms anaerobe or ingestion
via cytostome,
pinocytosis, or
phagocytosis
Ciliates Unicellular; cyst and Binary fission or Cilia Facultative Ingestion via
trophozoite forms conjugation anaerobe cytostome, food
vacuole
Sporozoa Unicellular, frequently Schizogony and None Facultative Simple diffusion
intracellular; multiple sporogony anaerobe
forms, including
trophozoites,
sporozoites, cysts
(oocysts), gametes
Helminths
Cestodes Multicellular; head Hermaphroditic No single Adults Absorption of
with segmented body organelle; usually nutrients from
(proglottids); lack of usually anaerobic intestines
digestive tract; head attachment
equipped with hooks to mucosa;
and/or suckers for possible
attachment muscular
motility
(proglottids)
Trematodes Multicellular; leaf Hermaphroditic; No single Adults Ingestion or
shaped with oral and Schistosoma organelle; usually absorption
ventral suckers, blind spp. has muscle anaerobic of body
alimentary tract separate sexes directed fluids, tissue,
motility or digestive
contents
Nematodes Multicellular; round, Separate sexes No single Adults Ingestion or
smooth, spindle organelle; usually absorption
shaped, tubular active anaerobic; of body
digestive tract; muscular larvae fluids, tissue,
possibility of teeth or motility possibly or digestive
plates for attachment aerobic content
160 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Laboratory Diagnosis of Parasitic Infections

Specimen Collection, Handling, and Transport
Essential to the management of parasitic infections is knowing the proper laboratory
diagnostic procedure to request. This includes knowledge of the proper specimen to collect.
The specimen varies depending on the portals of entry and exit of the parasite. For instance,
the most common portal of entry for parasites is the mouth. As such, the most common portal
of exit is the anus, in which case the proper specimen to collect is the stool. Daily shedding of
the diagnostic forms of the parasite may not occur. Thus, multiple specimens may be needed
for adequate detection. This will involve collection of three specimens, one specimen collected
every other day. For suspected cases of amoebiasis, up to 6 specimens collected within a period
of 14 days is recommended.
Timing of specimen collection is important. To demonstrate the motility characteristic of
protozoan parasites, fresh specimen must be used. The diagnostic stage for most protozoans
is the trophozoite, which is usually found in liquid stool. Therefore, it is recommended that
liquid stools be examined within 30 minutes after collection. Formed stools which usually
contain the cyst forms may be held for a maximum of 24 hours after collection. Preservatives
or fixatives may be added if the specimen cannot be examined right away. The preservatives
that may be used include formalin, polyvinyl alcohol, sodium acetate formalin, and modified
polyvinyl alcohol.
Stool specimens must be collected in a clean, water tight container and should be covered
tightly. Approximately 2–5 g of stool is recommended. No contamination with urine must be
allowed to avoid destruction of the parasite by the acidic urine. In addition, the stool should not
be collected from water from the toilet bowl since some parasites may be destroyed by water
(e.g., ova of schistosomes and trophozoites of amoeba).
Proper labelling of the container must be observed, accompanied by the fully accomplished
request form. Information other than patient general data may be included such as history of
travel and clinical findings. Once the specimen container is properly sealed, it must be placed
in a ziplock plastic bag for transport to the laboratory. Universal precautions must be observed
when handling all specimens. Gloves should be worn at all times.

Microscopic Examination
All fresh specimens submitted for examination must undergo microscopic examination,
which is divided into three stages—direct wet preparations, concentration technique, and
use of permanent stains. Ideally, the microscope to be used must be equipped with an ocular
micrometer since size (measured in microns or μm) is an important diagnostic feature
 Introduction to Parasitology 16

Direct wet preparation or direct wet mount

Purpose: To detect the presence of motile protozoan trophozoites; other stages detected include
cysts, oocysts, ova, and larvae of worms.
Principle: A small portion of unfixed stool is mixed with saline or iodine then studied under the
microscope.
Procedure:
1. Place small amount of unfixed stool on a glass slide.
2. Add a drop of 0.85% saline.
3. Mix using a wooden applicator stick.
4. Place a cover slip on the slide.
5. Examine slide using both low power and high power objectives of the microscope.
Variations include addition of a drop of iodine (Lugol’s or D’Antoni’s) to enhance the detail
of protozoan cysts. This is called direct iodine wet preparation.

Concentration methods
Purposes:
1. To aggregate parasites present into a small volume of the sample that enables the detection
of small numbers of parasites that might not be detected in direct wet preparations.
2. To remove debris and other contaminants that might interfere with the microscopic
examination.
Concentration techniques can be used on both fresh and preserved specimens. It is not
done if the purpose is to detect the motile trophozoites since the trophozoites do not survive
the procedure. It can be used to detect cysts, oocysts, ova, and larvae of nematodes. Two types of
concentration techniques are available—flotation and sedimentation.

Sedimentation (Formalin Ethyl Acetate Sedimentation Procedure)

(most widely used)
Principle: This is based on specific gravity – parasites are heavier than the solution used and
thus settle in the sediment of the tube while the fecal debris which are lighter will rise to the
upper layers of the test tube.
Procedure: Ethyl acetate is added to a saline washed formalin fixed sample in a test tube and
then centrifuged.
Advantage: It provides good recovery of most parasites and it is relatively easy to perform.
Disadvantage: The preparation contains more fecal debris than a flotation technique.
162 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Zinc sulfate flotation technique

Principle: This is based on the differences in specific gravity and the sample debris (in this
case heavier thus sinks to the bottom while the parasite is lighter and thus floats upward the
top of the tube). The zinc sulfate used has a specific gravity of 1.18–1.20 and is used as the
concentrating solution.
Advantage: It is able to remove more fecal debris, hence will yield a cleaner preparation.
Disadvantage: some helminth eggs are denser and may not float to the upper layer of the test
tube.

Permanent Stains
This serves as the final step in the microscopic examination for the detection of parasites.
A small amount of the fixed sample is placed on a slide glass and allowed to dry after which
it is stained. A cover slip is then placed after which a sealant is applied, thus allowing the
sample to remain intact for a longer period. It is designed to confirm the presence of cysts
and/or trophozoites of protozoans. Stains that may be used include Wheatly trichome
(most widely used), iron hematoxylin (to demonstrate morphology of intestinal protozoa), and
other specialized stains (e.g., modified acid fast stain to detect oocysts of Cryptosporidium).

Other Specimens and Laboratory Procedures

Duodenal Material
This may be collected using a nasogastric tube (NGT) or through the enteric capsule test
(Entero test). The collected duodenal fluid must be examined immediately to prevent rapid
deterioration of trophozoites, if present. A volume of > 2 mL is recommended. The sample
undergoes centrifugation prior to microscopic examination of the sediment.
In the Entero test, the patient is made to swallow a gelatin capsule that contains a coil
of yarn that is weighted. The yarn is released and is carried to the duodenum as the capsule
dissolves in the stomach. The free end of the yarn is attached to the patient’s neck or cheek
and after an incubation of 4 hours is pulled back out of the patient. The bile stained material
attached to the string is then examined microscopically with wet preparation followed by
application of permanent stains.

Sigmoidoscopy Material
Sigmoidoscopy is used to collect and examine material from the colon. This is helpful for
the diagnosis of infection with Entamoeba histolytica. Biopsy of colon material may be done
 Introduction to Parasitology 16

Cellophane Tape or Scotch Tape Preparation

This procedure is done to detect eggs of the pinworm Enterobius vermicularis. The female
parasite migrates to the anus at night where it lays its eggs. The procedure must therefore be
done first thing in the morning, before the patient defecates or washes. It may also be used to
detect the eggs of the tapeworm Taenia spp.

Blood
Examination of blood can detect the presence of blood borne parasites such as
Leishmania, Trypanosoma, Plasmodium and the filarial worms. Universal precautions and
asepsis must be observed during the collection and handling of blood specimen. Blood from
the fingertip or earlobe may be used (without anticoagulant) or from standard venipuncture
(with anticoagulant). In cases of suspected malaria infection, thick and thin blood smears
must be prepared and examined within 1 hour of collection. The thick smears serve for
screening purposes and used when parasites are few in number while the thin smears are best
to demonstrate the malarial parasites in the red blood cells, which is important for species
identification. The prepared smears may then be stained using Wright’s stain or Giemsa stain.

Cerebrospinal Fluid (CSF)

CSF may be used to diagnose certain amebic infections. It may also be used in patients
with African sleeping sickness. Similar to blood, the CSF must be immediately examined if
detection of parasite motility is desired. Wet preparations can be done to detect characteristic
morphologic forms of Naegleria, Acanthamoeba, and Trypanosoma as well as Toxoplasma gondii,
Taenia solium (cysticercosis), and Echinococcus.

Tissue and biopsy specimens

This may be utilized to detect the presence of Leishmania, Toxoplasma gondii, Trypanosoma,
Taenia solium, and Trichinella spiralis in tissues. In patients with suspected amebic liver abscess,
the abscess material taken from the liver is the specimen of choice.

Genitourinary Secretions
The specimen of choice for detecting the blood fluke Schistosoma haematobium is urine.
It may also be used to detect Trichomonas vaginalis, which may also be isolated from genital
secretions. Urine samples are centrifuged and the sediments examined for the presence of the
parasites. Genital secretions may be collected using a sterile cotton swab. Saline wet preparation
is then performed to demonstrate the trophozoite of the parasites.
164 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Others
1. Sputum – Paragonimus westermani, Strongyloides stercoralis (with hyperinfection),
E. histolytica, Ascaris lumbricoides larva, and the larvae of hookworms
2. Eye specimens – Acanthamoeba keratitis, Toxoplasma gondii, and Loa loa
3. Mouth scrapings and nasal discharge – E. gingivalis, Trichomonas tenax, Naegleria fowleri
4. Skin snips – skin fluid without bleeding obtained by making a small cut into the skin with a
razor blade; to detect motile microfilariae
5. Xenodiagnosis – special method for diagnosis of Chaga’s disease where an uninfected
reduviid bug (the vector) is allowed to take a blood meal from an infected patient and the
feces of the bug is then examined for the presence of Trypanosoma cruz
 Introduction to Parasitology 165

CHAPTER SUMMARY

• Symbiosis is a form of relationship where unlike organisms live together. Forms of

symbiotic relationships include commensalism, mutualism, and parasitism.

• Parasites are grouped based on the following: habitat (ectoparasites and endoparasites),
ability to live independently of the host (facultative and obligatory), and mode of living
(permanent, intermittent, erratic, incidental, and transitory).

• There are four types of hosts: definitive, intermediate, reservoir, and paratenic.
• The most common source of parasites is contaminated soil or water. Other sources
include: (1) food containing the parasite’s infective stage; (2) a blood sucking insect;
(3) a domestic or wild animal harboring the parasite; (4) another person and his or her
clothing, bedding, or the immediate environment he or she has contaminated; or (5) one’s
self (auto infection).

• The most common mode of transmission of parasites is through ingestion of

contaminated food and water (fecal oral transmission).

• Other means by which parasites are transmitted are through: (1) bite of an insect vector;
(2) skin penetration; (3) sexual intercourse; (4) transplacental transfer (mother to fetus);
and (5) mother’s milk (transmammary).

• The most common portal of exit of parasites is through the anus.

• Parasites produce disease through five basic mechanisms: (1) traumatic or physical
damage; (2) lytic necrosis; (3) toxic or allergic phenomena; (4) stimulation of host tissue
reaction; and (5) opening of pathways for entry of other pathogens into the tissues.

• Stool is the most common specimen used to detect presence of parasites. Other
specimens include urine, genital secretions, blood, sputum, CSF, and other sterile
body secretions.

• Microscopic
of parasites
examination of the stool specimen is the most widely used to detect presence
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 Introduction to Parasitology 167

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. It is a form of symbiotic relationship in which one organism benefits from the other
without benefitting or producing harm to the other:
a. Parasitism c. Commensalism
b. Mutualism d. B and C
2. Which of the following parasites may be transmitted through inhalation?
a. Ancylostoma c. Giardia
b. Enterobius d. Strongyloides
3. A type of host where the asexual stage of the parasite takes place:
a. Reservoir host c. Intermediate host
b. Paratenic host d. Definitive host
4. Infection with which among the following parasites can lead to development of
cancer of the liver?
a. Schistosoma japonicum c. Clonorchis sinensis
b. Plasmodium falciparum d. A and C
5. Which among the following parasites can be transmitted through sexual
intercourse?

a. Trichomonas vaginalis c. Enterobius vermicularis

b. Strongyloides stercoralis d. Balantidium coli
168 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Matching Type.

Parasite Mode of Transmission

6. Plasmodium a. Fecal oral transmission

7. Ascaris lumbricoides b. Auto infection
c. Bite of insect vector
8. Trichomonas
d. Sexual intercourse
9. Schistosoma e. Skin penetration
10. Entamoeba f. None of the abov
 CHAPTER

12 Protozoa

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. describe the general characteristics of the medically important parasitic protozoa; and
2. characterize and differentiate the parasitic protozoa as to their:
a. general features,
b. source of infection,
c. mode of transmission,
d. clinical manifestations,
e. treatment, and
f. prevention and control of infection.

Definition of Terms
Infective stage – refers to the stage of the parasite that enters the host or the stage that is
present in the parasite’s source of infection.
Pathogenic stage – refers to the stage of the parasite that is responsible for producing the organ
damage in the host leading to the clinical manifestations.
Encystation – process by which trophozoites differentiate into cyst forms.
Excystation – process by which cysts differentiate into trophozoite forms
170 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

General Properties of Protozoa

The Kingdom Protozoa consists of single celled eukaryotic organisms that are spherical to
oval or elongated in shape. The classification of these organisms is mainly based on the organ
of locomotion utilized. Not all protozoa are parasitic. Some are facultative parasites capable of
a free living state (e.g., Acanthamoeba and Naegleria). These normally reside in the soil or water
but can cause severe illness when they gain entrance into the central nervous system or the eyes.
Reproduction among the protozoa is relatively simple. Majority of protozoa divide by
means of binary fission (flagellates, ciliates, and amebae). Sporozoans reproduce through both
sexual and asexual means. Asexual reproduction is achieved through a process called merogony
or schizogony. Sexual recombination can occur, leading to antigenic and genomic variation.
Due to their small size, protozoan infections are most often diagnosed through
microscopic examination of body fluids, tissue specimens, or feces. Special stains may be used
to demonstrate the different protozoa. Most of the parasitic protozoa infections are diagnosed
by demonstrating the motile, feeding, dividing stage of the parasite called trophozoite, or the
dormant, non motile form called the cyst. The trophozoite is the motile (with pseudopods or
“false feet”) and feeding form and is the pathogenic stage. The cyst is the non motile form and
is the infective stage for most intestinal protozoan parasites, except for Trichomonas vaginalis
where cyst forms are not found.

Intestinal and Urogenital Protozoa

Subphylum Sarcodina: Entamoeba histolytica
Important properties and life cycle
Entamoeba histolytica is an intestinal and tissue ameba and is the only known pathogenic
intestinal ameba. Its life cycle consists of two stages—the non motile cyst (infective stage)
and the motile trophozoite (pathogenic stage). The trophozoite is found within the intestinal
and extra intestinal lesions, and in diarrheal stools. Cysts are usually found in non diarrheal,
formed stools
 Protozoa 171

a b
Ingested RBC Chromatoidal body
Nucleus Karyosome
Karyosome

Nucleus
Trophozoite Cyst

Figure 12.1 a Trophozoite of Entamoeba histolytica, as well as b comparison of trophozoite

and cyst morphology

Table 12.1 Comparison of E. histolytica trophozoite and cyst forms

Parameter Trophozoite Cys
Size range 8–65 μm 8–22 μm
Shape Irregular Spherical to round
Motility Yes (with finger like pseudopodia) No
Number of nuclei One One to four
Karyosome Small and central Small and central
Peripheral chromatin Fine and evenly distributed Fine and evenly distributed
Cytoplasm Finely granular Finely granular
Cytoplasmic inclusions Ingested red blood cells Chromatoid bars and diffuse
glycogen mass in young cysts

Epidemiology and Pathogenesis

Infection with Entamoeba histolytica is found worldwide but is more common in tropical
countries, especially in areas with poor sanitation. The parasite is primarily transmitted by the
fecal oral route through ingestion of the cyst from contaminated food and water. Water serves
as the major source of infection of the parasite. Sexual transmission may also occur when a man
has unprotected sex with a woman who has vaginal amoebiasis or through anal intercourse.
The ingested cyst undergoes excystation in the ileum where it differentiates into
a trophozoite (pathogenic stage). It then proceeds to colonize the cecum and colon.
The trophozoites may then undergo encystation and become converted into cysts, which are
then passed out with the feces. Trophozoites are usually recovered in the feces of patients
with active infection (diarrheic stools) while cysts are found in formed, non diarrheic stools.
The trophozoites of E. histolytica secrete enzymes that cause local necrosis producing the typical
“flask shaped” ulcer associated with the parasite. Invasion of the portal circulation may occur
leading to the development of abscess in the liver.
172 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Human ingests infective cyst

in contaminated food or water

Trophozoite
Organism excysts
in intestine—asexual
reproduction in colon
Encyst

Trophozoite passed
in liquid or soft
stool—not infective

Infective cyst Entamoeba histolytica

Hepatic abscess in formed stool only erodes intestinal wall

To liver via
circulatory system

Figure 12.2 Life cycle of Entamoeba histolytica

Disease: Amoebiasis
1. Acute intestinal amoebiasis – presents as bloody, mucus containing diarrhea (dysentery)
accompanied by lower abdominal discomfort, flatulence (release of gas), and tenesmus
(feeling of incomplete defecation). Chronic infection may occur, with symptoms such as
occasional diarrhea, weight loss, and fatigue. In some patients, a lesion called an amoeboma
may form in the cecum or in the rectosigmoid area of the colon, which may be mistaken
for a malignant tumor in the colon.
2. Extraintestinal amoebiasis – occurs when the parasite enters the circulatory system.
The most common extraintestinal form of amoebiasis is the amoebic liver abscess. This
is characterized by right upper quadrant pain, weight loss, fever, and a tender, enlarged
liver. Abscess found on the right lobe of the liver may penetrate the diaphragm and cause
lung disease (amoebic pneumonitis). Other organs that may become infected include the
pericardium, spleen, skin, and brain (meningoencephalitis)
 Protozoa 173

a b

Figure 12.3 a Solitary amebic liver abscess (arrow) and b resection of the abscess showing its
characteristic “anchovy sauce” appearance

3. Asymptomatic carrier state – occurs under the following conditions: (a) if the parasite
involved is a low virulence strain; (b) if the parasite load is low; and (c) if the patient’s
immune system is intact. In these cases, the patient presents with no symptoms but the
parasite reproduces and is passed out with the patient’s feces.

Laboratory Diagnosis
Diagnosis of intestinal amoebiasis is confirmed by the finding of trophozoites in diarrheic
stools or cysts in formed stools. The trophozoites characteristically contain ingested red blood
cells. The stool specimen should be examined within one hour of collection to see the motility
of the trophozoites. Serologic testing may be useful for the diagnosis of invasive amoebiasis.

Treatment
The drug of choice for symptomatic intestinal amoebiasis or hepatic abscess is
metronidazole. The alternative drug tinidazole is for both intestinal and extraintestinal
amoebiasis. Asymptomatic carriers should be treated with diloxanide furoate, metronidazole,
or paromomycin. Surgical drainage of amoebic liver abscess may be necessary if there is no
improvement with medical therapy.

Prevention and Control

The most important preventive measure is the observance of good personal hygiene.
This includes proper hand washing, especially for food handlers. Proper waste disposal should
be observed to avoid fecal contamination of water sources. The use of “night soil” (human feces)
for fertilization of crops must be avoided. Adequate washing and cooking of vegetables should
be observed
174 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Subphylum Mastigophora:
Giardia lamblia (Giardia intestinalis)
Important Properties and Life Cycle
Giardia lamblia is an intestinal protozoan that was initially known as Cercomonas intestinalis.
At present, the name Giardia intestinalis has gained popularity. Another name used is
Giardia duodenale.
The parasite also exists in a cyst form and a trophozoite form. The trophozoite is
pear shaped or teardrop shaped with four pairs of flagella and has a motility likened to a falling
leaf. The trophozoite has been described as resembling an old man with whiskers (“old man
facies”). It also possesses a sucking disc which the parasite uses to attach itself to the intestinal
villi of the infected human.
The cyst is typically oval and thick walled with four nuclei. The fully mature cyst contains
four nuclei with four median bodies. It divides through binary fission. Each cyst gives rise to
two trophozoites during excystation in the intestinal tract.

a b

Figure 12.4 a A typical trophozoite with four pairs of flagella and b an oval shaped cyst is
shown on the right photo

Epidemiology and Pathogenesis

Giardia lamblia has a worldwide distribution through contaminated water sources. The
disease can thus occur in outbreaks related to contaminated water supplies. About 50% of
infected individuals do not present with symptoms and serve as carriers. Other than humans,
many species of mammals may act as reservoirs. The infection is also common among
individuals engaging in oral anal contact. High incidence has been seen in daycare centers and
among patients in mental hospitals.
The parasite is primarily transmitted through ingestion of the cyst from fecally
contaminated water and food. The cyst enters the stomach and is stimulated by the gastric
acid to undergo excystation in the duodenum. The trophozoites then attach themselves to th
 Protozoa 175

duodenal mucosa through the sucking disks. Damage to the intestines is not due to invasion
of the parasite but because of inflammation of the duodenal mucosa, leading to diarrhea with
malabsorption of fat and proteins. The trophozoites may also infect the common bile duct
and gallbladder.

Figure 12.5 Life cycle of Giardia lamblia

Disease: Giardiasis
1. Asymptomatic carrier state – infection with the parasite is usually completely
asymptomatic. The infected individual unknowingly passes out the parasite with the feces
which can then contaminate water
176 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. Giardiasis (Traveler’s diarrhea) – infection is characterized by a non bloody, foul

smelling diarrhea accompanied by nausea, loss of appetite, flatulence, and abdominal
cramps. The symptoms may persist for weeks or months. Malabsorption of fat may
lead to the presence of fat in the stool (steatorrhea). Patients are usually afebrile.
Manifestations may vary depending on which nutrient becomes deficient due to the
resulting malabsorption. These may include deficiencies in fat soluble vitamins, folic acid,
and proteins. It is a self limiting infection, lasting one to two weeks. Relapses may occur,
especially in patients with IgA deficiency.

Laboratory Diagnosis
Diagnosis is made by the demonstration of the cyst or trophozoite (or both) in diarrheic
stools. Only cysts are isolated from the stools of asymptomatic carriers. If microscopic
examination of the stool is negative, string test may be performed which consists of making
the patient swallow a weighted piece of string until it reaches the duodenum. The trophozoites
adhere to the string and can be visualized after withdrawal of the string.

Treatment
As per recommendation of the Centers for Disease Control and Prevention in the United
States, the primary choice of treatments for G. lamblia infection are metronidazole, tinidazole,
and nitazoxanide.

Prevention and Control

The main preventive measure involves avoidance of fecal contamination of water supplies
through proper waste disposal. Drinking water should be boiled, filtered, or iodine treated
especially in endemic areas. Improvement of personal hygiene such as proper hand washing is
also recommended.

Subphylum Mastigophora:
Trichomonas vaginalis
Important Properties and Life Cycle
The parasite is a pear shaped organism
with a central nucleus, four anterior flagella,
and an undulating membrane. It exists Figure 12.6
Trophozoite of
only in the trophozoite form (infective and Trichomonas vaginalis
pathogenic). Source: Beards, 201
 Protozoa 177

Epidemiology and Pathogenesis

Trichomonas vaginalis is not an intestinal pathogen. It causes urogenital infections and
the main mode of transmission is through sexual intercourse. It has been isolated from the
urethra and vagina of infected women as well as the urethra and prostate gland of infected
men. Infection is highest among sexually active women in their thirties and lowest in post
menopausal women. Occasionally the parasite may be transmitted through toilet articles and
clothing of infected individuals. Infants may be infected as they pass through the infected birth
canal during delivery.
The parasite invades the vaginal mucosa of infected women, where they multiply through
binary fission. The trophozoites feed on local bacteria and leukocytes. In men, the most
common infection site is the prostate gland and the urethral epithelium.

Figure 12.7 Life cycle of Trichomonas vaginali

178 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Disease: Trichomoniasis
Infection in men – usually asymptomatic and men serve as the reservoir for infection in
women. In men who develop symptoms, the manifestations are those related to development of
prostatitis (inflammation of the prostate), urethritis (manifest as discharge), and other urinary
tract involvement. Persistent or recurring urethritis is the most common symptomatic form of
the infection.
Infection in women – also asymptomatic, some women may present with scant, watery
vaginal discharge. In more severe cases, the discharge may be foul smelling and greenish yellow
in color. This may be accompanied by itching (pruritus) and a burning sensation in the vagina.
The cervix appears very red, with small punctuate hemorrhages, giving rise to a strawberry
cervix. Other common symptoms include dysuria and increased frequency of urination.
Infection in infants – occurs as the infant passes through the infected birth canal of the
mother during vaginal delivery. The infected infants may manifest conjunctivitis or respiratory
infection.

Laboratory Diagnosis
Diagnosis is made by the finding of the characteristic trophozoite in a wet mount of vaginal
or prostatic secretions, urine, and urethral discharges.

Treatment
The drug of choice for treatment of trichomoniasis is metronidazole. All sexual partners
of an individual with the infection must be simultaneously treated to prevent “ping pong”
infections.

Prevention and Control

The best way to prevent infection is to practice safe sex. The use of condoms can limit the
transmission of the parasite. Health and sex education are important. Maintenance of the acidic
pH of the vagina may also be helpful.

Phylum Ciliophora: Balantidium coli

Important Properties and Life Cycle
Balantidium coli is morphologically more complex than E. histolytica. It has a primitive
mouth called a cytostome, a nucleus, food vacuoles, and a pair of contractile vacuoles.
The infective stage is the cyst and the pathogenic stage is the trophozoite, which invade
 Protozoa 179

the mucosal lining of the terminal ileum, cecum, and colon. It is the largest protozoan to
infect humans.
The trophozoites typically exhibit a rotary, boring motility (through cilia) and contain two
nuclei (a small dot like micronucleus adjacent to a kidney bean shaped macronucleus). The cyst
also contains two nuclei although the micronucleus may not be readily observable.

Epidemiology and Pathogenesis

The parasite has a world wide distribution. The most common and most important
reservoir is the pig. Monkeys may occasionally act as reservoirs of the parasite. The main
source of infection is water contaminated by pig feces and the mode of transmission is through
the fecal oral route. Person to person transmission via food handlers has been implicated
in outbreaks.
The cysts are found in contaminated water, which when ingested, undergoes excystation
in the small intestines. From there, the trophozoites travel to the large intestines where they
produce ulcers similar to those seen in amoebiasis. However, extra intestinal involvement is
not seen.

a b

Cytostome

Figure 12.8 Balantidium coli trophozoites characterized by their

large size (40 μm to more than 70 μm), and the presence of cilia
on the cell surface, which is particularly visible in a . The large
macronucleus is seen in b and a cytostome. The micronucleus is
less conspicuous.

Disease: Balantidiasis
Most infected individuals are asymptomatic. A dysenteric type of diarrhea resembling
amebic dysentery may occur in patients with high parasite load. Acute infections may manifest
with liquid stools containing pus, blood, and mucus while chronic infections may manifest
with a tender colon, anemia, wasting (cachexia), and alternating diarrhea and constipation.
Extraintestinal infection is rare and may involve the liver, lungs, mesenteric nodes, and
urogenital tract
180 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Laboratory Diagnosis
Diagnosis is based on the finding of trophozoites and cysts in the stool specimen. Due to
its large size, the parasite can be readily detected in fresh, wet microscopic preparations.

Treatment
The current recommended treatment of patients with balantidiasis involves two
and iodoquinol. Metronidazole may also be used as alternative to treat infecteddrugs—oxytetracycline

patients.

Prevention and Control

Preventive measures are similar to those for amoebiasis. These include maintenance of
sanitary hygiene, proper disposal of pig feces, and boiling of drinking water.

Blood and Tissue Protozoa

Subphylum Sarcodina: Acanthamoeba (Free living Amoeba)
Important Properties and Life Cycle
Acanthamoeba castellani, together with Naegleria, is a minor protozoan pathogen but unlike
Naegleria, Acanthamoeba usually causes infection in immunocompromisedpatients. It is a
free living amoeba that causes inflammation of the brain substance and its meningeal coverings
(meningoencephalitis). The parasite is found widely in soil, contaminated freshwater lakes, and
other water environment. It is able to survive in cold water. Like E. histolytica, the infective stage
is the cyst while the pathogenic stage is the trophozoite.

Epidemiology and Pathogenesis

There are two ways by which the parasite can be acquired—through aspiration or nasal
inhalation or through direct invasion in the eye. People acquire the infection usually while
swimming in contaminated water. Inhalation of the cysts from dust has also been shown to
occur. The trophozoites enter through the lower respiratory tract or through ulcers in the
mucosa or skin. The parasite then migrates through the bloodstream and invade the central
nervous system.
Eye infection with Acanthamoeba occurs primarily in patients who wear contact lenses.
The parasite has been recovered from contact lenses, lens cases, and contact lens solutions.
Tap water contaminated with the parasite is the source of infection for contact lens users
 Protozoa 18

Disease
1. Granulomatous amebic encephalitis – infection occurs primarily in immunocompromised
individuals. The parasite produces a granulomatous amebic encephalitis and brain
abscesses in immunocompromisedpatients. Symptoms develop slowly and may include
headache, seizures, stiff neck, nausea, and vomiting. The brain lesions may contain both
the trophozoites and the cysts. In rare instances, the parasite may spread and produce
granulomatous lesions in the kidneys, pancreas, prostate, and uterus.
2. Keratitis – infection of the cornea of the eye. Symptoms include severe eye pain and vision
problems. Loss of vision may occur due to perforation of the cornea.

Laboratory Diagnosis
Diagnosis is made by finding of both trophozoites and cysts in the cerebrospinal fluid as
well as brain tissue and corneal scrapings. Histologic examination of corneal scrapings may
also be done. Calcofluor white, a stain usually used to demonstrate fungi, may be used to
demonstrate the parasite in corneal scraping specimens.

Treatment
Pentamidine, Ketoconazole, or Flucytosine may be effective in the treatment of infection,
however, prognosis is poor even with treatment. For eye and skin involvement, topical
miconazole, chlorhexidine, itraconazole, ketoconazole, rifampicin, or propamidine may be used.
Propamidine has been documented to have the best success record.

Prevention and Control

Infection can be prevented through adequate boiling of water. Regular disinfection of
contact lenses is also advised. Contact lens wearers are also advised to avoid using homemade
non sterile saline solutions.

Subphylum Sarcodina: Naegleria

Important Properties and Life Cycle
Similar to Acanthamoeba, the parasite Naegleria is also classified as a free living protozoan.
It shares many characteristics with Acanthamoeba. The parasite is also found worldwide in
soil and contaminated water environment. Unlike Acanthamoeba, Naegleria can survive in
thermal spring water. The known pathogen worldwide is Naegleria fowleri, which is the only
amoeba with three identified morphologic forms—trophozoite, flagellate, and cyst forms.
182 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The trophozoite exhibits the typical amoeboid motility which is described as “slug like.”
The flagellate form is pear shaped and is equipped with two flagella that is responsible for
the parasite’s jerky or spinning movement. The non motile form is the cyst. The amoeboid
trophozoite form is however the only form that is known to exist in humans.

Epidemiology and Pathogenesis

Naegleria infection is usually acquired transnasally when swimming in contaminated
water. The parasite penetrates the nasal mucosa and cribriform plate, enters the central
nervous system, and produces a rapidly fatal meningitis and encephalitis (primary amoebic
meningoencephalitis). Unlike Acanthamoeba, the parasite produces infection in otherwise
healthy individuals, usually children. In some instances, the parasite may be acquired through
inhalation of dust containing the parasite. The entire life cycle of the parasite (amoeboid
trophozoite → flagellate trophozoite → amoeboid trophozoite → cyst form) occurs entirely in
the external environment.

Disease
1. Asymptomatic infection – the most common clinical presentation in patients with
colonization of the nasal passages.
2. Primary amoebic meningoencephalitis(PAM) – the result of colonization of the brain by
the amoeboid trophozoites leading to rapid tissue destruction. Patients initially complain
of sore throat, nausea, vomiting, fever, and headache. Patients eventually develop signs of
meningeal irritation (e.g., Kernig’s sign) as well as alterations in their senses of smell and
taste. If untreated, the patients may die within one week after onset of symptoms.

Laboratory Diagnosis
Diagnosis is based on the finding of the amoeboid trophozoites in the cerebrospinal fluid.

Treatment
Treatment is ineffective because of its rapidly fatal course. However, some patients
have been shown to recover from infection due to early detection and initiation of
treatment. Treatment of choice is Amphotericin B in combination with miconazole and
rifampicin (Murray, 2014)
 Protozoa 18

Prevention and Control

There is no known means of preventing Naegleria infection other than the prevention
of contamination of water sources. Adequate chlorination of swimming pools and hot
tubs is recommended.

Subphylum Mastigophora:
Hemoflagellates Leishmania spp.
Important Properties and Life Cycle
The life cycle of the parasite involves a vector, the female sandfly of the Phlebotomus
and Lutzomyia genera. Leishmania spp. are obligate intracellular parasites. It has three
morphologic forms—the amastigote, promastigote, and epimastigote. The infective stage is
the promastigote. The promastigote form may be seen only if a blood sample is collected and
examined immediately after transmission. Epimastigotes are found primarily in the vector.
The pathogenic stage and diagnostic form is the amastigote which is found primarily in tissue
and muscle, as well as the central nervous system within macrophages and in cells of the
reticuloendothelial system.
The typical amastigote is round to oval in shape and contains a nucleus, a basal
body structure called a blepharoblast, and a small parabasal body located adjacent to the
blepharoblast. Both the blepharoblast and parabasal body are collectively known as the
kinetoplast. The promastigote is long and slender, with a kinetoplast located in its anterior end,
and a single free flagellum extending from the anterior portion.

Epidemiology and Pathogenesis

The parasite has a worldwide distribution. Natural reservoirs include rodents, ant eaters,
dogs, and cats. In endemic areas, the parasite may be transmitted in a human vector human
cycle. There are three major strains of Leishmania which differ in the tissues affected
and the resulting clinical manifestations. These are Leishmania donovani (visceral
leishmaniasis), Leishmania tropica (cutaneous leishmaniasis), and Leishmania braziliensis
(mucocutaneous leishmaniasis).
184 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Figure 12.9 Life cycle of Leishmania spp.

Leishmania donovani complex

L. donovani is the causative agent of visceral leishmaniasis (also known as kala azar
or dumdum fever). The complex consists of (1) L. donovani chagasi which is mainly seen in
Central America (mainly Mexico, West Indies, and South America) and is transmitted by
the Lutzomyia sandfly; (2) L. donovani donovani found in parts of Africa and Asia (Thailand,
India, China, Burma, and East Pakistan) and is transmitted by the Phlebotomus sandfly;
and (3) L. donovani infantum, also transmitted by the Phlebotomus sandfly and is found mainly
in Mediterranean Europe, Near East, and Africa.
The promastigote is injected into the human host through bite of the sand fly. After entry
into the host, it loses its flagella, is engulfed by macrophages, and transforms into amastigotes.
The organs of the reticuloendothelialsystem (liver, spleen, and bone marrow) are the most
severely affected
 Protozoa 18

Disease: Visceral Leishmaniasis(Kala azar, Dumdum Fever)

After an incubation period of 2 weeks to 18 months, the disease begins with intermittent
fever, weakness, and weight loss. Massive enlargement of the spleen (splenomegaly) is
characteristic, leading to hypersplenism and resulting anemia. Hepatomegaly or enlargement
of the liver also occurs. In light skinned patients, hyperpigmentation of the skin may be seen
(kala azar means “black sickness” or “black fever”). Involvement of the bone marrow leads
to destruction of the cellular components with the corresponding clinical effects—anemia
due to destruction of red blood cells, bleeding tendencies due to reduction of platelets
(thrombocytopenia), and increased risk for secondary infection because of reduction of white
blood cell (leukopenia). Glomerulonephritis or inflammation of the glomeruli of the kidney
may also occur. The disease may be fatal if untreated.

Laboratory Diagnosis
The screening test is called the Montenegro skin test. This test is similar to the tuberculin
skin test for the diagnosis of tuberculosis. It is used as screening for large populations at
risk but is not used for diagnosis. Definitive diagnosis is done by demonstration of the
amastigote from Giemsa stained slides of specimen from blood, bone marrow, lymph nodes,
and biopsies of infected areas. Culture of blood, bone marrow, and other tissues may also be
done, which will show the promastigote forms. Serologic tests are now also available such as
indirect fluorescent antibody (IFA), enzyme linked immunosorbent assay (ELISA), or direct
agglutination test (DAT).

Treatment
The present recommended drug of choice is liposomal amphotericin B (Ambisome).
Sodium stibogluconate has also been found to be effective but the development of resistance
may occur. Other patients have shown favorable responses to gamma interferon in combination
with pentavalent antimony.

Prevention and Control

Control of the vector population is important in the prevention of infection. The use of
insect repellents, protective clothing, and installation of screens may be helpful. Prompt
treatment of infected humans is essential to help halt the spread of the disease.
186 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Leishmania braziliensis complex

L. braziliensis is the causative agent of mucocutaneous leishmaniasis which involves
skin, cartilage, and mucous membranes. Infection with L. braziliensis occurs most commonly
in Brazil and Central America, primarily in construction and forestry workers. The
complex consists of L. panamensis (Panama and Colombia), L. peruviana (Peruvian Andes),
and L. guyanensis (The Guianas, parts of Brazil and Venezuela).
Infection is transmitted by sandflies (Lutzomyia and Psychodopigus) through skin bite.
The promastigotes invade the reticuloendothelial cells where they transform into amastigotes
(diagnostic stage). Reproduction of the amastigotes result in tissue destruction. The amastigotes
are taken up by the vector during a blood meal and are transformed into promastigotes.

Disease: MucocutaneousLeishmaniasis
Mucocutaneous leishmaniasis, also called espundia, begins with a papule at the site of insect
bite, then forms metastatic lesions, usually at the mucocutaneous junction of the nose and
mouth. Disfiguring granulomatous, ulcerating lesions destroy the nasal cartilage (tapir nose)
but not the adjacent bone. Death can occur from secondary infections.

a b c

Figure 12.10 Clinical manifestations of infection with Leishmania: a cutaneous leishmaniasis by

L. tropica; b mucocutaneous leishmaniasis by L. braziliensis; and c enlarged spleen in patient
with visceral leishmaniasis caused by L. donovani.

Laboratory Diagnosis
Diagnosis is confirmed by demonstration of amastigotes in clinical specimen. Ulcer biopsy
specimens are used for the diagnosis of mucocutaneous leishmaniasis. Microscopic examination
of Giemsa stained ulcer biopsy specimens reveals the diagnostic amastigotes. Culture of
infected material may show the promastigotes. Serologic testing may also be done
 Protozoa 18

Treatment
At present, the most widely used drug for the treatment of mucocutaneous leishmaniasis
is sodium stibogluconate, although resistance has been shown to develop. Alternative drugs
include liposomal Amphotericin B and oral anti fungal drugs (fluconazole, ketoconazole,
and itraconazole).

Prevention and Control

The most important preventive measure is the control of the insect vector. If this cannot be
done, measures should be undertaken to protect individuals from sandfly bites by using netting,
window screens, protective clothing, and insect repellents. Prompt treatment can also help
prevent spread of the disease.

Leishmania tropica complex

Important Properties and Life Cycle
The complex consists of L. tropica, L. aethiopica, and L. major. These are the causative
agents of what is referred to as Old World cutaneous leishmaniasis. The life cycle of L. tropica
is similar to that of L. braziliensis. All three members of the complex are transmitted by the
Phlebotomus sandfly and primarily attacks the human lymphoid tissue of the skin.

Disease: Old World Cutaneous Leishmaniasis

The disease is also known as oriental sore, and Baghdad or Delhi boil. It is characterized
by one or several pus containing ulcers that may heal spontaneously. The initial lesion is a
small, pruritic red papule at the bite site. In patients with anergy and hypersensitivity responses,
spontaneous healing does not occur. Thick skin plaques with multiple nodules may develop,
especially on the limbs and face.

Laboratory Diagnosis
Microscopic examination of Giemsa stained slides of fluid aspirated from beneath the
ulcer bed is the usual diagnostic procedure of choice. Microscopic examination reveals the
typical amastigotes. Culture of specimen will show the promastigote form. Serologic tests are
also available.
188 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Treatment
The drug of choice is sodium stibogluconate. Steroids with application of heat to the
infected lesions may be used. Other alternative drugs are meglumine antimonite, pentamidine,
and oral ketoconazole. Paromomycin ointment may be helpful in the healing of the ulcers.

Prevention and Control

Preventive measures are the same as those for the different forms of leishmaniasis.
However, unlike the other Leishmania, a vaccine has been developed against L. tropica which is
currently undergoing clinical trials.

Trypanosomaspp.
Important Properties and Life Cycle
The trypanosomes are also hemoflagellates like Leishmania. The major difference
between the two lies in their diagnostic stages, which is the amastigote for Leishmania and the
trypomastigote for the trypanosomes. The trypomastigotes are curved, assuming the shape of
the letters C, S, or U. Unlike Leishmania, the kinetoplast of the trypomastigote is posteriorly
located, with the single large nucleus located anterior to it. The trypomastigotes are visible in
the peripheral blood.

Trypanosomacruzi
Epidemiology and Pathogenesis
The parasite is found primarily in South and Central America and is transmitted by
the bite of the reduviid or triatomid bud (Triatoma or “cone nose” bug or “kissing bug”).
It is usually transferred to a human host when the feces of the bug containing the infective
trypomastigotes is deposited near the bite site. The feces are then introduced into the bite site
when the host scratches the bite area. Other routes of transmission include blood transfusion,
sexual intercourse, transplacental transmission, and through the mucous membranes when
the bite site is near the eye or mouth. Humans and animals (domestic cats and dogs, and wild
species such as armadillo, raccoon, and rat) serve as reservoir hosts.
The trypomastigotes invade the surrounding cells and transform into amastigotes.
The amastigotes then reproduce leading to destruction of host cells. These are then transformed
back into trypomastigotes, which invade the blood, penetrate other cells in the body, and
transform back into amastigotes
 Protozoa 189

Different cell types may be affected. However glial cells, reticuloendothelialcells,

and especially myocardial cells are the most frequently affected. The disease is primarily seen
in rural areas because the reduviid bug lives in the walls of rural huts and feeds at night. Acute
infection is rarely seen in the United States. Chronic infection is now seen with increasing
frequency among immigrants from Latin America.

Disease: Chagas Disease (American Trypanosomiasis)

The acute phase of the disease begins with a nodule (chagoma) near the bite site and
unilateral swelling of the eyelid with conjunctivitis (Romana’s sign). The eyelid swelling may be
due to the bug feces being accidentally rubbed into the eye. This is accompanied by fever, chills,
malaise, myalgia, and fatigue. Patients may recover or may enter the chronic phase.
Hepatosplenomegaly,enlargement of lymph nodes (lymphadenopathy), and myocarditis
with cardiac arrhythmia characterize the chronic phase of Chagas disease. Cardiac muscle is the
most frequently and most severely affected tissue. Loss of tone of the colon and esophagus due
to destruction of the Auerbach’s plexus may lead to abnormal dilatation of these organs, called
megacolon and megaesophagus, respectively. CNS involvement may also be seen in the form of
meningoencephalitisand cysts. Death may occur due to cardiac failure and arrhythmias.

a b c

Figure 12.11 a A patient with chagoma on the lower lip, b the reduviid bug, and c Romana's sig
190 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Figure 12.12 Life cycle of Trypanosoma cruzi

Laboratory Diagnosis
Acute disease is diagnosed by the finding of trypomastigotes in thick or thin films of the
patient’s blood. Other diagnostic methods that can be used include bone marrow aspiration,
muscle biopsy, culture on special medium, and xenodiagnosis. Xenodiagnosis entails allowing
an uninfected laboratory raised reduviid bug to feed on an infected patient. After several weeks,
the intestinal contents of the bug are examined for the presence of the parasite. Serologic tests
can also be helpful. Both xenodiagnosis and serologic tests are useful in the chronic form of the
disease.

Treatment
The drugs of choice for treatment are benznidazole and nifurtimox but these are less
effective during the chronic phase of the disease. Alternative agents are allopurinol and
ketoconazole
 Protozoa 19

Prevention and Control

Prevention involves protection from the bite of the reduviid bug, improvement of housing
conditions, and insect control. Education regarding the disease and its transmission is
also helpful.

Trypanosomabrucei gambiense
and Trypanosomabrucei rhodesiense
Epidemiology and Pathogenesis
The two species are similar in morphology and life cycle. Their life cycles involve the tsetse
fly (Glossina) as the vector. Humans are the reservoir for T. brucei gambiense, while domestic
animals (especially cattle) and wild animals serve as the reservoir for T. brucei rhodesiense.
The infective and pathogenic stage is the trypomastigote.
The trypomastigotes spread from the skin to the blood then to the lymph nodes and the
brain. A demyelinating encephalitis occurs leading to the characteristic manifestations of the
disease. T. gambiense infection (West African or Gambian Sleeping Sickness) is chronic while
T. rhodesiense infection (East African or Rhodesian Sleeping Sickness) is more rapidly fatal.
The disease is endemic in sub Saharan Africa which is the natural habitat of the tsetse fly.
T. gambiense causes disease along the water courses in West Africa while T. rhodesiense causes
disease mostly in the arid regions of East Africa.

Disease: African Sleeping Sickness

The initial lesion is an indurated ulcer called chancre at the site of the insect bite.
Intermittent weekly fever and lymphadenopathy then develop. Enlargement of the posterior
cervical lymph nodes (Winterbottom’s sign) is commonly seen. Other manifestations seen
during this stage include red rash accompanied by pruritus, localized edema, and a delayed
pain sensation (Kerandel’s sign). The encephalitis is characterized by headache, insomnia, and
mood changes. Muscle tremors, slurred speech, and apathy follow, progressing to somnolence
(sleeping sickness) and coma. Untreated disease is fatal.
Trypanosoma brucei rhodesiense is more virulent than Trypanosoma brucei gambiense. Infection
with the parasite has a shorter incubation period. Winterbottom’ssign may not be seen.
There is no lymphadenopathy and CNS involvement occurs early in the course of the disease.
A rapid and fulminating disease may follow with the parasite spreading in the blood. Death is
seen usually within 9–12 months following infection in untreated patients and may be due to
glomerulonephritis and myocarditis.
192 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Laboratory Diagnosis
Microscopic examination of Giemsa stained slides of the blood, lymph node aspirations
and CSF will reveal the trypomastigotes during the early stages of the disease. Aspiration of the
chancre or enlarged lymph nodes may also reveal the parasites. Parasites are isolated from the
CSF of patients with CNS involvement. Serologic tests can also be helpful as well as detection
of the presence of IgM and proteins in the CSF of patients. The presence in the serum and/or
CSF of IgM is considered diagnostic.

a b c

Figure 12.13 a Typical trypanosomal chancre seen at bite site, b the tsetse fly, and c enlarged
cervical lymph nodes
Source: International Atomic Energy Agency, 2015 and Hudson, 2014

Figure 12.14 Life cycle of Trypanosoma bruce

 Protozoa 19

Treatment
Several drugs are available for the treatment of both East African and West African
Sleeping Sickness, which include melarsoprol, suramin, pentamidine, and eflornithine
(Zeibig, 2013). The choice of drug will depend on whether the patient is pregnant or not,
the age of the patient, and the stage of the disease.

Prevention and Control

Preventive measures involve protection against the bite of the fly. Use of netting and
protective clothing are recommended. Use of fly traps and insecticides may be helpful. Clearing
the forest around the villages are also helpful measures.

Subphylum Apicomplexa: Plasmodium spp.

Important Properties and Life Cycle
Malaria is caused by five plasmodia species: Plasmodium vivax, Plasmodium malariae,
Plasmodium ovale, Plasmodium knowlesi, and Plasmodium falciparum. The vector and definitive
host is the female Anopheles mosquito. The sexual cycle (sporogony) occurs primarily in
mosquitoes, and the asexual cycle (schizogony) occurs in humans (intermediate hosts).
The infective stage is the sporozoite from the saliva of the biting mosquito, which is taken up
by the liver cells. This is called the exoerythrocytic phase. Multiplication and differentiation
of sporozoites into merozoites occur during this stage. P. vivax and P. ovale produce a latent
form (called hypnozoite or sleeping form) in the liver, which is the cause of the relapse or
recrudescence seen in vivax and ovale malaria.
Merozoites (pathogenic stage) are released from liver cells and infect the red blood cells.
The parasite’s life cycle now enters the erythrocytic phase. These merozoites multiply and are
eventually released to infect other red blood cells. The periodic release of merozoites causes
the typical recurrent symptoms seen in malaria patients. Some merozoites then develop
into microgametocytes (male gametocytes) and macrogametocytes (female gametocytes).
The gametocyte containing red blood cells are ingested by the mosquito during feeding. Sexual
reproduction then ensues.
194 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Table 12.2 Comparison of morphological forms and characteristics of the different Plasmodium
species
P. falciparum P. malariae P. vivax P. ovale
Young • fine ring • thick ring; one • thick ring, • thick ring; one
trophozoite chromatin dot often irregular chromatin dot
• multiple infection amoeboid
• crescent shaped • similar
P. vivax
to
but appearance; • circular shape
mass at outer one chromatin
smaller
edge of RBC dot
(accole form);
1 to 2 small
chromatin dots
• only detected in
severe infection
Mature • ring enlarged; • round with • irregular, • round, compact
trophozoite slightly irregular central amoeboid
chromatin and
band forms;
pigment
(hemozoin)
distinct
Schizont 8 to 36 merozoites 6 to 12 merozoites 12 to 24 8 to 14 merozoites
in cluster or rosette arranged in merozoites with rosette
arrangement; ring rosettes or arranged arrangement
enlarged; only irregular clusters; irregularly
detected in severe central location
infections of brown green
pigment
Gametocyte • crescentic • oval or rounded • oval or rounded • oval or rounded
• male: reddish • male: diffuse • male: diffuse • male: diffuse
with diffuse chromatin chromatin chromati
chromatin
• female: bluish
with compact
chromatin
Size of Unchanged Unchanged or Enlarged Enlarged
infected smaller
red cell
Shape of Sometimes Unchanged Unchanged Often irregular
red cell irregular and with jagged
crenated edges
Stippling Sometimes present Rarely present Often present Always present
(Maurer’s dots) (Zieman’s dots) (Shuffner’s dots) (Shuffner’s dots)
 Protozoa 195

a b c d

Figure 12.15 Comparison of the trophozoite forms of the different Plasmodium species:
a P. falciparum; b P. vivax; c P. malariae; and d P. ovale

P. Vivax P. Ovale P. Malariae P. Falciparum

Ring Stage

Trophozoite

Schizont

Segmenter

sequestered
Gametocytes

Figure 12.16 Comparison of morphological forms of the different Plasmodium specie

196 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Epidemiology and Pathogenesis

Infection with plasmodia occurs worldwide. It occurs primarily in tropical and subtropical
areas, especially in Asia, Africa, and Central and South America. Sixty nine percent (69%) of
cases in the Philippines are due to Plasmodium falciparum while the remaining 31% are due to
Plasmodium vivax (World Malaria Report 2013). The primary vector is Anopheles flavirostris,
which breeds in clear, slow flowing streams near foot hills and forests. In the 2014 Asia Pacific
Malaria Elimination Network (APMEN) VI held in Makati City, Philippines, then Secretary
of Health Doctor Enrique Ona reported an 83% reduction in malaria cases from 2005 to 2013,
with a 92% decrease in malarial deaths. Secretary Ona also reported that of 53 known provinces
that are endemic for the disease, 27 have already been declared malaria free, which are: Cavite,
Batangas, Marinduque, Catanduanes, Albay, Masbate, Sorsogon, Camarines Sur, Iloilo, Aklan,
Capiz, Guimaras, Bohol, Cebu, Siquijor, Western Samar, Eastern Samar, Northern Samar,
Northern Leyte, Southern Leyte, Biliran, Camiguin, Surigao del Norte, Benguet, Romblon,
Batanes, and Dinagat Islands.
The main mode of transmission of malaria is the bite of the female mosquito vector.
However, the parasite can also be transmitted through blood transfusion (transfusion malaria),
intravenous drug abuse with sharing of IV needles (“main line malaria”), and transplacental
transmission (congenital malaria). Most of the pathologic findings result from the destruction
of red blood cells. P. falciparum and P. knowlesi can infect both young and old red blood cells
leading to high levels of parasitemia. P. vivax and P. ovale mainly infects young red blood cells,
while P. malariae infects old red blood cells.
Plasmodium knowlesi is a natural parasite of macaque monkeys throughout the Southeast
Asia region. Cases of infection have been noted in Thailand, Singapore, Brunei, Indonesia,
Myanmar, Vietnam, and the Philippines (Murray, 2014). The red blood cells infected
by P. knowlesi have normal morphology. All developmental stages of the parasite may be seen in
the peripheral blood
 Protozoa 197

Figure 12.17 Life cycle of Plasmodium spp.

Disease: Malaria
Paroxysms of malaria are divided into three stages: cold stage, hot stage, and the sweating
stage. These paroxysms are considered partially as allergic responses to the schizonts and to the
antigens released following the release of the merozoites. A malarial paroxysm presents with
abrupt onset of chills (rigors) accompanied by headache, muscle pain (myalgia), and joint pains
(arthralgia). This stage lasts for approximately 10–15 minutes or longer. Spiking fever lasting
2–6 hours follows, reaching up to 41 °C, accompanied by shaking chills, nausea, vomiting, and
abdominal pain. This is then followed by drenching sweats. Patients usually feel well between
febrile episodes. Splenomegaly is often present and anemia is prominent.
The timing of the fever cycle is 72 hours for P. malariae, in which symptoms recur every
4th day (quartan malaria). Malaria caused by P. vivax, P. ovale, and P. falciparum recur ever
198 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

3rd day (tertian malaria). P. falciparum causes malignant tertian malaria since it causes severe
infection which is potentially life threatening due to extensive brain (cerebral malaria) and
kidney damage. The dark color of the patient’s urine is due to kidney damage giving rise to the
term “black water fever.” P. vivax and P. ovale cause benign tertian malaria that is characterized
by relapses that can occur up to several years after the initial illness and is due to the latent
hypnozoites in the liver.
Most cases of P. knowlesi infection resembles infection in patients by other malarial
parasites. A small number of cases of patients develops severe infection. The severity of the
infection is due to the high parasitemia levels produced due to its ability to infect all stages of
red blood cells and its 24 hour erythrocyte cycle (quotidian malaria).

Laboratory Diagnosis
The diagnosis of malaria is based on examination of Giemsa stained or Wright stained
thick and thin smears of the blood. The thick blood smears are used for screening purposes
while the thin blood smears are used to differentiate the various Plasmodium species. The best
time to take blood films is midway between paroxysms of chills and fevers or before the onset
of fever. This is the time when the greatest number of intracellular organisms are present.
Characteristic trophozoites will be seen within the infected red blood cells. P. falciparum will
show characteristic crescent shaped or banana shaped gametocytes. Infection with P. falciparum
is highly considered if there are > 10 infected red blood cells consisting only of ring forms. For
P. malariae and P. knowlesi, demonstration of the characteristic rosette schizont is diagnostic.
P. knowlesi should be suspected if there is a higher average merozoite count of 16/red blood cell
as compared to 10–12/red blood cell of P. malariae. The presence of early trophozoite forms
and two to three parasites per red blood cell (similar to P. falciparum) is more suggestive of
P. knowlesi infection.

Treatment
The drugs of choice for acute malaria infection are chloroquine or parenteral quinine.
However, chloroquine does not affect the hypnozoites of P. vivax and P. ovale. For vivax and
ovale malaria, primaquine is given to destroy the hypnozoites. For chloroquine resistant strains
of P. falciparum other agents may be used including mefloquine + artesunate, artemether
lumafantrine, atovaquone proguanil, quinine, quinidine, pyrimethamine sulfadoxine
(Fansidar), and doxycycline (Murray, 2014). Artemisin based combination therapies (ACTs)
are now recommended for uncomplicated malaria and for chloroquine resistant vivax malaria.
Artesunate is the drug of choice for severe malaria, in combination with either amodiaquine,
mefloquine, or sulfadoxine pyrimethamine. P. knowlesi infection is managed similar to
P. falciparum due to its potential to produce severe infection
 Protozoa 19

Prevention and Control

Chemoprophylaxis of malaria for travelers to endemic areas consists of mefloquine or
doxycycline. Travelers to areas where the other plasmodia are found should take chloroquine
starting two weeks before arrival and continued for 6 weeks after departure, followed by a
2 week course of primaquine if exposure was high.
Other preventive measures include avoidance of the bite of the vector through the use of
mosquito netting, window screens, protective clothing, and insect repellants. The mosquitoes
usually bite from dusk to dawn, so protection is important during the night. Reduction of
mosquito population is also helpful, including the use of insecticide sprays, as well as drainage
of stagnant water in swamps and ditches.

Phylum Apicomplexa: Toxoplasma gondii

Important Properties and Life Cycle
The definitive host of the parasite is the domestic cat or other felines while humans and
other mammals serve as the intermediate hosts. The parasite develops in the intestinal cells of
the cat and passes to the tissues through the bloodstream. These are then passed in the cat’s
feces and mature into infective oocysts in the external environment. Infection in humans begins
with the ingestion of oocysts (infective form) in undercooked meat or from contact with cat
feces. In the small intestines, the oocysts rupture into trophozoites (tachyzoites or bradyzoites).
Tachyzoites are the rapidly multiplying forms responsible for the initial infection while
bradyzoites are shorter, slow growing forms seen in chronic infections.
200 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Conoid
Aprical polar ring

a Micronemes

Rhoptries

Dense granules b
Subpellicular microtubules
Inner membrane
complex with
underlying subpellicular
network (not shown)
Mitochondrion
Apicoplast
Nucleus

Endoplasmic reticulum

Plasma membrane

Posterior pole

Figure 12.18 a Tachyzoite and b bradyzoite of Toxoplasma gondii

Epidemiology and Pathogenesis

Infection by T. gondii occurs worldwide. Infection is usually sporadic but outbreaks
associated with ingestion of raw meat or contaminated water can occur. Individuals who are
severely immunocompromisedare more likely to develop severe disease.
The parasite can be transmitted in two ways: (1) ingestion of improperly cooked meat of
animals that serve as intermediate hosts, and (2) ingestion of oocyst from contaminated water.
Transplacental transmission may occur, with severe consequences on the fetus. Sharing of
needles by IV drug abusers as well as blood transfusion are less common modes of transmission
of the parasite
 Protozoa 201

Human ingests raw

Human ingests meat containing
infective cyst with
oocyst from bradyzoites
cat feces

Sporozoite
Congenital toxoplasmosis
released
penetrates
intestinal Formation of "cysts"
Infect fetus cell containing bradyzoites
in various organs

Tachyzoite
formed
Tachyzoites
cross
placental barrier Asexual Immune system response
reproduction
in cells
Other tissue
cells invaded
by tachyzoites

Pregnant woman Cells rupture

Hematogenous spread

Figure 12.19 Life cycle of Toxoplasma gondii

Disease: Toxoplasmosis
1. Infection in immunocompetentindividuals – usually asymptomatic. Acute infection may
manifest non specific symptoms such as chills, fever, headache, and fatigue. This may
be accompanied by inflammation of lymph nodes (lymphadenitis). Chronic infection
may manifest with lymphadenitis, hepatitis, myocarditis, and encephalomyelitis.
Chorioretinitis leading to blindness may also occur
202 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. Congenital infection – occurs in infants born to mothers who were infected during
pregnancy. The manifestations vary depending on when the infection was acquired.
Infection during the first trimester of pregnancy may result to miscarriage, stillbirth,
or severe infection (encephalitis, microcephaly, hydrocephalus, mental retardation,
pneumonia). If the infant acquires the infection during the last trimester, symptoms may
not develop until months to years after delivery. The most common manifestation is
chorioretinitis with or without blindness.
3. Infection in immunocompromisedhosts – usually manifest with neurologic symptoms
similar to patients with diffuse encephalopathy, meningoencephalitis,or brain tumors.
Reactivation of latent toxoplasma infection is common. Other sites of infection include
the lungs, eye, and testes.

Laboratory Diagnosis
Demonstration of high antibody titers through immunofluorescence assay is essential for
the diagnosis of toxoplasma infection. Microscopic examination of Giemsa stained preparations
will show the crescent shaped trophozoites during the acute infection. Cysts may be seen in the
tissues. Prenatal diagnosis can be done through ultrasonographyand amniocentesis with PCR
analysis of the amniotic fluid (method of choice).

Treatment
Infection in immunocompetenthosts is usually self limiting and does not require specific
therapy. The regimen of choice for immunocompromisedpatients, especially those with AIDS,
is initial high dose pyrimethamine plus sulfadiazine given for an indefinite period. Alternative
regimen for those who develop symptoms of drug toxicity is clindamycin plus pyrimethamine.
For pregnant women, clindamycin or spiramycin may be given.

Prevention and Control

The most effective preventive measure is through adequate cooking of meat. Pregnant
women should refrain from eating undercooked meat and should avoid contact with cats and
refrain from handling litter boxes. Cats should not be fed raw meat
 Protozoa 203

CHAPTER SUMMARY

• Protozoa are unicellular organisms that are spherical or elongated in shape.

• The classification of protozoa into phyla is based in part on their mode of locomotion,
namely: Sarcodina (pseudopods or false feet), Apicomplexa (no organ of locomotion),
Mastigophora (flagella), and Ciliophora (cilia).

• Important human pathogens of Subphylum Sarcodina are Entamoeba histolytica,

Acanthamoeba, and Naegleria.

• Subphylum Mastigophora is composed of flagellated members—the intestinal parasite

Giardia lamblia, the urogenital parasite Trichomonas vaginalis, and the hemoflagellates
Leishmania and Trypanosoma.

• Important members of the Subphylum Apicomplexa are Toxoplasma gondii and the
malarial parasite Plasmodium.

• There is only one significant human pathogen in the Subphylum Ciliophora which
is Balantidium coli. It produces infection similar to Entamoeba histolytica but does not
produce extraintestinal infection.

• Some protozoa are capable of a free living state. These are Acanthamoeba and Naegleria,
both of which can cause infection of the central nervous system.

• For most protozoa, reproduction is means of binary fission. In some, it is accomplished

by
through union of two cells (syngamy), while in others, sexual reproduction may be seen
(e.g., Plasmodium).

• The mode of transmission of protozoa may be varied. Intestinal and luminal protozoa
can be transmitted by person to person or through fecal oral means. Blood and tissue
protozoa may be spread through direct contact or through vectors (e.g., Anopheles
mosquito for malaria or reduviid bug for Trypanosoma). Congenital or transplacental
transmission may occur in infection with Toxoplasma gondii and Plasmodium.

• The infective stage for most protozoa is the trophozoite while the pathogenic stage is the
cyst, except for Trichomonas vaginalis which exists only in the trophozoite form.

• Diagnosis of protozoal infection is usually through microscopic examination of

Giemsa stained specimens. Diagnosis is confirmed by detecting the diagnostic forms of
the parasites
This page is intentionally left blank
 Protozoa 205

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. Which among the following parasites produce infection that is acquired by

ingestion of contaminated water?
a. Balantidium coli d. A and B only
b. Giardia lamblia e. A, B, and C
c. Leishmania donovani
2. Which among the following parasites is transmitted by the bite of an insect vector?
a. Plasmodium c. Entamoeba
b. Trichomonas d. Toxoplasma
3. Malignant tertian malaria is caused by which among the following?
a. P. vivax d. P. falciparum
b. P. ovale e. P. knowlesi
c. P. malariae
4. Infection with which among the following parasites is through sexual transmission?
a. Leishmania c. Trichomonas
b. Plasmodium d. Entamoeba
5. Which among the following protozoal diseases is associated with intestinal and
extraintestinal involvement?
a. Amoebiasis c. Malaria
b. Giardiasis d. Trypanosomiasis
206 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. Visceral leishmaniasis is caused by which among the following parasites?

a. L. donovani c. L. braziliensis
b. L. tropica d. B and C
7. Dysentery and steatorrhea are associated with which of the following parasites?
a. Entamoeba c. Balantidium
b. Giardia d. Toxoplasma
8. Which of the following is the insect vector for Leishmania?
a. Anopheles mosquito c. Tsetse fly
b. Aedes mosquito d. Triatoma or reduviid bug
9. Transplacental transmission is associated with which among the following parasites?
a. Toxoplasma c. Entamoeba
b. Trichomonas d. Trypanosoma
10. East African Sleeping Sickness is caused by:
a. Trypanosoma cruzi c. Trypanosoma brucei rhodesiense
b. Trypanosoma brucei gambiense d. B and
 CHAPTER

13 Cestodes

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. describe the general characteristics of the medically important cestodes; and
2. characterize and differentiate the different cestodes as to their:
a. general features,
b. source of infection,
c. mode of transmission,
d. clinical manifestations,
e. treatment, and
f. prevention and control of infection.

General Properties of Cestodes

Cestodes are classified under the subkingdom Metazoa, phylum Platyhelminthes. These
parasites are considered as primitive worms. They do not possess a digestive system nor a
nervous system. They absorb nutrients and eliminate waste products through their outer
surface called the tegument. Commonly known as tapeworms, these parasites are flat and
consist of three distinct regions—the head, neck, and body (proglottids). The head contains
an organ of attachment called the scolex, which may consist of either hooks, suckers, or sucking
grooves. In some species, the scolex has a fleshy extension called a rostellum to which hooks may
be attached.
The body is divided into multiple segments (hence, the name tapeworm) called proglottids.
A series of proglottids is called strobila (plural strobili). All cestodes are hermaphroditic
(self fertilizing) with each proglottid containing both male and female reproductive organs
208 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Each proglottid, therefore, is capable of laying eggs (now called a pregnant proglottid
or gravid segment).
The neck serves as the region of growth and connects the head to the body of the worm.
The worm grows by adding new proglottids from the neck. The oldest proglottids are found at
the most distal part of the body of the parasite.
A typical cestode life cycle is divided into three stages—egg, larva, and adult worm. For the
majority of cestodes, the egg contains an embryo called the oncosphere, which represents the first
larval or motile stage. It is equipped with small hooks (called hooklets) that eventually enable the
parasite to pierce the wall of the intestines. The eggs are excreted in the feces of infected hosts
and are transmitted to the intermediate hosts (cattle, pig, or fish). Infection in humans is usually
acquired through ingestion of the undercooked or raw flesh of the intermediate host containing
the infective larvae. After ingestion, the ingested larvae are transformed into adult worms in
the intestines of the infected host. The adult worm then undergoes self impregnation with the
gravid segment rupturing to release the eggs in the intestines. These eggs are then passed out to
the external environment during defecation.

Intestinal Cestodes
Taenia saginata (Beef Tapeworm)
Important Properties and Life Cycle
The intermediate host is cattle where the eggs enter the blood vessels within the cattle’s
intestines. The eggs are then transported to the skeletal muscles of the cattle where they
develop into cysticerci (larvae). Infection with the beef tapeworm is acquired by ingestion of
improperly cooked or raw beef containing the infective larva (called cysticercus). These larvae
then mature into adult worms (pathogenic stage) in the small intestines within a period of
approximately three months. These tapeworms are known to achieve a length of as much as
10 meters. Humans serve as the definitive hosts.
The eggs of Taenia saginata are usually indistinguishable from the eggs of the pork
tapeworm Taenia solium. Both species may be differentiated by the appearance of their scolices
and the structures of their proglottids. The scolex of Taenia solium contains a rostellum while
that of Taenia saginata does not. Taenia saginata proglottid is rectangular and contains more
uterine branches (about 15–30) in comparison with Taenia solium which is square in appearance
containing about 7–15 uterine branches
 Cestodes 209

Intermidiate Host
(Cattle)

Infection by ingestion of
Definitive Host undercooked contaminated meat
(Man) containing the cysticercus larvae.
Figure 13.1 Life cycle of the beef tapeworm Taenia saginata

Epidemiology and Pathogenesis

Taenia saginata infection is common in areas of the world where beef is routinely eaten,
especially undercooked beef. It has been found to be endemic in Eastern Europe, Russia,
Eastern Africa, and Latin America (Centers for Disease Control and Prevention). The adult
worms do not produce significant damage in the small intestines.

Disease: Taeniasis
Majority of patients are asymptomatic. Those with high worm burden may complain
of diarrhea, abdominal pain, loss of appetite with resultant weight loss, and body malaise.
The gravid proglottids may reach the anus where egg laying may occur resulting in itchiness in
the anal region (pruritus ani).

Laboratory Diagnosis
Examination of fecal specimen from infected patients is the procedure of choice. Eggs
or gravid proglottids may be recovered from the stool although eggs are less often found than
the proglottids
210 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Treatment
The drug of choice against the adult worm is praziquantel.

Prevention and Control

Proper waste disposal and sanitation practices as well as the adequate cooking of beef
are the main preventive measures for taeniasis. Freezing of beef meat for approximately
10 days may kill the encysted larvae. Prompt treatment of infected persons help prevent spread
of the disease.

Taenia solium (Pork Tapeworm)

Important Properties and Life Cycle
Infection with the pork tapeworm is acquired through ingestion of improperly cooked
or raw pork meat which contains the infective larva called cysticercus cellulosae. Unlike the
beef tapeworm, Taenia solium infection can also occur following the ingestion of food or
water contaminated with human feces that contain the eggs of the parasite. Therefore, unlike
the beef tapeworm, Taenia solium has two infective stages—eggs and larvae. Autoinfection
may also occur. Pigs serve as the intermediate host while humans serve as both intermediate
and definitive hosts.
There are two scenarios that can occur depending on which infective stage entered the
human host. In cases where infection is acquired through ingestion of undercooked or raw
pork meat, the infective stage is the larval form which transforms into adult worm in the
intestines of infected individuals. In this instance, humans serve as the definitive hosts. On the
other hand, ingested worm eggs hatch in the small intestines, burrow through the wall of the
intestines into a blood vessel, and disseminate to various organs. In this instance, humans serve
as intermediate hosts.

Table 13.1 Comparison of scolex and gravid segments of Taenia saginata and Taenia solium
Characteristic Taenia saginata Taenia soliu
Scolex
Number of suckers Four Four
Rostellum Absent Present
Hooks Absent Present; double crown
Gravid Proglottid
Appearance, shape Rectangular Somewhat square
Number of uterine branches 15–30 7–15
on each side of uterus
 Cestodes 211

Epidemiology and Pathogenesis

T. solium infection is more prevalent in underdeveloped communities with poor sanitation
and where people eat raw or undercooked pork. Higher rates of illness have been seen in people
in Latin America, Eastern Europe, sub Saharan Africa, India, and Asia (Centers for Disease
Control and Prevention).
Adult worms produce little damage in the intestines. Encysted larvae may produce
damage in the tissues where they disseminate. For instance, in the brain, they may manifest as
space occupying lesions. Although the larvae may encyst in various tissues of the body, they
evoke little inflammatory response. However, when the encysted larvae die, they may release
substances that may induce an allergic reaction in the host which may potentially be fatal due
to the development of anaphylactic shock.

Cysticercus in
Scolex lungs, brain, eyes
attaches
to intestine Circulation

Humans Onchosphere

Gravid Autoinfection
Egg Embryonated
Cysticercus in muscle eggs or
proglottids
ingested
in feces
Proglottid
Circulation Swine

Embryonated eggs
Onchosphere or proglottids ingested

Figure 13.2 Life cycle of the pork tapeworm Taenia solium

Disease
1. Taeniasis – the disease produced by the adult worm. Most cases are asymptomatic
but in the presence of high worm burden, manifestations may be similar to beef
tapeworm infection.
2. Cysticercosis – the result of larval encystation in various tissues of the body. The most
common involvement is that of the skeletal muscles where patients may complai
212 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

of muscle pain. Cyticercosis of the brain (neurocysticercosis) is the most feared and
most severe involvement. It may present with symptoms associated with increased
intracranial pressure such as seizures, headache, and vomiting. Ocular cysticercosis may
lead to visual disturbances due to development of inflammation of the uvea (uveitis) and
retina (retinitis).

Laboratory Diagnosis
Microscopic examination of stool specimen from infected persons is the diagnostic
procedure of choice in patients with taeniasis. Demonstration of ova or proglottids may
help establish the diagnosis. The demonstration of the typical morphology of the scolex can
differentiate pork tapeworm from beef tapeworm. For cysticercosis, diagnostic procedure
depends on demonstration of the cyst in tissue, through biopsy or CT scan.

Treatment
The drug of choice for treatment of intestinal infection is praziquantel. For cysticercosis,
praziquantel may also be effective but it is usually not recommended for ocular and
CNS involvement. Alternative drugs include albendazole, paromomycin, and quinacrine
hydrochloride. Surgical removal of the larvae may be necessary. Anti convulsants may be given
in cases of neurocysticercosis.

Prevention and Control

Important preventive measures for pork tapeworm infection are the same as that for beef
tapeworm and include proper waste disposal and sanitary measures, thorough cooking of pork
meat, and the prompt treatment of infected persons to prevent the spread of the parasite.

Diphyllobothriumlatum (Broad Fish Tapeworm)

Important Properties and Life Cycle
The longest of the tapeworms, the fish tapeworm can reach a length of about 13 meters. Its
eggs consist of ciliated larvae called coracidia (s. coracidium). One end of the egg is occupied by
a lid structure called an operculum. Its scolex contains a pair of long sucking grooves. The gravid
segments contain a uterine structure that is centrally located and assumes a rosette formation.
Human infection with D. latum is through ingestion of improperly cooked or raw fish
containing the plerocercoid (infective stage), the precursor larval stage. After ingestion, the
plerocercoid attaches to the intestinal mucosa and matures into the adult worm. The adul
 Cestodes 213

worm self fertilizes and the eggs are passed out with the stool. If the eggs come to contact
with fresh water, the coracidium hatches and is ingested by the first intermediate host, a
tiny crustacean called a copepod (Cyclops sp.). After ingestion, the coracidium develops into
the larval stage called the procercoid. The copepod is then eaten by a freshwater fish (second
intermediate host) where the procercoid develops into the plerocercoid. Definitive hosts for the
parasites are humans and other fish eating mammals such as dogs, cats, bears, and seals.

Figure 13.3 Life cycle of Diphyllobothrium latum

Epidemiology and Pathogenesis

D. latum infection occurs in countries where raw freshwater fish is consumed. Little damage
is produced in the small intestines of the human hosts. In some individuals, the parasite may
compete with the host for vitamin B12, leading to a deficiency of this vitamin
214 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Disease: Diphyllobothriasis
1. Asymptomatic disease – the most common presentation among most individuals infected
with the parasite.
2. Diphyllobothriasis – may manifest with symptoms of gastrointestinal involvement, which
may include diarrhea and abdominal discomfort. When the adult worm attaches itself to
the jejunum and ileum, the patient may develop deficiency of vitamin B12, leading to
anemia similar to pernicious anemia and is characterized as megaloblastic anemia resulting
from lack of maturation of red blood cells.

Laboratory Diagnosis
Diagnosis is based on finding of the characteristic eggs and/or the proglottids (less
frequent) in a stool specimen.

Treatment
The drug of choice for the treatment of diphyllobothriasis is praziquantel. An alternative
drug is niclosamide.

Prevention and Control

Preventive measures include proper sanitary procedures, thorough cooking of fish prior
to consumption, and the prompt treatment of infected individuals to prevent spread of the
parasite. Freezing of the fish for 24–48 hours at –18 °C can kill all larvae.

Hymenolepis nana (Dwarf Tapeworm)

Important Properties and Life Cycle
H. nana is different from the other tapeworms because it does not require an obligatory
intermediate animal host. The eggs are directly infectious and humans get the infection
after the accidental ingestion of the eggs of the parasite. This may occur after ingestion
of fecally contaminated food or water. One may also acquire the eggs by touching one’s
mouth with contaminated fingers or through ingestion of contaminated soil. Accidental
ingestion of rice or flour beetles containing the infective larvae and that may have gotten
into food is another way by which the infection may be acquired. Rodents serve as additional
source of infection
 Cestodes 215

Once the eggs (infective stage) gain entrance into the human host after ingestion of
contaminated food and water, the eggs transform into cysticercoid larvae. The larvae mature
into adult worms capable of self reproduction. Eggs are released after disintegration of the
gravid segments. There are two pathways for the eggs—the eggs may be passed to the outside
environment through the feces or some of the eggs may remain inside the human host. Those
that remain inside the human host hatch into larvae and mature into adult worms, thereby
starting a new cycle within the human host. This type of re infection is called autoinfection.

Epidemiology and Pathogenesis

The dwarf tapeworm is the most common tapeworm recovered in the United States. It has
a worldwide distribution and is also found in East Asia and the Philippines. It is common in
areas with inadequate sanitation and hygiene. Children and persons living in crowded areas are
at risk of developing infection. The parasite produces little damage in the small intestines.

= Infective Stage
= Diagnostic Stage

Oncosphere hatches
5 Cysticercoid develops
3 Humans and rodents are
infected when they ingest in intestinal villus
cysticercoid infected arthropods.

Cysticercoid
develops in
9 Autoinfection can occur
if
insect eggs remain in the intestine.
The eggs then release the
4 Scolex
hexacanth embryo, which
Embryonated egg penetrates the intestinal villus
ingested by humans continuing the cycle.
from contaminated
food, water, or hands

Adult ileal 7
portion of small
Egg ingested
by insect intestin
2

Eggs can be released through the

8 genital atrium of the gravid proglottids.
Gravid proglottids can also disintegrate
1 Embryonated egg in feces releasing eggs that are passed in stools.

Figure 13.4 Life cycle of Hymenolepis nana

216 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Disease: Hymenolepiasis
Most patients are asymptomatic. In cases of high worm burden, patients may complain of
nausea, weakness, loss of appetite, diarrhea, and abdominal pain. In young children with heavy
infection, anal itchiness (pruritus ani) may occur leading to headaches due to difficulty sleeping.
It can be confused with a pinworm infection. Autoinfection may lead to hyper infection
syndrome which can result in secondary bacterial infection and spread of the worms to other
tissues of the body.

Laboratory Diagnosis
Diagnosis is established by finding of the characteristic eggs in stool specimen.

Treatment
Praziquantel is the drug of choice. Niclosamide can be an alternative drug.

Prevention and Control

Important preventive measures include proper hygiene and waste disposal, control of
transport host population, and rodent control. Proper storage of grains and flour must be
observed to prevent infestation with flour and grain beetles. Prompt treatment of infected
individuals must be instituted to prevent the spread of the parasite.

Extra Intestinal Cestode

Echinococcusgranulosus
(Dog Tapeworm or Hydatid Tapeworm)
Important Properties and Life Cycle
Infection with E. granulosus is primarily a zoonotic type of infection. Dogs are the most
important definitive hosts while sheep are usually the intermediate hosts. Humans are
considered as accidental and dead end hosts. The eggs of E. granulosus are identical to those of
Taenia spp. and are thus not diagnostic. The diagnostic stage of the parasite is its larval form,
which is encased in a cyst wall and is called the hydatid cyst.
Infection is acquired after ingestion of eggs (infective stage) from food and water
contaminated by dog feces or through contact with contaminated dog feces. Eggs transform
into larvae in the intestines, penetrate the intestines, and migrate through the bloodstrea
 Cestodes 217

to different tissues in the body, particularly the liver and the lungs. The hydatid cyst
(pathogenic stage) then develops in the infected tissues. Dogs acquire the parasite by eating the
visceral organs of the intermediate host.

4
Adult flea harbours
the infective cysticercoid. Humans, normally children,
7 acquire the infection by
5 ingesting the infected flea.
Cysticercoid
Host is infected
by ingesting fleas
Infected larval containing cysticercoid.
stage develop
into adult flea.

Oncosphere Cysticercoid
Scolex attaches
Oncospheres hatch from in intestine
the eggs and penetrate Animals can transmit the
the intestinal wall of the 3 infected fleas to humans.
larvae. Cysticercoid larvae
develop in the body cavity. Gravid proglottids are
passed intact in the feces
or emerge from perianal
1 region of either animal
or human hosts. 8 Adult in small intestin
2
Egg packets containing Each proglottid contains egg packets
embryonated eggs that are held together by an outer
are ingested by larval embryonic membrane (see 2 ).
stage of flea. The proglottids disintegrate and
release the egg packets.
= Infective Stage
= Diagnostic Stage

Figure 13.5 Life cycle of Echinococcus granulosus

Epidemiology and Pathogenesis

E. granulosus infection is common in Africa, Europe, Asia, the Middle East, Central and
South America, and in rare cases, North America (Center for Disease Control and Prevention).
The embryos develop into large, fluid filled hydatid cysts, which act as space occupying lesions.
In addition, the cyst fluid contains antigens that can sensitize the host. Rupture of the cyst,
either spontaneously or during trauma or surgical removal, may lead to the release of these
antigens leading to anaphylaxis and widespread dissemination of the parasite.
218 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Disease: Echinococcosis, Hydatid Cyst Disease,

Hydatid Disease, Hydatidosis
Most patients are asymptomatic during the early stages of the disease. However, as the cysts
enlarge, necrosis of the infected tissues occur. Involvement of the liver may result in obstructive
jaundice. Patients with lung involvement may manifest with cough, chest pain, and shortness of
breath. Other organs that may be infected include the spleen, kidneys, heart, bone, and central
nervous system, including the brain and eyes (Center for Disease Control and Prevention). Cyst
rupture may lead to anaphylactic shock leading to death of the patient.

Laboratory Diagnosis
There are several ways by which E. granulosus infection can be diagnosed. These
include (1) examination of biopsy specimen; (2) serologic tests (e.g., ELISA or indirect
hemagglutinationtest); and (3) radiography to demonstrate the hydatid cysts (e.g., CT scan or
ultrasound). Care should be exercised when doing biopsy to prevent rupture of the cyst.

Treatment
In cases when surgery is possible, removal of the cyst has been considered as the treatment
of choice. However, medical management alone may prove effective, especially if the cyst is
located in inaccessible areas. Drugs that have been proven effective include mebendazole,
albendazole, and praziquantel.

Prevention and Control

Improvement of personal hygiene practices, prevention of contamination of food and
water with dog feces, avoidance of feeding pet dogs with contaminated viscera, and the
prompt treatment of infected canines and humans are some measures to prevent the spread of
the parasite. Chemoprophylaxis should be given to dogs in endemic areas. Health education
is essential
 Cestodes 219

CHAPTER SUMMARY

• Cestodes are primitive worms that do not possess a digestive system nor a nervous
system. They absorb nutrients and eliminate wastes through their outer covering called
the tegument.

• Aandtypical cestode consists of a head containing the organ of attachment (scolex), a neck,
the body which is divided into segments called proglottids.

• All cestodes are hermaphroditic and capable of self reproduction. Eggs are released
disintegration of the gravid proglottids and are released to the outer environment with
by

the feces.

• The major mode of transmission for all cestodes is through ingestion of the infective
stage, usually the eggs, found in contaminated water, soil, or food.

• The major intestinal cestodes are Taenia saginata (beef tapeworm), Taenia solium
(pork tapeworm), Diphyllobothrium latum (broad fish tapeworm), and Hymenolepis nana
(dwarf tapeworm).

• Both beef and pork tapeworm infections are acquired through ingestion of raw or
undercooked beef or pork meat.
» Cattle serve as the intermediate hosts for the beef tapeworm while pig or swine serve
as the intermediate hosts for the pork tapeworm.
» The infective stage for T. saginata is the cysticercus larva while for T. solium both the
egg and the larva (cysticercus cellulosae) serve as the infective stages.
» The adult worms serve as the pathogenic stage for both beef and pork tapeworms.
Larvae may also serve as the pathogenic stage for pork tapeworm if the infective stage
is the egg.
» The adult worm for both tapeworms produces the disease taeniasis. The larval form
of the pork tapeworm encysts in tissues leading to the development of cysticercosis,
the most severe form of which is neurocysticercosis.

• D.intermediate
latum is unique among the tapeworms because it has two intermediate hosts. The
host is the copepod, a tiny crustacean which is ingested by the second
first

intermediate host, the freshwater fish.

• There is no obligatory animal intermediate host for E. granulosum. Unlike the other
tapeworms, humans only serve as accidental and dead end hosts for the parasite
This page is intentionally left blank
 Cestodes 221

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Matching Type.

Column A Column B
1. Dogs serve as the definitive host a. Taenia saginata
2. Has two intermediate hosts b. Taenia solium
c. Diphyllobothrium
3. Autoinfection can occur latum
4. Acquired through ingestion of raw beef d. Hymenolepis nana
5. Has no obligatory intermediate host e. Echinococcus
granulosu
6. Obstructive jaundice may occur
7. A tiny crustacean serves as intermediate host
8. Megaloblastic anemia may develop
9. Cysticercosis develops after ingestion of eggs
10. Humans serve as dead end hosts

Identification.

11. The infective stage for D. latum.

12. The organ of attachment of cestodes.
13. The usual intermediate host for E. granulosus.
14. The most commonly recovered tapeworm in the U.S.
15. Anal itchiness occurs in infection with this cestode.
This page is intentionally left blank
 CHAPTER

14 Trematodes

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. describe the general characteristics of the medically important trematodes; and
2. characterize and differentiate the different trematodes as to their:
a. general features,
b. source of infection,
c. mode of transmission,
d. clinical manifestations,
e. treatment, and
f. prevention and control of infection.

General Properties of Trematodes

Trematodes, commonly known as flukes, belong to the class Trematoda or Digenea.
They may be hermaphroditic or dioecious (reproduce via separate sexes). Most flukes are
hermaphroditic except Schistosoma spp. (blood flukes). Morphologically, flukes are fleshy,
leaf shaped worms. Unlike tapeworms, flukes have a digestive tract. In general, flukes have two
muscular suckers—an oral type, which is the beginning of an incomplete digestive system and a
ventral sucker which serves for attachment.
The eggs of the trematodes vary in appearance and are the primary morphologic stage
that are usually recovered from humans. Some eggs may possess a lid like structure that can
flip open to release its contents. This lid like structure is called operculum and is present in the
eggs of Fasciolopsis and Fasciola. In Schistosoma spp., the eggs of the various members may be
differentiated based on the presence and location of spines
224 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Unlike in cestode infections, humans never serve as intermediate hosts for the flukes.
In general, flukes have two intermediate hosts except for the blood flukes where there is
only one intermediate host. Common to all trematodes, the first intermediate hosts are
mollusks (snails and clams) where asexual reproduction takes place. The second intermediate
host varies depending on the parasite. Sexual reproduction of flukes occurs in humans.
In most cases, humans acquire the infection through ingestion of undercooked or raw second
intermediate host. Skin penetration by the infective larvae is the major mode of transmission
for blood flukes.

Blood dwelling Flukes

Schistosomaspp.
Important Properties and Life Cycle
Three schistosomes are frequently associated with human disease, namely: Schistosoma
mansoni, Schistosoma japonicum, and Schistosoma haematobium. Blood flukes are dioecious.
Known as the “romantic parasites,” the male and female worms are usually in a state of
copulation (en copula). Female worms are usually larger than the male worms. The schistosomes
are also obligate intravascular parasites.
The eggs are found in fresh water contaminated with the feces or urine of infected
humans. Once in the water, eggs develop into a miracidium, that will then locate a snail as
its host, where it transforms into cercariae. Infection is acquired through skin penetration by
the fork tailed cercaria (larval form). The parasite migrates into the bloodstream where they
undergo maturation.
The location of the adults varies by species. For Schistosoma japonicum and Schistosoma
mansoni, after skin penetration, the worms enter the veins surrounding the intestinal tract
(superior and inferior mesenteric and portal vein for S. japonicum; inferior mesenteric for
S. mansoni). Schistosoma haematobium worms localize in the veins surrounding the urinary
bladder. The adult worms lay thousands of eggs per day. The eggs produce enzymes that enable
them to travel through the tissue. The eggs then find their way into the colon (for S. japonicum
and S. mansoni) or into urine (for S. haematobium) from which they are excreted
 Trematodes 225

Figure 14.1 General life cycle of Schistosoma spp.

Epidemiology and Pathogenesis

Schistosoma mansoni and Schistosoma haematobium are both distributed throughout
Africa. S. mansoni is also found in South America while S. haematobium is also prevalent in
the Middle East. Schistosoma japonicum is endemic in Indonesia, some parts of China, and
Southeast Asia, including the Philippines. It is the only schistosome for which domestic
animals (e.g., water buffalo and pigs) act as important reservoirs.
Most of the findings are caused by the presence of eggs in the liver, spleen, or walls of
the gut or the urinary bladder, depending on which species is causing the infection. Eggs of
S. japonicum in the liver may induce granuloma formation leading to fibrosis and portal
hypertension, as well as damage the walls of the small and large intestines. Eggs of S. mansoni
may damage the walls of the distal colon. Eggs of S. haematobium may induce granuloma and
fibrosis in the walls of the urinary bladder
226 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Disease: Schistosomiasis (Bilharziasis)

1. Asymptomatic infection – the most common form of the disease. Chronic infection may
become symptomatic.
2. Early acute infection – characterized by pruritic papules seen at the site of entry of the
parasite. This is called “swimmer’s itch” or “clam digger’s itch.” This is followed after
2–3 weeks by fever and chills, abdominal pain, cough, bloody diarrhea, and weight loss.
Painful urination (dysuria) and blood in the urine (hematuria) may also occur in patients
infected with S. haematobium.
3. Katayama fever – a systemic hypersensitivity reaction to the migrating schistosomes,
usually associated with S. japonicum. It is characterized by a rapid onset of fever, myalgia,
body malaise, cough, diarrhea, and eosinophilia occurring 1–2 months after exposure to
the parasite. Swollen lymph nodes (lymphadenopathy) and enlargement of the liver and
spleen (hepatosplenomegaly)may also occur. It can lead to hepatic dysfunction, leading
to portal hypertension. The most common cause of death in this case is internal bleeding
from ruptured esophageal varices.
4. Associated conditions – include development of nephrotic syndrome in S. japonicum
and S. haematobium infection. Infection with S. mansoni or S. japonicum may predispose
to repeated Salmonella infections. S. japonicum is associated with the development of
hepatocellular carcinoma or liver cancer while S. haematobium has been implicated in the
development of cancer of the urinary bladder.

Figure 14.2 Swimmer’s itch

following skin penetration
by the cercariae of Schistosoma spp.
Source: Cornellier, 200
 Trematodes 227

Laboratory Diagnosis
Diagnosis relies on demonstration of characteristic eggs in the feces or rectal biopsy
specimen for S. mansoni or S. japonicum, or urine for S. haematobium. S. mansoni eggs have a
large lateral spine while S. japonicum eggs have a rudimentary spine. The eggs of S. haematobium
have large terminal spines.

a b c

Figure 14.3 Schistosoma ova: a S. mansoni; b S. haematobium; and c S. japonicum

Treatment
The recommended drug for all three species is praziquantel. An alternative drug for
S. mansoni is oxamniquine. Anti malaria drugs such as artemether and artemisinins have also
been proven effective.

Prevention and Control

There are two objectives of schistosomal control: (a) control of transmission through snail
control, health education, and provision of sanitary facilities and water supply; and (b) control
of disease. Chemotherapy using praziquantel is the main thrust of the Philippine program for
schistosomiasis control (Department of Health). In order to prevent infection, swimming in
endemic areas should be avoided.

Tissue dwelling Flukes

Clonorchis sinensis (Asian Liver Fluke, Chinese Liver Fluke)
Important Properties and Life Cycle
There are three morphologic stages of the parasite—egg, larva, and adult. The egg have
an operculum surrounded by a thick rim called the shoulder. The first intermediate host is
the freshwater snail while the second intermediate host is a freshwater fish. Within the egg
is the developed miracidium, that is released once the egg comes into contact with fresh water
228 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The miracidium penetrates the first intermediate host and develops into a sporocyst that
contains numerous larval stages called the rediae. The larvae are then released into the water
where they transform into cercariae. The cercariae enter a freshwater fish where they encyst
to become the metacercariae. The larvae excyst in the duodenum, enter the biliary ducts,
and differentiate into adults. The adult worms produce eggs that are excreted in the feces.
Humans acquire the infection by ingesting raw or undercooked freshwater fish containing the
infective metacercariae.

Metacercariae in flesh or
skin of fresh water fish are
ingested by human host. = Infective Stage
4 = Diagnostic Stage

Free swimming cercariae

encyst in the skin or flesh
of fresh water fish. 3
5 Excyst in
duodenum

Eggs are ingested

by the snail. 2

6 Adults in
biliary duc

Miracidia Sporocysts Rediae Cercariae 1 Embryonated eggs

2a 2b 2c 2d passed in feces

Figure 14.4 Life cycle of Clonorchis sinensis

Epidemiology and Pathogenesis

Clonorchis sinensis is found in Asia including Korea, China, Taiwan, Vietnam, Japan, and
Asian Russia (Center for Disease Control and Prevention). The parasite does not usually
cause significant lesions, however, parasites that inhabit the bile ducts can damage the
biliary tract. Patients who develop infection with the parasite are at higher risk of developing
cholangiocarcinoma or cancer of the bile ducts. The egg has also been associated with the
development of gallstones (stones in the gall bladder or cholelithiasis).
 Trematodes 22

Disease: Clonorchiasis
Most patients are asymptomatic. In heavy worm burden, patients may manifest a fever,
upper abdominal pain, anorexia, hepatomegaly, diarrhea, and eosinophilia. Liver dysfunction
may also occur in chronic infection associated with heavy worm burden.

Laboratory Diagnosis
Diagnosis is established by finding the characteristic eggs in stool specimen or duodenal
aspirates.

Treatment
The drug of choice for treatment is praziquantel. An alternative drug is albendazole.

Prevention and Control

Infection can be prevented by thorough cooking of fish prior to consumption. Other control
measures include health education, proper waste disposal to avoid contamination of bodies of
fresh water, and prompt treatment of infected persons.

Fasciola hepatica (Sheep Liver Fluke)

Important Properties and Life Cycle
The Fasciola hepatica eggs possess an operculum similar to Clonorchis sinensis and is also
equipped with shoulders. The first intermediate host for the parasite is the snail while the
second intermediate hosts are edible aquatic plants (kangkong and watercress). Humans acquire
the infection by ingesting raw edible aquatic plants or by drinking water contaminated by
metacercariae (infective stage). Upon ingestion, the metacercariae excyst in the duodenum or
jejunum, releasing the young flukes. These young flukes wander over the viscera until they reach
the liver capsule. The parasite then burrows through the liver parenchyma until it finally enters
the bile ducts where they mature.
The adult worms (pathogenic stage) live in the biliary passages of the liver. Immature eggs
are carried by the bile into the intestines and subsequently excreted with feces. The eggs mature
in the water and infect the first intermediate host. The cercariae escape the snail host, usually at
night, then encyst on the surface of aquatic plants, forming metacercariae. The natural host for
the completion of the life cycle is the sheep, however the parasite may also be found in cattle.
In sheep raising countries, ingestion of raw sheep liver containing the adult worm also serves as
an additional mode of transmission. Humans serve as accidental hosts.
230 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Figure 14.5 Life cycle of Fasciola hepatica

Epidemiology and Pathogenesis

The Fasciola hepatica is found worldwide, especially in sheep and cattle raising countries,
and where humans consume raw watercress such as Asia, Europe, and the Middle East.
The stages of the disease correspond with the migration of the parasites. The acute or invasive
phase corresponds to the migration of the parasite through the liver parenchyma, which leads
to traumatic and necrotic lesions in the liver. The severity of the destruction is proportional to
the number of metacercariae ingested. The chronic phase corresponds to the localization of the
adult worms to the bile ducts. The worm can obstruct the bile duct and stimulate inflammation.
During migration from the intestines to the liver, the parasite may wander to other sites
(e.g., lungs, subcutaneous tissues, brain, or orbit) where abscesses may develop
 Trematodes 23

Disease: Fascioliasis or Sheep Liver Rot

Migration of the larval worm through the liver irritates the organ, manifesting as
tenderness and hepatomegaly. Characteristic clinical features include right upper quadrant pain,
fever and chills, and marked eosinophilia. Hepatitis may develop with biliary obstruction. Some
worms may cause necrotic foci in the liver. Ingestion of raw sheep liver may lead to temporary
lodgment of the adult worm in the pharynx leading to suffocation.

Laboratory Diagnosis
Diagnosis rests on finding of eggs in stool specimen, although the appearance of the
eggs of F. hepatica may be indistinguishable from the eggs of another fluke, Fasciolopsis buski.
Examination of a sample of the patient’s bile may aid in the differentiation. If the eggs are
present in bile then this is indicative of F. hepatica. Other tests that can be performed include
ELISA and the Enterotest (discussed in Chapter 11).

Treatment
The treatment of choice is dichlorophenol (bithionol). An alternative drug is
triclabendazole.

Prevention and Control

Preventive measures include proper human waste disposal, improvement of hygiene, control
of snail population, and avoidance of consumption of raw aquatic plants and contaminated
water. In endemic areas it is highly recommended to boil water before consumption or use.
Avoidance of ingesting of raw sheep liver is also important, as well as prompt treatment of
infected individuals.

Paragonimus westermani (Oriental Lung Fluke)

Important Properties and Life Cycle
Similar to other trematodes, the first intermediate host for Paragonimus is a snail while
the second intermediate hosts are crabs or crayfish. Humans acquire the infection by ingesting
raw or undercooked crabs or crayfish that contain the infective encysted metacercaria. The
larva excysts in the small intestines, migrate through the intestinal wall, through the peritoneal
cavity, into the diaphragm then into the lung parenchyma where they mature. The adult worms
enter the bronchioles and are then coughed up or swallowed. Eggs in the sputum or feces reach
fresh water, hatch, and penetrate the first intermediate host, where they differentiate into free
swimming cercariae. The cercariae leave the snail host and encyst in freshwater crabs that are
eaten by humans.
232 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Cercariae invade the crustacean

5
and encyst into metacercariae.
4a 4b 4c

Sporocysts Rediae Cercariae

Humans ingest 6
inadequately
cooked or pickled
4 crustaceans containing
metacercariae.
7

Excyst in
3 duodenum
Miracidia hatch
and penetrate snail 8

Adults in cystic
cavities in lungs
lay eggs which
are excreted
in sputum.
2 Embryonated eggs Alternately, eggs
are swallowed
= Infective Stage
1 Unembryonated eggs and passed
with stool.
= Diagnostic Stage
Figure 14.6 Life cycle of Paragonimus westermani

Figure 14.7 A freshwater crab that has

been implicated in the transmission of
Paragonimus westerman
 Trematodes 23

Epidemiology and Pathogenesis

P. westermani infection occurs most commonly in Asia, in countries like China, the
Philippines, Japan, Vietnam, South Korea, Taiwan, and Thailand (Centers for Disease Control
and Prevention). Pigs, monkeys, and other animals that eat crayfish and crabs serve as reservoir
hosts. The worms exist in a fibrous capsule within the lung which communicates with a
bronchiole. Within this cyst is blood tinged purulent material containing eggs. Secondary
bacterial infection frequently occurs.

Disease: Paragonimiasis (Pulmonary distomiasis, Endemic hemoptysis,

Parasitic hemoptysis)
The early stages of the disease are usually asymptomatic. Patients may later experience
symptoms related to pulmonary involvement including cough productive of blood tinged
sputum (hemoptysis), fever, and chest pain. The sputum has a foul, fishy odor and is most
pronounced in the morning. The disease may mimic pulmonary tuberculosis. In rare cases,
the immature flukes may migrate to the brain leading to cerebral paragonimiasis, which may
manifest as seizures, visual disturbances, and reduced motor skill precision.

Laboratory Diagnosis
Diagnosis is made by demonstration of the characteristic eggs in sputum or feces (when
sputum is swallowed). A chest x ray may be done which may show a ring shadowed opacity
with several contiguous cavities giving the appearance of a cluster of grapes.

Treatment
The drug of choice for treatment is praziquantel. An alternative drug is bithionol.

Prevention and Control

Preventive measures include adequate and thorough cooking of freshwater crabs or crayfish,
health education, control of snail population, and elimination of reservoir hosts. Prompt
treatment of infected persons is also important to prevent the parasite from spreading.

Fasciolopsis buski (Large Intestinal Fluke)

Important Properties and Life Cycle
The eggs of F. buski and F. hepatica are mophologically identical however they differ only
in size. The adult worms of both parasites are also similar in appearance except that shoulders
234 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

are present in F. hepatica and not in F. buski. Similar to the other tissue dwelling trematodes,
the first intermediate host is a snail while the second intermediate hosts are edible aquatic
plants (e.g., watercress and lotus).
Humans acquire the parasite by ingestion of raw or inadequately cooked aquatic vegetation
that carries the encysted metacercariae. The metacercariae excysts in the duodenum and
attaches to the intestinal wall where they attain maturity. The adult worms lay eggs, that are
released together with the feces into water, where they hatch and infect the first intermediate
host. The eggs develop into cercariae which encyst as metacercariae on the surface of the
aquatic plants. Other animals such as pigs and dogs may also serve as the reservoir hosts.

6 Metacercariae
on water plant
4a 4b 4c ingested by
Sporocysts Rediae Cercariae 5 Free swimming
cercariae
humans or pigs,
causing infection

in snail tissue

4 Snail
7 Excyst in
duodenum

3
7
Miracidia hatch,
penetrate snail 8

2 Embryonated eggs
in water
8
= Infective Stage 1 Unembryonated eggs Adults in
passed in feces small intestin
= Diagnostic Stage

Figure 14.8 Life cycle of Fasciolopsis buski

 Trematodes 23

Epidemiology and Pathogenesis

F. buski is the largest intestinal fluke that can infect humans. Infection with the parasite
is common in Asia and the Indian subcontinent, particularly in areas where pigs are raised
and where freshwater aquatic vegetation is ingested raw. No locally acquired cases in humans
or pigs have been reported in the Philippines. Pathologic changes are due to damage to the
intestinal mucosa by the adult fluke.

Disease: Fasciolopsiasis
Most infected persons are asymptomatic. However, with heavy worm burden, patients
may experience abdominal discomfort with inflammation and bleeding in the affected area.
Ulcerations may occur and symptoms may mimic those of duodenal ulcer. Patients may also
suffer from malabsorption. Intoxication may result from absorption of worm metabolites by the
host, leading to allergic symptoms such as edema of the face, abdominal wall, and lower limbs.
Profound intoxication can result in death.

Laboratory Diagnosis
Diagnosis is made by demonstration of the eggs in stool specimen. Examination of bile
samples and duodenal aspirates may help differentiate F. buski eggs from those of F. hepatica.

Treatment
The drug of choice for treatment is praziquantel.

Prevention and Control

Adequate washing and cooking of aquatic plants before consumption can help prevent
infection. Other measures include proper disposal of human waste, control of snail population,
and prompt treatment of infected persons.
236 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• Trematodes, also known as flukes, are worms that possess a primitive digestive tract.
• Trematodes
flukes.
may be divided into two classes—tissue dwelling flukes and blood dwelling

» Tissue dwelling flukes include Clonorchis sinensis, Fasciola hepatica, Paragonimus

westermani, and Fasciolopsis buski.

› All tissue dwelling flukes have two intermediate hosts.

› The first intermediate host is always a snail.
› Second intermediate hosts include freshwater fish, crabs or crayfish, and edible
aquatic plants.
› All are transmitted by ingestion of raw or inadequately cooked second intermediate
host.

› The infective stage is the encysted metacercaria.

» Blood dwelling flukes are the schistosomes which are composed of three
japonicum, Schistosoma mansoni, and Schistosoma haematobium. species—Schistosoma

› Blood flukes do not have a second intermediate host. The intermediate host is the
freshwater snail.

› The major mode of transmission is skin penetration by the infective fork tailed
cercaria.

› The pathogenic stage is the adult worm except for S. japonicum where the eggs also
serve as the pathogenic stage.

› S.Thejaponicum has predilection for the superior and inferior mesenteric veins.
adult female migrates to the portal vein where egg laying may occur. S. mansoni
adult worms localize to the inferior mesenteric veins while S. haematobium worms
localize to the veins around the urinary bladder.

• Patients with S. japonicum infection are at a higher risk for development of liver cancer
while those with S. haematobium infection are more prone to develop cancer of the
urinary bladder.

• sinensis and Fasciola hepatica can lead to development of obstructive jaundice.

Clonorchis

• buski produce disease manifestations that mimic duodenal ulcer.

Fasciolopsis

• Paragonimus westermani produce symptoms that mimic pulmonary tuberculosis

 Trematodes 237

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. The first intermediate host common to the trematodes is the:

a. Fish c. Snail
b. Crab d. Water plant
2. Which among the following is the second intermediate host of P. westermani?
a. Fish c. Snail
b. Crab d. Water plant
3. Which among the following schistosomes has predilection for the urinary
bladder?

a. S. japonicum c. S. mansoni
b. S. haematobium d. A and C only
4. Which among the following is a characteristic of blood flukes?
a. Transmitted by skin penetration
b. Second intermediate host is a snail
c. Capable of self reproduction
d. A, B, and C
e. A and C only

5. Cholangiocarcinoma is associated with which among the following flukes?

a. Clonorchis sinensis c. Paragonimus westermani
b. Fasciola hepatica d. Schistosoma japonicum
238 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. The lid like structure seen on the eggs of F. buski and F. hepatica is called:
a. Miracidium c. Rediae
b. Operculum d. Shoulder
7. The specimen of choice for diagnosis is the sputum for which among the
following?

a. F. buski c. P. westermani
b. F. hepatica d. S. japonicum
8. Katayama fever is associated with:
a. S. japonicum c. F. hepatica
b. P. westermani d. C. sinensis
9. Schistosoma japonicum is associated with development with cancer of the:

a. Bile ducts c. Liver

b. Colon d. Urinary bladder
10. The drug of choice for most trematode infection is:
a. Bithionol c. Praziquantel
b. Mebendazole d. Triclabendazol
 CHAPTER

15 Nematodes

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. describe the general characteristics of the medically important nematodes; and
2. characterize and differentiate the different nematodes as to their:
a. general features,
b. source of infection,
c. mode of transmission,
d. clinical manifestations,
e. treatment, and
f. prevention and control of infection.

General Properties of Nematodes

Nematodes or roundworms are unsegmented, bilaterally symmetrical worms with
elongated, cylindrical bodies. The life cycle of these parasites consists of three
egg or ova, larva, and adult worms. The body covering is called the cuticle. stages—embryonated

Underneath the layer of cells that secrete the cuticle are long muscles that allow the worm to
move its body from side to side. These parasites have separate sexes, with the female worm
being larger than the male worm.
Adult worms are equipped with a complete digestive system, a simple nervous system, an
excretory system, and a reproductive system. The digestive system consists of three
stomodeum (mouth, esophagus, and buccal cavity), intestines, and anus (called proctodeum)
structures—the
240 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The nervous system is made up of two nerves that run along the length of the body of
the worm on both the dorsal (back) and ventral (front) sides. Both nerves are connected to a
nerve center found at the head of the worm. Nematodes have a sensory organ called amphid
which is usually located in the anterior end of the head region of the worms. In some
(e.g., the aphasmids Trichuris and Trichinella), the amphids are found in the posterior head
region. Some nematodes are equipped with a pair of caudal chemoreceptors called phasmids.
These are Ascaris, Necator, and the filarial worm Wuchereria. Unlike the more primitive worms,
nematodes are equipped with excretory canals along each side of the body for the elimination of
waste materials.
Most patients with nematode infection are asymptomatic. The severity of the disease
depends on the worm burden and the host’s immunity. The nematodes may be divided into
three groups based on their primary location in the body—intestinal nematodes, the intestinal
tissue nematodes, and the blood tissue nematodes. The intestinal nematodes important
in the Philippines include Enterobius, Ascaris, Trichuris, Necator, Ancylostoma, Strongyloides,
and Capillaria. Ascaris, Trichuris, and Necator are the most prevalent in the Philippines. With
the exception of Capillaria, the major source of infection for the intestinal nematodes is soil
contaminated with human feces. Ascaris, Enterobius, and Trichuris are transmitted through
ingestion of the embryonated ova. Necator, Ancylostoma, and Strongyloides are transmitted
by skin penetration. Capillaria philippinensis, transmitted by ingestion of undercooked or raw
infected fish, is endemic in certain areas in the Philippines, particularly Northern Luzon.
The blood tissue nematodes of significant medical importance particularly in the
Philippines are the filarial worms Wuchereria and Brugia. The filarial worms, found in
specific locales in the Philippines, are transmitted by the bite from arthropod vectors
(usually mosquitoes). The muscle worm, Trichinella is an intestinal tissue nematode acquired by
ingesting improperly cooked or raw pork meat containing the worm’s encysted larva.

Table 15.1 Summary of intestinal nematodes

Parasite/ Mode of
Site of Infection Diagnosis Treatment
Disease Transmission
Enterobius Lumen of cecum, Ingestion of Scotch Pyrantel pamoate,
vermicularis colon eggs; self Tape test; mebendazole
(Pinworm) contamination microscopy
or autoinfection for eggs
Trichuris Cecum, colon Ingestion of eggs Stool exam for Mebendazole,
trichiura from fecally eggs albendazole
(Whipworm) contaminated soil
or food
Ascaris Small intestines; Ingestion of Stool exam for Albendazole,
lumbricoides larvae through eggs from fecally eggs; sputum mebendazol
(Common lungs contaminated soil exam for larvae
roundworm) or food
 Nematodes 241

Parasite/ Mode of
Disease Site of Infection Transmission Diagnosis Treatment
Ancylostoma Small intestines; Larvae in soil Stool exam for Albendazole,
duodenale, larvae through penetrate skin eggs; sputum mebendazole
Necator skin, lungs exam for larvae
americanus
(Human
hookworms)
Strongyloides Small intestines; Larvae in soil Stool exam, Ivermectin,
stercoralis larvae through penetrate skin; sputum exam albendazole
(Threadworm) skin, lungs autoinfection or bronchial
(rare) lavage for
larvae
Adapted from Jawetz, Melnick & Adelberg’s Medical Microbiology 25th ed. 2012, p. 686

Table 15.2 Summary of blood and tissue nematodes

Parasite/ Site of Infection
Mode of
Diagnosis Treatment
Disease Transmission
Trichinella Adults in small Eating Serology and Albendazole +
spiralis intestines for 1–4 undercooked, muscle biopsy steroids (for severe
(Muscle worm) months; larvae infected pork or (larvae) symptoms)
encysted in other animal
muscle tissue
Wuchereria Adult worms in Bite of Blood smear Diethylcarbamazine
bancrofti, lymph nodes, mosquitoes for microfilariae
Brugia malayi lymphatic ducts transmit larvae
(Filarial
worms)
Adapted from Jawetz, Melnick & Adelberg’s Medical Microbiology 25th ed. 2012, pp. 686–687

Intestinal Nematodes
Ascaris lumbricoides (Large Intestinal Roundworm)
Important Properties and Life Cycle
Ascaris lumbricoides is the largest intestinal roundworm infecting humans. The adult
worm is creamy white in color with an outer covering of cuticle. Humans acquire infection
through ingestion of food or water contaminated with human feces containing the infective
embryonated ova. Upon entry into the small intestines, larvae are released from the eggs,
penetrate the intestinal wall, enter the blood to go initially to the liver, and finally localizes
to the lung. In the lungs, the larvae gain entrance into the air sacs and migrate into the
bronchioles. The larvae are then coughed up with the sputum which is swallowed thereb
242 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

returning the worm to the intestines. The larvae mature into adult worms in the small
intestines, where they lay their eggs that are eliminated with the feces. The eggs are capable of
surviving in soil, sewage, or water for several years.

= Infective Stage

= Diagnostic Stage

4
7

5
Feces

Fertilized

Unfertilized

Figure 15.1 Life cycle of Ascaris lumbricoides

Epidemiology and Pathogenesis

Ascaris infection is considered as the most common helminth infection worldwide.
The parasites are more common in areas characterized by warm climates and poor sanitation.
Areas that use human feces as fertilizer or where children defecate directly on the ground are
highly susceptible to infection with the parasite. Young children are the most affected when
they play in soil contaminated with human feces.
Adult worms produce little damage in the intestines. However, since the adult worms
obtain nourishment from ingested food, they may contribute to development of malnutrition.
The major damage occurs during larval migration to the lungs where inflammation occurs
 Nematodes 243

Disease: Ascariasis
Asymptomatic infection is usually seen with low worm burden. The patient may not
be aware of the presence of the parasite until the adult parasite is passed out with the
feces. Symptomatic infection occurs due to migration of the parasite through the host.
During larval migration, the larvae may induce allergic reactions, manifesting as asthmatic
attacks accompanied by eosinophilia (called Loeffler’s syndrome). Penetration of the lung
capillaries by the larvae as they enter the air sacs can lead to pneumonia.
The presence of multiple adult worms in the intestines can lead to abdominal pain (most
common complaint), vomiting, fever, and abdominal distention. Mature worms may entangle
with each other forming a mass that can cause intestinal obstruction. In addition, due to the
erratic nature of the mature worms, the adult parasite can travel to different organs of the body.
An adult worm can obstruct the appendix leading to appendicitis. Other organs that can be
obstructed include the liver and the bile ducts. Due to the tough, flexible body of the worm,
it may cause perforation of the intestines, leading to peritonitis which can be fatal. Secondary
bacterial infections may also occur in the damaged tissues.

Figure 15.2 a Comparison of male and female ascaris adult worms. Take note of the curved
posterior portion of the male worm. b Two children with massive ascariasis, with worms coming
out of the mouth and nose of the child on the left, and out of the anus of the child on the right
244 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Laboratory Diagnosis
Diagnosis is established by finding of the eggs in a stool specimen. In cases of heavy worm
burden, the adult worm may be present in the stool or be regurtitated. Larvae may be recovered
from the sputum during the pulmonary phase of the disease.

Treatment
Drugs that have been proven effective are mebendazole, albendazole, and pyrantel pamoate.

Prevention and Control

The preventive measures are measures used to prevent other parasitic infections such as
proper disposal of human feces, health education of the population, and improved personal
hygiene. It is also recommended to avoid using human feces as fertilizer. A program of mass
chemotherapy is recommended especially for children and in areas with high incidence of
parasitism.

Enterobius vermicularis (Pinworm, Seatworm)

Important Properties and Life Cycle
The egg of E. vermicularis is typically oval and flat on one side. The adult worms are small
and yellowish white in color. The common name pinworm is based on the appearance of a
clear, pointed tail of the adult female that resembles a pinhead. Humans acquire the infection
through ingestion of the eggs of the worm. As it reaches the small intestines, larvae emerge
from the eggs and mature into adult worms in the large intestines. Once the female becomes
impregnated, it migrates to the perianal region where egg laying occurs, usually at night.
The infective eggs may dislodge from the body due in part to intense scratching of the anal
area, and deposit in dust, soil, linens, and clothing. Some infective pinworm eggs may migrate
back into the host body rather than be dislodged leading to a retroinfection. Autoinfection
occurs as a result of hand to mouth transmission. It is also possible to ingest the eggs through
breathing them in, as the eggs are so small.

Epidemiology and Pathogenesis

Pinworm infection occurs worldwide, especially in temperate regions. People at risk
for pinworm infection are children, their caretakers, institutionalized persons, and those
in unsanitary and crowded living conditions. Deposition of eggs in the anal area incite a
hypersensitivity reaction that leads to the prominent manifestation of the disease—anal
itchiness. Vigorous scratching of the anus can lead to development of mucosal breaks in th
 Nematodes 245

anus which can become secondarily infected. Like Ascaris, some pinworms may obstruct the
appendix leading to appendicitis.

Embryonated eggs
ingested by human
2

Larvae hatch
3
in small intestine

1
Eggs on perianal folds Adults in lumen
Larvae inside the eggs
of ceum 4
mature within 4 to 6 hours.

= Infective Stage
= Diagnostic Stage 5 Gravid migrates
to perianal region
at night to lay eggs

Figure 15.3 Life cycle of Enterobius vermicularis

Disease: Enterobiasis
Most cases of enterobiasis are asymptomatic. The most common manifestation is intense
itching with inflammation in the anal area (pruritus ani) or the vaginal area which occurs most
frequently at night. Other symptoms may include intestinal irritation and mild nausea. Since
the itchiness occurs at night, infected persons may be deprived of sleep and become irritable
246 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

a b

Figure 15.4 a Pinworm parasite, b adult worms in the anal area,

and c child with pinworm infection

Laboratory Diagnosis
Definitive diagnosis is established by demonstration of the eggs or adult females using
the Scotch Tape method or cellophane tape method (see Chapter 11). The small size of the
eggs may make recovery from stool difficult. Several samples may be necessary to confirm
the diagnosis.

Treatment
Drugs of choice for treatment are albendazole, mebendazole, or pyrantel pamoate.
It is recommended that household members also undergo treatment as pinworm infection is
considered a group infection.

Prevention and Control

Good personal hygiene, clipping of fingernails, thorough washing of beddings, and prompt
treatment of infected persons contribute to the control and prevention of the parasite spreading
to other individuals. To avoid ingestion and/or inhalation of the eggs, it is recommended to
thoroughly clean the house using a damp mop in areas including the floor under the beds,
the windowsills, and overdoors
 Nematodes 247

Trichuris trichiura (Whipworm)

Important Properties and Life Cycle
The eggs of the human whipworm have a characteristic barrel or football shape with a
prominent hyaline plug at each end of the egg. The anterior end of the adult worm appears
colorless while the posterior end is pinkish in color. The male worm has a recognizable curled
tail. The posterior end is larger and resembles the handle of a whip while the anterior end
resembles the whip itself.
Humans acquire the infection through ingestion of food or water contaminated by human
feces containing the infective eggs. The larvae emerge from the eggs in the small intestines,
become immature adults, and migrate to the colon where complete maturation and mating
occurs. Thousands of eggs are produced each day, which are then passed in the feces.

4 Embryonated eggs are ingested.

3 Advanced cleavage
= Infective Stage

= Diagnostic Stage

Larvae hatch
in small intestine

2 2 cell stage

1 Unembryonated eggs
passed in feces.

6 Adults in cecum
Figure 15.5 Life cycle of Trichuris trichiur
248 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Epidemiology and Pathogenesis

The whipworm is the third most common roundworm affecting humans. Infection is
seen worldwide, especially in tropical countries and areas with poor sanitation practices.
The parasite, like Ascaris, is seen in locales where human feces is used for fertilizer and where
humans defecate directly on the soil. Children are at highest risk for development of infection
when they play in contaminated soil. Infection is acquired primarily by ingesting food or water
contaminated by human feces containing the infective eggs. Adult worms burrow their hair like
anterior ends into the intestinal mucosa but do not cause significant anemia.

Disease: Trichuriasis
Severity and occurrence of manifestations of trichuriasis are related to the intensity
of the worm burden. Heavy infection in children resemble manifestations of ulcerative
colitis, a chronic inflammatory condition of the colon that has an autoimmune etiology.
Manifestations may include chronic dysentery (bloody, mucoid diarrhea), severe anemia,
or growth retardation. Rectal prolapse and hyperperistalsis are also seen in infected children.
Rectal prolapse occurs due to irritation and straining during defecation. Manifestations
in adults resemble those of inflammatory bowel disease and include abdominal pain and
tenderness, weakness, and dysentery.

b c

Figure 15.6 a Adult whipworm, b rectal prolapse seen in patients with heavy worm burden,
and c typical football or barrel shaped egg with plugs on both end
 Nematodes 24

Laboratory Diagnosis
Diagnosis is confirmed by demonstrating the presence of characteristic eggs in stool
specimens.

Treatment
Drugs of choice for treatment are mebendazole or albendazole.

Prevention and Control

Effective preventive and control measures include health education, proper sanitation,
good personal hygiene, and avoidance of use of human feces as fertilizer.

Ancylostoma duodenale (Old World Hookworm)

and Necator americanus (New World Hookworm)
Important Properties and Life Cycle
There are two common species of hookworms, Ancylostoma duodenale and the Necator
americanus, both of which share the same four stages in the life cycle—eggs, rhabditiform
larvae, filariform larvae, and adults. The eggs of the two hookworms vary only in size.
The rhabditiform larva is the immature, newly hatched larva. It is an actively feeding form
that consists of a long oral cavity called buccal cavity or buccal capsule, and a small genital
primordium. The filariform larva is the non feeding, infective larva that has a distinct pointed
tail. The adult worms of the two hookworms are differentiated by the appearance of their buccal
capsule. The N. americanus buccal capsule is equipped with a pair of cutting plates while that of
A. duodenale consists of teeth.
Unlike the other intestinal roundworms, the infective stage for hookworms is the larva and
transmission through skin penetration by the filariform larva. The feet or legs are the usual sites
of penetration. After penetration, the larvae are carried by the blood to the lungs, migrate to
the air sacs, pass up the bronchi and trachea, are coughed up and then swallowed with sputum,
similar to the larval migration phase of Ascaris lumbricoides. Once in the small intestines,
the larvae mature into adult worms and attach themselves to the intestinal wall using their
cutting plates or teeth. The adult worms feed on blood from the capillaries of the intestinal villi.
Mating occurs in the small intestines, where thousands of eggs are laid each day. The eggs are
then passed out with the feces.
250 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

4
Filariform larva
penetrates skin

3
Filariform larva

2 Rhabditiform
larva hatches
Adults in small intestine

= Infective Stage
1
= Diagnostic Stage
Eggs in feces

Figure 15.7 Life cycle of hookworms

Epidemiology and Pathogenesis

Hookworms are found worldwide, especially in tropical countries. Walking barefoot on soil
puts one at risk of acquiring the infection. Irritation of the skin at the site of penetration may be
seen, as well as inflammatory reactions in the lungs during the larval phase. The major damage
to the host is due to chronic blood loss at the site of attachment in the small intestines.

Disease: Hookworm Infection

Penetration of the skin by the filariform larvae produces a pruritic papule or vesicle.
This is called “ground itch.” Pneumonia with eosinophilia may occur during the lung phase.
The presence of adult worms in the intestines can manifest nausea, vomiting, and diarrhea.
As the worm feeds on blood, a microcytic, hypochromic anemia akin to iron deficiency anemia
may occur. Intestinal sites may be secondarily infected by bacteria
 Nematodes 251

Figure 15.8 Skin irritation at the site of penetration of hookworm filariform larva (ground itch)

Laboratory Diagnosis
Stool examination will show the characteristic thin shelled eggs. Occult blood in the stool
and blood eosinophilia are frequent findings. Peripheral blood smear will show microcytic,
hypochromic anemia. Larvae may be recovered from sputum.

Treatment
The recommended drugs for treatment are mebendazole and pyrantel pamoate. Iron
replacement therapy is recommended for the anemia. In severe cases, blood transfusion may be
necessary.

Prevention and Control

The preventive measures are similar to those for Ascaris lumbricoides and the other intestinal
roundworms. Wearing shoes or any protective footwear is also important, especially in endemic
areas.

Strongyloidesstercoralis (Threadworm)
Important Properties and Life Cycle
The eggs of Strongyloides stercoralis are similar to those of hookworms except for two
features—Strongyloides ova are smaller and contain well developed larvae. The rhabditiform
larva of Strongyloides differ from that of hookworms in having a longer buccal cavity and
a smaller genital primordium. Like hookworms, the infective stage is the filariform larva
252 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The S. stercoralis differs from hookworm filariform larva in that the former has a longer
esophagus and a notched tail, while the hookworm filariform larva’s tail pointed.
Strongyloides stercoralis is unique among the intestinal roundworms for having two distinct
life cycles—one within the host and a free living cycle in soil. Humans acquire the infection
through three possible means. The first is through direct skin penetration by the infective
filariform larva, as that of hookworm acquisition. This direct mode of transmission marks the
beginning of the human cycle. The direct or human cycle resembles that of the hookworm life
cycle where a lung phase also occurs. It differs from the hookworm cycle in that it is usually
the rhabditiform larvae that are passed out with the feces instead of the eggs. The rhabditiform
larvae transform directly to the infective filariform larvae in warm, moist soil.
In the second, the indirect mode, rhabditiform larvae are passed out in the feces which
transform into filariform larvae in the soil. These mature into adult, free living, non parasitic
adult worms. This is the free living cycle which occurs in soil. The adult female worm lays eggs
that develop into rhabditiform larvae, which transforms into the infective filariform larvae that
can then enter a host to start a direct life cycle.
Infection may also occur through autoinfection. This occurs when the rhabditiform larvae
develop into filariform larvae in the intestines of the infected person. These then enter the
lymphatic system or the bloodstream of the infected host, thus starting a new cycle.

Epidemiology and Pathogenesis

Threadworn infection occurs worldwide but is more common in tropical, sub tropical,
and warm, temperate areas. The parasite is frequently seen in agricultural areas where there is
constant contact with soil. As in hookworm infection, irritation at the site of skin penetration
also occurs (ground itch) similar to a hookworm infection. The larvae in the lungs can produce
an inflammatory reaction similar to Ascaris. The adult worms in the small intestines can initiate
an inflammatory reaction on the intestinal wall, resulting in diarrhea. This is especially seen in
autoinfection, where significant damage can occur in the intestinal mucosa which may lead to
secondary bacterial infection and sepsis
 Nematodes 253

Figure 15.9 Life cycle of Strongyloides stercoralis

Buccal canal

Esophagus b
Esophagus intestine
jucture
a

Genital primordium

Strongyloides stercoralis Hookworm Hookworm Strongyloides stercoralis

Figure 15.10 Comparison of the a rhabditiform larvae and b filariform larvae of Strongyloides
stercoralis and hookworm
254 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Disease: Strongyloidiasis (Cochin China Diarrhea)

Patients with light infection are usually asymptomatic. Like hookworm infection, skin
irritation at the site of entry is seen. Migration of the larvae into the lungs lead to pneumonitis
just like that of hookworm and Ascaris infections. The presence of numerous adult worms
in the intestines lead to diarrhea and abdominal pain. In some patients, the parasite can
stimulate recurrent allergic reactions resulting to urticaria and eosinophilia. In patients with
very high worm burden, which is seen in autoinfection, malabsorption syndrome may occur
due to involvement of the biliary ducts, pancreas, small intestines, and colon. This can lead to
steatorrhea (fat in the stool) and resulting nutrient deficiencies, epigastric pain and tenderness,
and increasing diarrhea. These symptoms constitute a hyper infection syndrome. In some
instances, disease presentation is mistaken for peptic ulcer disease.
Autoinfection can also lead to development of chronic infection, increasing the
risk of developing hyper infection syndrome, which can prove fatal in patients who
are immunocompromised(commonly in patients under corticosteroid therapy or other
immunosuppressivetherapies). Aside from the potentially fatal electrolyte abnormalities,
fatal complications of the hyper infection syndrome include bacterial sepsis, peritonitis, and
endocarditis.

Figure 15.11 The pathognomonic of Strongyloides stercoralis infection, typically appearing as skin
lessions resulting from the migration of larva migrans in the bloodstrea
 Nematodes 25

Laboratory Diagnosis
Eggs, although not commonly present, may be recovered from stool of patients with heavy
worm burden who have severe diarrhea. The usual diagnostic method is through the recovery of
the rhabditiform larva in fresh stool samples. It is recommended that three sample collections
be done, one per day for three days, as the larvae may occur in “showers” with many seen in one.
Examination of duodenal aspirates may also yield the larvae. Larvae may also be recovered from
sputum during the lung phase of the parasite’s life cycle. Striking eosinophilia may occur in a
massive infection. Serologic tests such as ELISA have already been developed.

Treatment
The drug of choice for treatment is ivermectin with mebendazole and thiabendazole as
alternative drugs.

Prevention and Control

Preventive and control measures for Strongyloides are similar to those for hookworms. These
include thorough health education of the population at risk, proper sanitation and sewage
disposal, wearing of protective footwear, and prompt treatment of infected individuals.

Capillaria philippinensis (Pudoc worm)

Important Properties and Life Cycle
The parasite was first described in the Philippines in 1963, when the first human case
died from the infection. From 1967–1968, outbreaks of the infection occurred leading to the
death of more than a hundred infected individuals. Unlike the other intestinal roundworms,
migratory fish eating birds are the natural hosts. Typically, the unembryonated eggs are passed
out to the external environment with the feces of the birds or infected humans, usually in fresh
water. The eggs become embryonated and are ingested by freshwater fish (usually bagsit in
the Ilocos region). The larvae encyst in the tissues of the fish. Humans acquire the infection
by eating improperly cooked or raw freshwater fish. Once in the small intestines, the larvae
mature into adult worms that burrow into the wall of the intestines, where the worms lay eggs.
Some of the eggs may become embryonated in the intestines which leads to development
of autoinfection.
256 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Figure 15.12 Life cycle of Capillaria philippinensis

Epidemiology and Pathogenesis

Capillaria philippinensis is endemic in the Philippines, especially in the Ilocos region. Cases
have also been seen in Thailand. The parasite is also found in other parts of the Philippines
including Zambales and Southern Leyte. The large number of worms that develop within the
infected host is responsible for the pathology seen. Adult worms can cause micro ulcers in the
intestinal mucosa that if severe, can lead to malabsorption syndrome
 Nematodes 257

Disease: Intestinal Capillariasis

Intestinal capillariasis is characterized by abdominal pain with a gurgling stomach
(borborygmus) and chronic diarrhea. The chronic diarrhea leads to weight loss which
is aggravated by the accompanying loss of appetite (anorexia), nausea, and vomiting.
Malabsorption of fat, carbohydrates, and proteins as well as electrolyte abnormalities
can be fatal.

a b

a
Figure 15.13 Egg of Capillaria philippinensis in unstained wet mount of stool and b longitudinal
section of C. philippinensis adult worm taken from an intestinal biopsy specimen

Laboratory Diagnosis
Diagnosis is confirmed by demonstration of the characteristic eggs in stool specimens.
In high worm burden, larvae as well as adult worms may also be demonstrated in stool.

Treatment
The drug of choice for treatment is albendazole, with mebendazole as alternative, especially
for adult patients. Chemotherapy is given for at least 20 days in order to totally eradicate the
parasite. Relapses may occur if the treatment regimen is not followed. Patients with severe
infection with electrolyte loss and malabsorption must be managed with electrolyte replacement
and a high protein diet.

Prevention and Control

Preventive measures include adequate and thorough cooking of seafood before
consumption, especially in endemic areas. Other measures include proper human waste
disposal, health education, and prompt treatment of infected persons
258 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Blood and Tissue Nematodes

Wuchereria bancrofti (Bancroft’s Filarial Worm)
and Brugia malayi (Malayan Filarial Worm)
Important Properties and Life Cycle
W. bancrofti and B. malayi are both mosquito borne parasites. Both have two important
morphologic forms—the adult worm and the larvae (called microfilariae). The adult male
is usually the size of the female worm. Both are threadlike in appearance with creamy white
color. The microfilariae have a delicate transparent covering called a sheath. Humans acquire
the infection through the bite of a mosquito vector. The infective larvae migrate to the
tissues, mature, and localize in the lymphatics, subcutaneous tissues, or internal body cavities.
Migration of the parasites exhibit periodicity, where the parasite is present in the bloodstream
during specific times of the day, which corresponds to the feeding schedule of the mosquito
vector. Migration may occur at night (nocturnal), during the day (diurnal), or with no clear cut
timing (sub periodic).

Figure 15.14 Life cycle of Wuchereria bancrofti, which is similar to that of Brugia malay
 Nematodes 25

Epidemiology and Pathogenesis

Majority of filarial worm infections worldwide are caused by Wuchereria bancrofti. Infections
in Asia are frequently due to Brugia malayi. In the Philippines, bancroftian filariasis is more
common. Mosquito vectors for W. bancrofti include Culex spp., Anopheles spp., Aedes spp., and
Mansonia spp. The typical vectors for B. malayi are Mansonia and Aedes mosquitoes. In rural
areas in the Philippines, the major vector is Anopheles minimus falvirostris. In urban areas, the
parasite is transmitted chiefly by Culex spp., which can breed in latrines, sewage, and ditches.
Fourty four (44) provinces in the Philippines have been identified by the Department of
Health (DOH) as endemic. These provinces are mainly in the Southern portion of the
Philippines. To date, the following provinces have been declared filaria free: Southern Leyte,
Sorsogon, Biliran, Bukidnon, Romblon, Agusan del Sur, and Dinagat Islands.
The manifestations of filariasis are due to the obstruction of the lymphatic vessels by the
adult worms, causing edema of the limbs. The adult worms cause inflammatory and fibrotic
reactions. Microfilariae cause less severe pathology.

Disease: Filariasis
Symptoms of filariasis may vary depending on the species. The clinical course may be
divided into three stages—asymptomatic, acute, and chronic.
1. Asymptomatic stage is characterized by the presence of thousands of microfilariae
in the peripheral blood. Adult worms may be found in the lymphatic system without
clinical manifestations of filariasis.
2. Acute stage of infection is marked by fever, with inflammation of the lymph nodes
(lymphadenitis), particularly those of the male genitalia (in bancroft’s filariasis) and
of the extremities (due to Brugia). In females, involvement of the lymphatics of the
breast may be seen. Recurrent attacks are characterized by epididymitis (inflammation
of the epididymis), orchitis (inflammation of the testes), retrograde lymphangitis,
and localized inflammation of the arms and legs. The acute stage is also called
adenolymphangitis. Transient swellings of subcutaneous tissues may also occur called
Calabar swellings.
3. Chronic filariasis develops slowly after several years of infection. Manifestations
include chronic edema and repeated acute inflammatory episodes. The edema
and fibrosis gradually lead to lymphatic obstruction of the legs and genitalia
(especially the scrotum). The enlarged extremity hardens with loss of skin elasticity
producing elephantiasis. Obstruction of the lymphatics of the tunica vaginalis of the
testes lead to accumulation of edema fluid in the scrotum (called hydrocele). Hydrocele,
chronic epididymitis, and lymphedematous thickening of the scrotal skin are
commonly seen in bancroft’s filariasis. Deformities resulting from Malayan filariasis
260 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

are not as severe and include enlargement of the epitrochear, inguinal, and axillary
lymph nodes. In more advanced cases of Malayan filariasis, elephantiasis of one or
more limbs, usually involving the area below the knee may occur however the scrotum
is rarely involved.

Figure 15.15 Cases of elephantiasis involving the lower extremities

Laboratory Diagnosis
Examination of Giemsa stained peripheral blood smear is the diagnostic method of choice
demonstrating the microfilariae. In light infections, the blood specimen (approx. 1 mL) may
be immersed in 10 mL of a 2% formalin solution to lyse the red blood cells. Optimal sampling
collection is at night, especially for species that demonstrate nocturnal periodicity (usually
Wuchereria). The ideal times for specimen collection are between 9:00 pm and 4:00 am, the peak
periods for the appearance of the mosquito vectors. Antigen detection methods and serologic
tests have been developed as alternative diagnostic methods.

Treatment
The recommended drugs for treatment are diethylcarbamazine (DEC) and ivermectin
in combination with albendazole. Both DEC and ivermectin are effective in killing the
microfilariae, however, higher doses are necessary to kill the adult worms. Microsurgery may be
necessary to remove the obstructing parasite from the lymphatics. Other supportive measures
include anti inflammatory drugs to reduce the inflammation. The use of elastic bandages or
elevation of the involved limbs may help reduce the size of the involved limb
 Nematodes 26

Prevention and Control

The WHO Division of Control of Tropical Diseases recommend mass treatment in
endemic areas. In the Philippines, a Filariasis Control Program was implemented in 2001
which entailed mass treatment in endemic areas using a combination of DEC and albendazole.
This resulted in the elimination of infection in some endemic areas. Other measures include
the use of mosquito nets and repellents, the use of insecticides to control the mosquito vectors,
wearing of protective clothing, and thorough health education of the population.

Intestinal Tissue Nematode

Trichinella spiralis (Muscle Worm, Trichina Worm)
Important Properties and Life Cycle
There are two important morphologic forms of the parasite—larva and adult worm.
The larvae have a coiled appearance and encysts in muscle tissues, surrounded by striated
muscle cells called nurse cells. The adult worms are small and rarely recovered. The usual, natural
host is the pig but any mammal can be infected. Humans are accidental hosts and acquire the
infection by ingesting of raw or improperly cooked pork meat containing the encysted larva.
The larvae are released from the cysts with exposure to gastric acid and pepsin, after which they
invade the mucosa of the small intestines where they mature into adult worms. After mating,
the gravid female “gives birth” to the larvae in the intestinal submucosa. Among the nematodes,
the life cycle of the muscle worm has no egg stage. The larvae then migrate through the
bloodstream and localize to striated muscles where they undergo encystation.
262 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

= Infective Stage

= Diagnostic Stage

Meat scraps/
cannibalism
1

Predation/
scavenging
3

5 Circulation

Figure 15.16 Life cycle of Trichinella spiralis

Epidemiology and Pathogenesis

Infection with T. spiralis is seen worldwide, especially in parts of Europe and the United
States where meat can be eaten raw. Aside from the pig, other animals that may be infected
include deer, bear, walrus, and rodents (rats). The severity of the symptoms depends on
the intensity of the infection. Patients harboring a hundred or more worms are usually
symptomatic. Encystation of the larvae may lead to inflammation, then granuloma formation,
which can later become calcified
 Nematodes 263

Disease: Trichinosis, Trichinellosis

Trichinosis may be divided into three phases—enteric phase, invasion phase, and convalescent
phase. These correspond to the incubation and intestinal invasion stage (enteric phase),
the larval migration and muscle invasion stage (invasion phase), and the encystation and
encapsulation stage of the larva (convalescent phase).
The enteric or intestinal phase may manifest with diarrhea, abdominal pain, and vomiting.
In the invasion phase, potentially any organ with striated muscles may be the target of the
parasite. Symptoms may include periorbital and facial edema, conjunctivitis, fever, muscle pain
(myalgia), splinter hemorrhages, rashes, and peripheral eosinophilia. Involvement of the heart
can lead to life threatening myocarditis. During the convalescent phase, the manifestations start
to decline. The disease is self limiting, hence full recovery is expected. Rare causes of death are
congestive heart failure and respiratory paralysis.

a b

Figure 15.17 Encysted larva in muscle tissue stained with hematoxylin

and eosin at a 200x and b 400x magnifications

Laboratory Diagnosis
Definitive diagnosis is done by demonstrating the encysted larvae in muscle biopsy
specimen. Blood examination results include eosinophilia, leukocytosis, and elevated serum
muscle enzyme levels (lactate dehydrogenase, aldolase, creatine phosphokinase). Serologic
tests are available. False negative results may be seen during early infection, hence it is often
necessary to perform multiple tests
264 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Treatment
The disease is self limiting and therefore does not require medication. Supportive measures
include bed rest as well as the giving of analgesics and anti pyretics to relieve muscle pain and
fever. Corticosteroids may be given for severe infections. Thiabendazole may be given during
the early stages of the disease, especially during the first week, to kill the adult worms. The drug
has no effect on the migrating larvae.

Prevention and Control

Health education is important in preventing infection. It is also important to thoroughly
and adequately cook meat before consumption. Freezing meat may also kill the encysted
larvae. Avoidance of feeding pork scraps to hogs may help break the life cycle of the parasite.
Other measures include strict meat inspection and keeping pigs and other farm animals in
rat free pens
 Nematodes 265

CHAPTER SUMMARY

• Nematodes or roundworms are the most developed among the parasites. They are
dioecious and are equipped with digestive, reproductive, excretory, and nervous systems.
The male worms are usually smaller than the female worms.

• The life cycle of most nematodes consists of three morphologic forms—ova, larvae, and
adult worms, except for Trichinella spiralis, where there is no egg stage.

• The nematodes can be divided into three—intestinal nematodes, blood tissue nematodes,
and the intestinal tissue nematodes.

• The intestinal nematodes include Ascaris lumbricoides, Enterobius vermicularis, Trichuris

trichiura, Strongyloides Capillaria philippinensis, and the hookworms
stercoralis, Ancylostoma
duodenale and Necator americanus.

» The most common intestinal nematode is the giant intestinal roundworm Ascaris.
» Ascaris, Enterobius, Trichuris, and Capillaria are transmitted through ingestion.
Capillaria is transmitted through ingestion of freshwater fish while Ascaris, Enterobius,
and Trichuris are transmitted through ingestion of fecally contaminated food and
water.
» Infection with Strongyloides and the hookworms are acquired through skin penetration
by the infective larvae.
» The infective stage for Ascaris, Enterobius, and Trichuris is the embryonated egg while
the infective stage for hookworms, Strongyloides, and Capillaria is the larva.
» Autoinfection may occur with Enterobius, Strongyloides, and Capillaria.
» Infection with Enterobius is considered as a group infection.

• The blood tissue nematodes are the filarial worms Wuchereria bancrofti and Brugia malayi.
» Both filarial worms are transmitted by the bite of a mosquito vector.
» The infective stage for both is the microfilariae which often exhibit periodicity.
» Involvement of the scrotum leading to hydrocele is more common in bancroft’s
filariasis. In addition, deformities resulting from elephantiasis are more common with
Wuchereria infection.
» Diagnosis is established by examination of the peripheral blood smear and
demonstration of the microfilariae. Optimal time for specimen collection is at night.
• Theingestion
by
intestinal tissue nematode is represented by Trichinella spiralis. Infection is acquired
of improperly cooked or raw pork meat. The pathology is due to encystation
of the larva in striated muscles. Humans are accidental hosts, the disease is self limiting
and therefore does not require drug treatment
This page is intentionally left blank
 Nematodes 267

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date

Multiple Choice.

1. Larval migration to the lungs or lung phase is seen in the life cycle of which
among the following nematodes?
a. Ascaris d. A, B, and C
b. Ancylostoma e. A and B only
c. Necator
2. Infection with which among the following is acquired through skin penetration?
a. Trichuris, Strongyloides, Enterobius
b. Ancylostoma, Necator, Strongyloides
c. Ascaris, Trichinella, Capillaria
d. Enterobius, Necator, Strongyloides
3. Hydrocele with edema of the extremities is characteristic of which disease?
a. Ascariasis c. Filariasis
b. Trichinellosis d. Trichuriasis
4. Rectal prolapse is associated with infection with which among the following
parasites?
a. Ascaris c. Trichuris
b. Enterobius d. Strongyloides
5. The infective stage is the larva for which among the following parasites?
a. Ascaris c. Enterobius
b. Ancylostoma d. Trichuris
268 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Matching Type.

Column A Column B
6. Trichinella spiralis a. Threadworm
b. Hookworm
7. Trichuris trichiura
8. Strongyloides stercoralis
c. Pinworm
d. Whipworm
9. Enterobius vermicularis e. Muscle worm
10. Necator americanus f. Giant intestinal
roundwor
 CHAPTER
Infections
16 of the Skin

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. identify the different types of skin rashes or lesions;
2. recognize common skin infections based on clinical manifestations;
3. differentiate the characteristics of the causative organisms of each skin infection; and
4. discuss the laboratory diagnosis, treatment, and prevention of each skin infection.

Types of Skin Lesions

1. Macules – flat lesions characterized by change in color of the affected skin
2. Papules – raised lesions, solid in consistency of less than 5 mm in diameter
3. Plaques – flat with elevated surface (plateau like) with more than 5 mm diameter
4. Nodules – rounded raised lesions more than 5 mm in diameter
5. Urticaria (wheals or hives) – annular or ring like papules or plaques with pinkish color
6. Vesicles – circumscribed fluid filled lesions less than 5 mm in diameter
7. Bullae – circumscribed fluid filled lesions more than 5 mm in diameter
8. Pustules – circumscribed, exudate filled lesions
9. Purpura – skin lesions due to bleeding into the skin
a. Petechiae – less than 3 mm diameter
b. Ecchymosis – more than 3 mm diameter
10. Ulcer – crater like lesion that may involve the deeper layers of the epidermis and dermis
11. Eschar – necrotic ulcer covered with a blackened scab or crus
270 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

a b

Figure 16.1 a Confluent, erythematous macular rashes and b papular

rashes on the nape of a patient

Bacterial Skin Infections

Staphylococci: Staphylococcus aureus
Important Properties
S. aureus is a common pathogen in humans. They are gram positive cocci usually arranged
individually, in pairs, short chains, or in grape like clusters. It is found in the skin and the
nasopharynx. On culture they produce gray to golden yellow colonies. Golden yellow colonies
are best produced when cultured at 20 °C–25 °C. It is coagulase positive, which differentiates
it from other species of Staphylococcus. Staphylococci also produce catalase which differentiates
it from Streptococci. The organism produces enzymes and toxins responsible for its invasiveness
and pathogenicity.

Mode of Transmission
Skin infections are transmitted through direct contact with a person having purulent
lesions, from hands of healthcare or hospital workers, and through fomites like bed linens and
contaminated clothing.

Clinical Findings
1. Folliculitis – a pyogenic (pus producing) infection involving the hair follicle. It is
characterized by localized painful inflammation and heals rapidly after draining the pus.
2. Furuncle – an extension of folliculitis and is also known as boil. It is characterized by larger
and painful nodules with underlying collection of dead and necrotic tissue.
3. Carbuncle – represents a coalescence of furuncles that extends into the subcutaneous
tissue with multiple sinus tracts.
4. Sty or hordeolum – folliculitis occurring at the base of the eyelids
 Infections of the Skin 271

5. Impetigo – infection is common in young children and primarily involves the face
and the limbs. Initially it starts as a flattened red spot (macule) which later becomes a
pus filled vesicle that ruptures and forms crust (honey colored crust). It may be caused by
both S. aureus and S. pyogenes.
6. Staphylococcal Scalded Skin Syndrome (Ritter’s disease) – primarily a disease found
in newborns and young children. It is manifested by sudden onset of perioral erythema
(redness) that covers the whole body within two days. When slight pressure is applied
over the skin, it causes displacement of the skin. This is known as positive Nikolsky
sign. Bullae and cutaneous blister formation will soon follow and will later undergo
desquamation. Antibodies against the exfoliative toxin are produced within 7 to 10 days
enabling the skin to become intact again. The toxin responsible for these manifestations is
the exfoliative toxin. Only the outer layer of the epidermis is affected hence there will be
no scarring.

a b c

Figure 16.2 Clinical infections with Staphylococcus aureus include a staphylococcal scalded skin
syndrome, b impetigo, and c carbuncle
Source: CNX OpenStax, 2016; Åsa Thörn, 2011; and Drvgaikwad, 2008

Laboratory Diagnosis
Laboratory identification includes microscopic examination of Gram stained specimen
(gram positive cocci) and culture (gray to golden yellow colonies). The qualities of
microorganism is catalase positive and coagulase positive.

Treatment and Prevention

The treatment of choice is beta lactam antibiotics like penicillin. However, S. aureus readily
develops resistance to penicillin and other penicillin derivatives like methicillin and nafcillin.
Oxacillin is the only penicillin derived antibiotic that has remained active against S. aureus.
The incision and drainage of localized skin and soft tissue infection is also necessary
272 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Staphylococcus epidermidis
S. epidermidis is part of the normal flora of the skin and is commonly associated
with “stitch abscess,” UTI, and endocarditis. It also causes infections in individuals with
prosthetic devices.

Streptococci: Streptococcus pyogenes

S. pyogenes are gram positive cocci arranged in pairs or in chains when seen under the
microscope. It belongs to group A Streptococci based on Lancefield classification. It is beta
hemolytic (cause complete hemolysis of blood). Its major virulence factor is M protein which is
anti phagocytic. S. pyogenes also produces enzymes and toxins responsible for the pathogenesis
of infections caused by the organism. Some strains are encapsulated which protect them from
phagocytosis and may be associated with severe systemic infections.

Mode of Transmission
Soft tissue infections are acquired through direct contact with infected persons or
through fomites.

Clinical Findings

1. Pyoderma (Impetigo) – a purulent skin infection that is localized and commonly involves
the face, and the upper and lower extremities. It starts as vesicles then progresses to
pustules. The lesions rupture and form honey colored crusts. There may be enlargement
of the regional lymph nodes but no sign of systemic infection.
2. Erysipelas (St. Anthony’s Fire) – follows a respiratory tract or skin infection caused
by S. pyogenes. Patients manifest with localized raised areas associated with pain,
erythema, and warmth. It is grossly distinct from normal skin. There is accompanying
lymphadenopathy and systemic manifestations.
3. Cellulitis – involves the skin and subcutaneous tissue. Unlike erysipelas, the infected and
the normal skin are not clearly differentiated. It is also manifested as local inflammation
with systemic signs.
4. Necrotizing Fasciitis – involves the deep subcutaneous tissue and is also known as
“flesh eating” or streptococcal gangrene. It starts as cellulitis then becomes bullous and
gangrenous. It spreads to the fascia then the muscle and fat. It may become systemic and
cause multi organ failure leading to death
 Infections of the Skin 273

Complications
Acute glomerulonephritis and rheumatic fever are non suppurative, immune mediated
complications. Acute glomerulonephritis is more commonly associated with skin infections
while rheumatic fever is usually associated with S. pyogenes throat infection.

a b c

Figure 16.3 a “Strawberry tongue” seen in Scarlet Fever, b characteristic honey colored crustin
of impetigo; and c “flesh eating” necrotizing fasciitis
Source: Åsa Thörn, 2011 and Smuszkiewicz et al., 2008

Laboratory Diagnosis
1. Microscopy – Gram stain of samples of infected tissue will show gram positive cocci
in pairs and chains associated with leukocytes.

2. Culture – positive beta hemolysis in blood agar.

3. Bacitracin test – antibiotic susceptibility test with (+) zone of inhibition of growth around
the Bacitracin disc.

Treatment and Prevention

The drug of choice is penicillin. In case of penicillin allergy, macrolides (erythromycin,
azithromycin) or cephalosporins are alternate drugs. The drainage of pus and thorough
debridement of infected tissues must also be done.

Pseudomonas aeruginosa
P. aeruginosa are gram negative bacilli arranged in pairs that are encapsulated. They are
capable of producing water soluble pigments (e.g., pyocyanin – blue). It is an opportunistic
pathogen, a common cause of nosocomial infections (hospital acquired) and resistant to
most antibiotics. The virulence of P. aeruginosa can be attributed to adhesins (flagella, pili,
LPS, alginate), toxins (exotoxin A, pigments), and enzymes.
274 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Mode of Transmission
A common mode of transmission is through the colonization of previously injured skin.

Clinical Findings
P. aeruginosa is commonly associated with colonization of burn wounds and characterized
by blue green pus that exudes a sweet grape like odor. Other skin infections are folliculitis,
and secondary infections in individuals with acne and nail infections resulting from immersion
in contaminated water. It is also the most common cause of inflammation of the bone and
cartilage of the foot called osteochondritis following a penetrating injury.

Laboratory Diagnosis
Gram stain demonstrates gram negative bacilli arranged individually or in pairs. Culture
shows flat colonies with green pigmentation and characteristic sweet, grape like odor. Oxidase
test is positive.

Treatment and Prevention

P. aeruginosa is resistant to most antibiotics hence culture and sensitivity must be done.
Preventive measures for control of P. aeruginosa should be focused on preventing contamination
of sterile hospital equipments and instruments, and cross contamination of patients by hospital
personnel.

Clostridium perfringens
C. perfringens are gram positive bacilli that are anaerobic and rarely produce endospores.
It produces four lethal toxins namely: alpha, beta, iota, and epsilon toxins. Of the four toxins,
alpha toxin is the most lethal because it acts as a lecithinase that cause lysis of erythrocytes,
platelets and leukocytes. This toxin also causes massive hemolysis and bleeding and tissue
destruction. It is widely distributed in nature and particularly associated with soil and water
contaminated with feces.

Mode of Transmission
C. perfringens is commonly transmitted by the colonization of the skin following physical
trauma or surgery
 Infections of the Skin 275

Clinical Findings

C. perfringens causes soft tissue infections like cellulitis, suppurative myositis, and
myonecrosis or gas gangrene. Gas gangrene is a life threatening infection following physical
trauma or surgery characterized by massive tissue necrosis with gas formation, shock, renal
failure, and death within two days of onset.

Laboratory Diagnosis
Diagnosis is based on microscopic detection of gram positive bacilli in pairs and growth in
culture under anaerobic condition.

Treatment and Prevention

Surgical wound debridement and high dose penicillin therapy are the main approaches to
the management of the disease.

a b

Figure 16.4 a Gram stain of Clostridium perfringens showing centrally located spores and
b infection that led to myonecrosis or gas gangrene
Source: Schröpfer et al., 2008

Bacillus anthracis
B. anthracis are gram positive bacilli arranged individually or in pairs or long serpentine
chains giving them the characteristic “bamboo fishing rod” or “Medusa head” appearance. It is
aerobic, spore forming, and encapsulated. Virulence of the organism is due to its polypeptide
capsule which is responsible for evading phagocytosis and toxins—edema toxin and lethal
toxin—both of which inhibit the host’s immune responses. B. anthracis can also be used
in bioterrorism
276 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Mode of Transmission
B. anthracis is transmitted through inoculation into open skin from either the
soil or infected animal products, ingestion of infected meat or milk and inhalation of
aerosolized spores.

Clinical Findings
Anthrax is a disease of herbivores. There are three forms of anthrax—cutaneous,
gastrointestinal, or pulmonary anthrax. The skin infection, cutaneous anthrax, is the
most common form. It is characterized by painless papules at the site of inoculation that
become ulcerative, and later develops necrotic eschar. This is also associated with painful
lymphadenopathy and edema.

Laboratory Diagnosis
The peripheral blood contains a large number of B. anthracis which is easily seen on
Gram stain. Spores are only observed on culture in low carbon dioxide tension. Demonstration
of the spores can be done using Dorner stain or Wirtz Conklin stain.

Treatment and Prevention

Antibiotics like penicillin or doxycycline are the drugs of choice. In resistant cases,
ciprofloxacin is recommended. Prevention is through vaccination of animals and individuals
at risk such as animal handlers, veterinarians, military personnel, and those working
in slaughterhouses.

Fungal Skin Infections

Superficial Mycoses
Tinea versicolor (Pityriasis versicolor)
Tinea versicolor is caused by Malassezia furfur (Pityrosporum orbiculare). M. furfur is a normal
flora of the skin particularly in areas rich in sebaceous glands. The infection is worldwide in
distribution but more common in tropical regions. The lesions are irregular, discrete hypo or
hyperpigmented macules depending on the skin color of the affected individual. The lesions are
scaly with a dry, chalky appearance, and usually appear on the face, neck trunk, and arms
 Infections of the Skin 277

Diagnosis is made by microscopic visualization of “spaghetti and meatballs” appearance

of furfur with an alkaline stain (10% KOH or NaOH). It can also be demonstrated with
M.
Periodic Acid Schiff stain (PAS stain) or hematoxyllin eosin stain (H & E stain). Treatment
includes application of keratolytic agents containing selenium disulfide or salicylic acid and
topical antifungal drugs like ketoconazole.

a b Figure 16.5 a Malassezia furfur

spore (Yuping, 2016). Using
modified Dixon’s agar and b direct
microscopy of skin scrapings
demonstrating characteristic clusters
of thick walled round, budding
yeast like cells and short angular
hyphal forms
Source: Yuping et al., 2016

Tinea nigra
The Tinea nigra infection is caused by Hortaea werneckii (formerly Exophiala werneckii),
a dematiaceous fungus that produces melanin and grows as mold producing annelids or
annelloconidia. The lesions involve the palms and soles and are described as gray to black,
well demarcated macules. The infection is common in the tropical and subtropical regions, and
is more frequently seen in adolescents, young adults, and females.
Diagnosis is made by direct microscopic examination of skin scrapings with potassium
hydroxide and culture using Sabouraud’s dextrose agar medium. Treatment is similar to the
treatment for tinea versicolor.

a b

Figure 16.6 a Brownish lesions seen

on the palm of patient with tinea
nigra and b hypopigmented lesions
in a patient with tinea versicolor
Source: Dermatology Atlas and DermNet N
278 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Cutaneous Mycoses or Dermatophytosis

Cutaneous mycoses are fungal infections involving the keratinized structures of the body
like the skin, hair, and nails. The infection is caused by a group of fungi collectively referred
to as Dermatophytes. Dermatophytes produce keratinase, an enzyme capable of breaking
down keratin. They are keratinophilic and keratinolytic. The three genera that cause these
infections are: (1) Microsporum which infect the hair and nails only; (2) Trichophyton which
infect the skin, hair and nails; and, (3) Epidermophyton which infect only the skin and nails.
Microsporum produces both microconidia and macroconidia with predominance of
macroconidia, while Trichophyton predominantly produces microconidia. Epidermophyton, on the
other hand, produces smooth walled macroconidia in bundles of two or three. Based on their
natural habitat, Dermatophytes are classified as zoophilic (from animals to humans), geophilic
(from the soil to humans or animals), and anthropophilic (from humans to humans).
The infections are referred to as tinea or ringworm. The name of the infection reflects the
anatomic site involved, namely: (1) tinea pedis, also known as athlete’s foot (feet); (2) tinea
capitis (scalp); (3) tinea corporis (body); (4) tinea cruris or jock itch (groin); (5) tinea manus
(hands); (6) tinea barbae (beard); and (7) tinea unguium, also known as onychomycosis (nails).
Tinea involving the skin usually present with “ring worm” pattern. The lesions have reddish
border with central clearing. There may be inflammatory scaling. Hair invasion can be ectothrix
(arthroconidia are outside the hair shaft), endothrix (arthroconidia are inside the hair shaft),

a b c

d e

Figure 16.7 Cutaneous mycoses: a tinea unguium (Heilman, 2010), b tinea capitis, c tinea pedis,
d tinea corporis, and e tinea cruris or jock itch
Source: Heilman, 201
 Infections of the Skin 279

or favic (formed inside the hair but with “honeycomb” pattern or resembling a favic chandelier)
depending on the dermatophyte species causing the infection.
Specimens for diagnosis are skin or nail scrapings, or hair cuttings from the affected areas.
More fungi can be obtained from the borders of the lesion rather than the center. Diagnosis is
based on the clinical appearance of the lesions, direct microscopic examination and culture.
Treatment involves administrating antifungal drugs such as azoles (miconazole,
clotrimazole, econazole).

Subcutaneous Mycoses
The infection initially involves the deeper layers of the dermis and subcutaneous tissue
then later the bones. The mode of transmission is through traumatic inoculation into the skin.
The infections are relatively rare with the exception of sporotrichosis. Other infections are
chromoblastomycosis,phaeohyphomycosis, zygormycosis and mycetoma (Madura foot).
Sporotrichosis, also known as rose gardener’s disease, is caused by a dimorphic fungus,
Sporothrix schenckii, that is found in the soil and decaying vegetation. The infection initially
presents as a small nodule which may later become ulcerative and pustular. Two weeks later,
painless, subcutaneous nodules along the lymphatic drainage develops. Sometimes they may
present as verrucous lesions and often misdiagnosed as malignancy of the skin.
Chromoblastomycosis is characterized by verrucous nodules or plaques. The infection is
insidious and may become chronic. The etiologic agents are all dematiaceous fungi namely:
Exophiala, Fonseca, Cladosporium, Phialophora, and Rhinocladiella.
Mycetoma or Madura Foot may be caused by true fungi (Eumycotic Mycetoma) or
Actinomycetes (Actinomycotic mycetoma). The common causes of eumycotic mycetoma are
Phaeoacremonium, Madurella, Curvularia and Fusarium. It frequently involves the feet and
hands. The infection is characterized by the clinical triad of tumefaction, granules, and draining
sinus. Diagnosis is primarily based on the characteristics of the granules.

a b c

Figure 16.8 a Ulcerated lesion of sporotrichosis following course of draining lymphatics,

b Madura foot, c wart like lesions of chromomycosis
Source: Alfalah, 200
280 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Viral Infections of the Skin

Warts
Etiologic Agent
Warts are caused by a DNA virus, Human Papillomavirus (Family Papovaviridae). There are
at least 70 serotypes. The virus is capable of causing malignant transformation of the infected
cell.

Mode of Transmission
HPV infection is acquired by (1) direct contact through mucosal or skin breaks; (2) sexual
contact; and (3) upon passage through infected birth canal.

Clinical Findings

1. Skin warts (common, plantar, and flat warts) – benign, self limiting proliferation of the
skin that undergoes spontaneous resolution. These warts may be flat, dome shaped, or
plantar.
HPV types 1–4 are the most common isolates from the lesions. Frequently affected
sites are the hands and feet and common among children and adolescents.
2. Genital and anogenital warts – also known as condylomata acuminata (singular
condyloma acuminatum).

a b c

d e

Figure 16.9 Various types of warts: a dome shaped, b plantar, c periungal,

d flat (plane), and e filifor
 Infections of the Skin 281

a b

Figure 16.10 a Periungual warts caused by HPV types 1–4 and b venereal wart
(condyloma acuminata) due to HPV types 6 and 11

Laboratory Diagnosis
Diagnosis is based on gross appearance of the lesions and histologic appearance on
microscopic examination that includes hyperkeratosis.

Treatment and Prevention

Treatment is removal of the lesion by: (1) surgical excision; (2) cryosurgery;
(3) electrocautery; (4) application of caustic agents like podophyllin; and (5) interferon for
genital warts. Avoiding contact with infected tissue prevents spread of the infection.

Herpes Simplex Infections

Etiologic Agent
The etiologic agents are Herpes simplex virus types 1 and 2, DNA viruses under the family
of Human Herpesviridae. These viruses are capable of latency in the neurons and are capable of
recurrent infections.

Modes of Transmission
HSV is present in oral and genital secretions and vesicle fluid. It can be transmitted
through (1) oral contact (kissing); (2) fomites (sharing of glasses, toothbrushes and other
saliva contaminated materials); (3) sexual contact; (4) transplacental (during pregnancy);
and (5) during childbirth
282 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Clinical Findings

1. Gingivostomatitis – the primary infection, primarily caused by HSV 1. It presents as

vesicles that rupture and ulcerates. Lesions are seen in the buccal mucosa, palate, gingivae,
pharynx, and the tongue. The most striking feature is gingivitis. Gingivostomatitis is
common during childhood.
2. Herpes labialis (Fever Blister or Cold Sore) – represents recurrent mucocutaneous HSV
infection. This is caused by HSV 1 and 2. Lesions are usually located at the vermillion
borders of the lips. Lesions are vesicular, they rupture then form an ulcer and later form
crusts. Recurrences are less severe than the primary infection and often occur on the
same site.
3. Herpetic whitlow – HSV infection involving the fingers and caused by both HSV
types 1 and 2.
4. Eczema herpeticum – HSV infection occurring in children with eczema. This only shows
that HSV can be an opportunistic pathogen. It can also cause a superimposed infection
in burns.
5. Herpes gladiatorum – HSV infection of the body and is usually acquired during
wrestling or playing rugby.

a b c

Figure 16.11 Herpes simplex infections of a the mouth (herpetic gingivostomatitis), b the fingers
(herpetic whitlow), and of c the penis (herpes genitalis)
Source: Heilman, 2010 and SOA AIDS Amsterdam

Laboratory Diagnosis
Diagnosis is based mainly on the clinical presentation of the infection. Diagnosis can be
made based on histopathologic changes and using the Tzanck smear to demonstrate the
characteristic inclusion bodies known as the Cowdry type A inclusions. Cell culture is also
diagnostic but seldom requested.

Treatment and Prevention

The recommended drug is acyclovir. There is currently no available vaccine for HSV
 Infections of the Skin 283

CHAPTER SUMMARY

• Skin infections may present in different forms of lesions.

• Skin infections may be caused by a variety of infectious agents—bacteria, viruses, or fungi.
• or clusters. It aureus
Staphylococcus are gram positive cocci, arranged individually, in pairs, short chains
is part of the normal flora of the skin and nasopharynx and a common
human pathogen.
» S. aureus causes skin infections that may be pyogenic like folliculitis and toxin
mediated infections like scalded skin syndrome.

• epidermidis a common cause of stitch abscess.

Staphylococcus is

• hemolysis on blood are gram positive cocci arranged in pairs or chains and causes beta
Streptococcus pyogenes
agar.

» S. pyogenes causes impetigo, erysipelas, cellulitis and necrotizing fasciitis.

• Pseudomonas aeruginosa is a gram negative bacillus and produces water soluble pigments
like pyocyanin and is resistant to most antibiotics.

• Myonecrosis is a severe skin infection caused by Clostridium perfringens, a gram negative,

anaerobic bacillus that rarely produces spores.

• Anthrax is a disease of herbivores caused by Bacillus anthracis, a gram negative, aerobic,

spore forming bacillus.
» Cutaneous anthrax is characterized by eschar formation.
• Majority of severe bacterial skin infections are toxin mediated.
• Fungal infections of the skin may be superficial only involving the outer epidermis,
or it may be cutaneous involving the deeper layers of the epidermis and dermis, or it may
involve the subcutaneous tissues and lymphatics.

• Warts are benign skin infections that may be resolved spontaneously caused by Human
Papilloma viruses, which are capable of malignant transformation.

• Herpes simplex virus causes infections of the skin and mucous membranes. It is
capable of latency and recurrent infections
also
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 Infections of the Skin 285

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Case No. 1: A 3 month old boy was brought to a physician because of vesicles and bullous
skin lesions with desquamation. The condition started a week prior to consultation as perioral
erythema that later involved the whole body. A positive Nikolsky sign was elicited when slight
pressure was applied over the affected skin and the skin was displaced from the underlying
tissue. The patient later developed vesicular lesions all over the body.

1. What is the most probable diagnosis?

a. Fever blister c. Impetigo
b. Scalded Skin Syndrome d. Erysipelas
2. What is the most probable causative agent for this condition?
a. Staphylococcus aureus c. Pseudomonas aeruginosa
b. Streptococcus pyogenes d. Clostridium perfringens
3. What toxin is responsible for the manifestations of this disease?
a. TSST 1 c. Exfoliative toxin
b. Erythrogenic toxin d. Lethal toxin
4. The probable etiologic agent is described as:
a. Gram positive diplococci, lancet shaped
b. Gram positive cocci in clusters
c. Gram positive cocci, beta hemolytic
d. Gram positive bacilli, anaerobi
286 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Case No. 2: A 15 year old girl consulted a physician because of dark brown to blackish macules
over the right palm. During the physical examination, the lesions appear scaly and dry. There is
no other manifestation that bothers the patient except for the cosmetic problem.

5. This is probably a case of:

a. Tinea corporis c. Tinea nigra
b. Tinea versicolor d. Leprosy
6. The most likely etiologic agent is:
a. Malassezia furfur c. Microsporum canis
b. Exophiala werneckii d. Mycobacterium leprae
7. This case can be diagnosed by:
a. Wet mount of skin scraping using KOH
b. Wood’s lamp
c. Gram staining
d. Acid fast staining

Matching Type.

Column A Column B
8. Tinea corporis a. groin
b. body or trun
9. Tinea unguium
10. Tinea cruris
c. nails
 CHAPTER
Infections of the
17 Respiratory Tract

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. recognize common respiratory tract infections based on clinical manifestations;
2. describe the characteristics of the causative organisms of each respiratory tract infection;
3. explain how the different respiratory tract infections are transmitted; and
4. discuss the appropriate laboratory diagnosis, treatment, and prevention of each infection.

Infections involving the respiratory tract are classified as upper respiratory tract infections
and lower respiratory tract infections. The upper respiratory tract is from the nose down to the
larynx including the sinuses while the lower respiratory tract includes the bronchial tree and the
lungs. Respiratory tract infections may be caused by a myriad of organisms—viruses, bacteria
and fungi.
There are several risk factors that promote the development of respiratory tract infections.
The most critical of the factors is the quality or condition in the environment. Most preventable
infections can be attributed to poor environmental standards such as poor housing conditions,
overcrowding, and air pollution both inside and outside the home.
Respiratory tract infections are transmitted by person to person which may involve direct
spread via droplet nuclei (e.g., sneezing, talking, or coughing resulting in discharge of airborne
particles from the respiratory tract of the infected person), or through indirect spread via articles
or hands contaminated with the person’s infectious secretions
288 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Diseases of the Upper Respiratory Tract

Common Cold
The common cold (acute rhinitis) is considered as a common illness in both children and
adults. Its incidence and seasonal occurrence tend to be predictable. For instance, in tropical
countries, the average occurrence of the illness increases during the rainy season. It commonly
occurs in school settings, especially with the proliferation of day care centers.
Close personal contact is necessary for the transmission of colds. The greatest concentration
of the etiologic agent (most commonly due to rhinovirus) is in the nasal secretions and the
greatest source of infection is through sneezing, nose blowing, and contamination of external
surfaces with nasal secretions.
The manifestations of the common cold may be varied. In older children, its onset may be
accompanied by a fever. More commonly, the onset is characterized by sneezing with breathing
watery nasal discharge (runny nose or rhinorrhea/coryza). The degree of rhinorrhea increases
with manifestations of nasal congestion. Nasal obstruction due to congestion may interfere with
breathing, eating, and sleeping. After 1–3 days, the nasal secretions may become thicker and
purulent. Overall, viruses remain to be the most common etiologic agents of the common cold.

Table 17.1 Infectious agents associated with the common cold

Category Agents
Common viruses that usually Rhinovirus (most common)
cause the common cold Parainfluenza viruses
Respiratory syncytial virus
Coronavirus
Common infectious agents that Adenoviruses
occasionally cause illness with Enteroviruses
common cold symptoms Influenza viruses
Reoviruses
Mycoplasma pneumoniae
Illnesses with initial symptoms Coccidioides immitis
suggestive of the common cold Histoplasma capsulatum
Bordetella pertussis
Chlamydia psittaci
Coxiella burnetti
Source: Lecture Guide in Microbiology, Dept. of Microbiology, College of Medicine,
Our Lady of Fatima University, 201
 Infections of the Respiratory Tract 28

Antibiotics have no role in the management of the common cold. Management is mainly
symptomatic. Paracetamol can be given for fever. The true efficacy of decongestants has not
been determined yet. In some instances, use of decongestants may cause rebound vasodilation
which can further contribute to the congestion.

Rhinoviruses
Rhinoviruses are the main cause of the common cold. They are non enveloped RNA viruses
that have more than 100 serologic types. They primarily affect the nose and conjunctiva. The
virus can withstand adverse environmental conditions and can survive the external environment
for many hours but are killed by gastric acid when swallowed.

Coronaviruses
Coronaviruses are the second most common cause of the common cold. Unlike rhinoviruses,
coronaviruses are enveloped RNA viruses. Infection occurs worldwide and the virus is mainly
transmitted by respiratory aerosol. In 2002, a new disease, an atypical pneumonia called SARS
(Severe Acute Respiratory Syndrome) emerged and coronavirus was implicated as the etiologic
agent (CoV SARS). The civet cat was identified as the likely reservoir of CoV SARS.
The common cold caused by coronavirus has the same manifestations as that caused by
rhinovirus. On the other hand, SARS is a severe form of atypical pneumonia characterized
by fever, non productive cough, dyspnea, and hypoxia. Chills, rigors, malaise, and headache
commonly occur. The incubation period ranges from 2 to 10 days.
There is no antiviral therapy or vaccine available. There have been attempts of using
a combination of ribavirin and steroids in the treatment of SARS but the efficacy is still
undetermined.

Adenoviruses
Adenoviruses are non enveloped DNA viruses that cause a variety of upper and lower
respiratory tract diseases such as pharyngitis, conjunctivitis, common cold, and pneumonia.
They also cause keratoconjunctivitis, hemorrhagic cystitis, and gastroenteritis.

Mode of Transmission
1. Aerosol droplet – transmission of pathogens through the air
2. Fecal oral – most common mode of transmission among young children and their families
3. Direct inoculation of conjunctivae by fingers – transmission may occur when coming into
contact with contaminated surfaces
290 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Clinical Findings

1. Upper respiratory tract infections

a. Nasopharyngitis – characterized by the swelling of the nasal passages and the back of
the throat
b. Pharyngoconjunctivalfever – acute respiratory disease characterized by fever, sore
throat, coryza (runny nose), and conjunctivitis
2. Lower respiratory tract infections
a. Bronchitis – inflammation of the brocial tubes
b. Atypical pneumonia – infection caused by different bacteria than the common ones
that cause pneumonia
3. Hemorrhagic cystitis – characterized by hematuria and dysuria.
4. Gastroenteritis – with non bloody diarrhea in children younger than 2 years of age
Laboratory Diagnosis
Diagnosis can be confirmed by isolation of the virus in cell culture and detection of 4 fold
increase in antibody titer.

Treatment and Prevention

There is no antiviral therapy. Live, non attenuated adenovirus vaccine for serotypes 4, 7,
and 21 has been developed but was used only by the military. The vaccines were administered
orally and were not available for civilian use. The vaccine is no longer used now.

Pharyngitis
Pharyngitis refers to inflammation of the mucous membranes of the pharynx. The clinical
diagnostic category includes tonsillitis, tonsillopharyngitis, and nasopharyngitis.

Nasopharyngitis
Nasopharyngitis is a common illness of childhood, occurring more commonly during the
cold weather months. The most common cause are adenoviruses, frequently causing infection in
adolescent and young adults in military training. Other viruses that can also cause the disease
are influenza and parainfluenza viruses. Clinical manifestations are varied but fever occurs in
nearly all cases. It is an acute, self limited disease lasting 4 to 10 days. Other symptoms depend
on the specific etiologic agent
 Infections of the Respiratory Tract 29

Diagnosis is based on clinical manifestations. Management is mainly supportive. The use

of throat lozenges, aseptic mouthwash, and decongestants have no role in the management of
the infection.

Tonsillopharyngitis
Tonsillopharyngitis is inflammation involving both the pharynx and the tonsils. It has a
seasonal occurrence and usually involves children 5–10 years of age, with a secondary peak
at 12 and 18–20 years of age. The most common cause is Streptococcus pyogenes. It is obtained
primarily by direct contact with large droplets or respiratory secretions.
The disease manifests with sudden onset of fever, sore throat, headache, nausea, malaise,
and pain. There is marked tonsillo pharyngeal erythema. The gold standard for diagnosis
is culture of specimen obtained by swab of the posterior pharyngeal and tonsillar regions.
Complications include sinusitis, otitis media, peritonsillar and retropharyngeal abscess, acute
rheumatic fever, and acute glomerulonephritis.
It is generally a self limited disease. However, because of the possible sequelae, antibiotic
treatment is necessary. The drug of choice is penicillin. Erythromycin or Clindamycin may be
given as an alternative for patients allergic to penicillin.

Scarlet Fever
This infection occurs in association with streptococcal pharyngitis and is caused by
Streptococcus pyogenes strains producing streptococcal pyrogenic exotoxin or formerly known as
erythrogenic toxin. The toxin causes a hypersensitivity reaction producing a pinkish red rash on
the skin. The rash blanches when pressed and is best observed in the abdomen and pastia lines
or skin folds. The tongue has a spotted, strawberry like appearance and will eventually become
very red and enlarged. As the disease progresses the skin peels off similar to a sunburn.
The drug of choice for treatment is penicillin G. Since the disease is transmitted primarily
by inhalation of infective droplets from an infected person, control measures are directed mainly
at the human source.

Sinusitis
Sinusitis is inflammation of the mucosal lining of one or more of the paranasal sinuses.
It is common in children with allergies, or adenoids and enlarged tonsils, dental infections,
and in children with chronic ear infections. The principal pathogens in all age groups are
Haemophilus influenzae and Streptococcus pneumoniae, both of which will be discussed later under
lower respiratory tract infections.
292 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The clinical features are age dependent. In young children, manifestations may include
persistent rhinorrhea (nasal discharge) with a daytime cough that is worse at night. They also
manifest with periorbital edema, post nasal drip, and foul smelling breath. In older children
or adults, manifestations may include headaches, dental and facial pain with tenderness over
the involved sinuses on palpation.
Specific diagnosis involves culture with specimen taken from the infected sinus. X ray
may also be done to demonstrate the involved sinus. Antimicrobial therapy is done to achieve
clinical improvement and sterilization of sinus secretions, it is given for 7 days or more. Other
measures include: (1) normal saline washes to liquefy secretions and enhance mucociliary
transport; (2) use of anti histamines if allergic rhinitis is contributory; (3) corticosteroids
to reduce inflammation but must be used with caution because of risk of superinfection;
and (4) surgical drainage.

Otitis Externa
Otitis externa is inflammation involving the external ear. The more common cause
in tropical countries is Pseudomonas aeruginosa. Other causes are Staphylococcus aureus,
Proteus vulgaris, Klebsiella, and Escherichia coli. Itching and pain are prominent and intense pain
is felt when the tragus is pulled. Periaural edema and complete obliteration of the canal may be
seen in severe infection.
Diagnosis is made based on clinical presentation. Management includes flushing or
irrigation of the external auditory canal with 3% hypertonic saline. If there is no evidence of
infection, use of topical corticosteroid cream is sufficient. In the presence of overt infection,
Neosporin cream must be applied three times a day. Preventive measures include minimizing
swimming and exposure to water and minimizing excessive cleaning of the ears.

Otitis Media
Otitis media refers to inflammation of the mucoperiosteal lining of the middle ear.
Two thirds of cases are caused by bacteria with Streptococcus pneumoniae as the most common
and Haemophilus influenzae as the second most common cause. Viral causes include respiratory
syncytial virus, influenza virus, adenovirus, and rhinovirus.
The condition begins with non specific signs and symptoms of fever, irritability, headache,
anorexia, and vomiting. Cough and coryza usually occur prior to the signs of ear infection.
The most common specific manifestation is otalgia (ear pain). Other signs and symptoms
include otorrhea (ear discharge), hearing impairment, and tinnitus (ringing in the ears).
Complications that may arise if not properly managed include perforation of the tympanic
membrane, mastoiditis, hearing loss, meningitis, and brain abscess. Management involves taking
antibiotics for the specific etiologic agent
 Infections of the Respiratory Tract 29

Croup (Laryngitis, Laryngotracheitis,

Laryngotracheobronchitis)
Croup is a term used to denote several respiratory illnesses characterized by varying degrees
of inspiratory stridor, cough, and hoarseness (“seal bark”) resulting from obstruction in the
region of the larynx. Majority of cases occur within the first 3 years of life. The major causes of
croup are the Parainfluenza viruses. It can also be caused by the Respiratory Syncitial Virus. Both
are enveloped RNA viruses belonging to the family Paramyxoviridae.
Croup is transmitted mainly by respiratory droplets. In addition to croup, other respiratory
diseases caused by Parainfluenza viruses include the common cold, pharyngitis, laryngitis, otitis
media, bronchitis, and pneumonia. The diagnosis of croup is based on clinical manifestations.
There is no antiviral drug or vaccine available.

Influenza
Influenza, more commonly known as the “flu,” initially involves the upper respiratory tract.
However, it later progresses to involve the lower respiratory tract. It is caused by the influenza
viruses which are members of the Family Orthomyxoviridae. There are three immunologic
types of influenza viruses: influenza A, influenza B, and influenza C. Only influenza A and
influenza B cause infections to humans and it is usually a mild viral infection. The incubation
period is 1–3 days and is communicable during the prodromal phase until three days after the
onset of symptoms.
Influenza A is responsible for most cases of epidemics and pandemics. It is also the cause
of influenza in birds (avian flu), pigs, horses, and seals which are sources of new strains of
Influenza A. Some of the strains isolated are similar to those causing disease in the human
population. Influenza B mainly causes epidemics and infection is restricted to humans.
Influenza viruses undergo antigenic changes, with the exception of Influenza C. There
are two types of antigenic variations that involve the two surface antigens of
(HA) and neuraminidase (NA) antigens namely, antigenic drift and antigenic
influenza—hemagglutinin

shift. Antigenic drift is a minor change and is due to accumulation of point mutations in the
gene resulting in amino acid changes involving the H ag. This is seen in both Influenza A and
Influenza B. Antigenic drift is responsible for the occurrence of epidemics. Antigenic shift is
a major change that involves rearrangement of the gene segments involving the H ag or N ag
resulting in the development of new strains. It is responsible for pandemics and occurs only
in Influenza A.
A new strain, H5N1, is now identified as the cause of the avian flu and is currently being
closely monitored for fear that it can lead to the development of a pandemic. H5N1 is not a
product of antigenic drift or antigenic shift. It is considered as a re emerging virus which has
294 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

existed since the 1950s. Before the development of vaccines for influenza, millions of people
died from the infection.
From 1919 to 1920, the great “Spanish flu” pandemic caused by influenza A(H1N1) caused
the death of at least 20 million people within a year. The latest pandemic was attributed to
a novel virus similar to the Spanish Flu A(H1N1). This is the Mexican swine flu A(H1N1)
which is a quadruple re assortant virus resulting from the recombination of two strains from
birds, one from swine and one from human.
In 1957, the “Asian flu” caused by influenza A(H2N2) and in 1968, the “Hong Kong flu”
caused by influenza A(H2N2) together killed more than 1.5 million people. The individuals
at risk of influenza are children, the elderly, immunocompromisedpeople, people in nursing
homes, smokers, and those with underlying cardiac or respiratory conditions like asthma.

Mode of Transmission
The virus is transmitted by airborne respiratory droplets during breathing, coughing and
talking.

PB1, PB2, PA
(RNA polymerase)
HA (hemagglutinin)

M2 (ion channel)

M1 (matrix protein)
NA (neuraminidase)

Lipid bilayer

NEP
NP (nucleocapsid protein)
Segmented (–) strand RNA gene
Figure 17.1 Diagrammatic representation of the structure of influenza virus showing the
hemagglutinin (HA) and neuraminidase (NA) antigens on the envelope of the virus, as well as the
segmented genom
 Infections of the Respiratory Tract 29

Clinical Findings

1. Uncomplicated influenza – symptoms appear abruptly consisting of chills, headache, and

dry cough, followed by high fever, generalized muscular pains, malaise, and anorexia.
Symptoms in children are like those of adults although the fever may be higher in
children. Febrile convulsions may occur.
2. Pneumonia – complicates influenza and may be viral, secondary bacterial, or a combination
of the two. This usually occurs in the elderly and debilitated patients, especially those with
underlying chronic disease. The common bacterial pathogens implicated are S. aureus,
S. pneumoniae, and H. influenzae.

3. Reye’s Syndrome – a complication usually associated with Influenza B and varicella

zoster infections. It is an acute encephalopathy that occurs in children and adolescents
and is associated with the intake of aspirin or salicylate.

Laboratory Diagnosis
Diagnosis of influenza are made on clinical grounds. However, laboratory tests are
available for confirmation, including isolation of the virus and identification of viral antigens in
patient’s cells.

Treatment and Prevention

Amantadine and its derivative rimantadine are used for both treatment and prevention
but only for Influenza A. The newer drugs zanamivir (Relenza) and oseltamivir (Tamiflu)
are now being used for the treatment of both Influenza A and B. To be effective, they must
be administered very early in the disease. Relenza and GS4104 have been proven effective in
preventing multiplication of the virus and the virus’ resistance to the drug is less frequent.
The main mode of prevention is the vaccine, consisting of Influenza A and B viruses. It is
given annually, especially to high risk groups such as elderly individuals and hospital personnel.
Protection lasts for one year.

Diphtheria
The disease is caused by Corynebacterium diphtheriae, gram positive, non spore forming
rods that are club shaped and arranged in V or L shaped forms, giving rise to a “Chinese
character” appearance. The bacteria are transient flora of the throat and are non toxigenic
strains. Toxigenic strains produce diphtheria toxin which is responsible for the disease.
296 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

a b c

Figure 17.2 a Gram stain of Corynebacterium diphtheria demonstrating some bacilli assuming
L, V, or X formation and clinical features of diphtheria showing b the typical pseudomembrane
and c “bull neck” appearance of the neck, respectively
Source: Dileepunnikri, 2014

Mode of Transmission
Humans are the only natural hosts of the corynebacterium diphtheria. The main mode
of transmission is by air borne droplets of a carrier or patient or through the discharge from
cutaneous lesions. Prolonged direct contact is required for transmission of the infection. Since
the bacteria are resistant to drying, contaminated materials may serve as a reservoir of the
infection. The incubation period is 2–5 days and patients are communicable up to three weeks
following onset of the manifestations.

Clinical Findings
Inflammation begins in the respiratory tract leading to sore throat and fever. The most
prominent sign is the thick, gray, adherent pseudomembrane over the tonsils and throat.
Extension of the membrane into the larynx and trachea causes airway obstruction manifesting
as dyspnea. The obstruction may even cause suffocation that is relieved by intubation or
tracheostomy. Do not attempt to remove the membrane because it is tightly adherent to the
underlying tissue and cause massive bleeding. Damage to the heart may manifest as arrhythmia
(irregularity in rhythm). Nerve weakness or paralysis may also occur, especially involving the
cranial nerves. There is massive enlargement of the cervical lymph nodes giving the neck a “bull
neck” appearance. Manifestations tend to subside spontaneously.

Laboratory Diagnosis
Swabs from the nose, throat, or other suspected lesions must be obtained before
antimicrobial drugs are given. Gram staining and microscopic examination of the
Gram stained specimen can help identify the organism. Definitive diagnosis can be mad
 Infections of the Respiratory Tract 29

by culturing a specimen using Loeffler’s medium. Colonies of C. diphtheriae from culture

must be tested for toxin production through the ELEK test, which is a test for pathogenicity
of C. diphtheriae.

Treatment and Prevention

Management involves prompt administration of antimicrobial agents and diphtheria
antitoxin to neutralize the exotoxin. The recommended antibiotics are penicillin G or
erythromycin.
The major aims of prevention are to limit the distribution of the toxigenic strains in the
population and to maintain a high level of active immunization. Infected patients must be
isolated. Children must receive an initial course of immunizations and boosters. Diphtheria
toxoid is given in combination with tetanus toxoid and acellular pertussis vaccine (DTaP) at
ages 2 months, 4 months, and 6 months. Boosters are given at 15 to 18 months and 4 to 6 years
of age.

Diseases of the Lower Respiratory Tract

Chronic Bronchitis
Individuals with functional and anatomic abnormalities of the respiratory tract
(e.g., COPD) are subject to attacks of chronic bronchitis, an inflammatory condition
involving the bronchi. This is a recurrent bacterial infection that is often precipitated by viral
infections. The most common etiologic agents are Streptococcus pneumoniae (Pneumococci) and
Haemophilus influenzae. Other causative agents include adenovirus, Bordetella pertussis, and
parainfluenza virus.

Bronchiolitis
Bronchiolitis is a severe inflammatory condition involving the bronchioles.

Respiratory Syncytial Virus (RSV)

RSV is an RNA virus that belongs to the Family Paramyxoviridae. It can cause a variety of
respiratory tract infections ranging from mild common colds to bronchiolitis and pneumonia.
It is the major cause of bronchiolitis in infants and young children. Almost all children by age
2 years had already been infected by this virus. RSV infections are prevalent during winter and
epidemics occur yearly.
298 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Mode of Transmission
RSV is spread by large droplets and direct contact. The main points of entry of the virus
into the host are through the nose and eyes.

Clinical Findings

1. Bronchiolitis
2. Common cold
3. Influenza like illness
4. Pneumonia
5. Otitis media
Laboratory Diagnosis
Viral genome can be detected by RT PCR. Viral antigens can be detected by enzyme
immunoassay or immunofluorescence.

Treatment and Prevention

The antiviral drug ribavirin has been approved for the treatment of RSV bronchiolitis.
Supportive care is essential, consisting of the removal of secretions and administration of
oxygen. Administration of immune globulin may be beneficial. Control measures to prevent
nosocomial outbreaks include contact isolation through hand washing and restriction of visitors.

Pneumonia
Pneumonia is an infection involving the lung parenchyma. Most cases of pneumonia are
caused by bacteria. Pneumonia may be classified as typical pneumonia or atypical pneumonia.
Atypical pneumonia is also referred to as “walking pneumonia,” usually with milder
manifestations than typical pneumonia and caused by other bacteria that are not common
causes of pneumonia. These atypical organisms cannot be stained by Gram stain and do not
grow in cultures using common media.
Pneumonia may also be classified based on the areas of the lower respiratory tract affected.
If entire lobes of the lungs are involved, it is referred to as lobar pneumonia. If the alveoli are not
involved and the inflammation is confined to the interstitial spaces, it is interstitial pneumonia.
If the bronchus and the alveoli of the lungs adjacent to the bronchi are involved, then it is
called bronchopneumonia
 Infections of the Respiratory Tract 299

Acute bacterial pneumonia typically presents with abrupt onset of fever and chills,
cough productive of purulent sputum, and pleuritic chest pain. Pleuritic chest pain occurs if
there is involvement of the pleural membranes. The patients appear ill and usually manifest
with tachypnea (rapid breathing) and tachycardia (rapid heart rate). The most common causes
of community acquired pneumonia are Streptococcus pneumoniae (most common), Mycoplasma
pneumoniae, and Legionella pneumophila. Other causes are Haemophilus influenzae, Staphylococcus
aureus, and Klebsiella pneumoniae in chronic alcoholics. Nosocomial pneumonia, on the other
hand, is most often caused by Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.
Viral pneumonia is characterized by inflammation of the interstitial spaces. The
manifestations may be less severe as that of bacterial pneumonia and physical examination may
only reveal rales on auscultation. It is most commonly caused by respiratory syncytial virus,
parainfluenza viruses, influenza viruses, adenoviruses, measles virus, and varicella zoster virus.
Diagnosis can be made by Gram stain and microscopic examination of sputum specimen.
However, the gold standard for diagnosis is still through culture of the blood or sputum.
Table 17.2 summarizes some characteristics of the more common and important bacterial
pathogens causing pneumonia.

Table 17.2 Characteristics and treatment of different types of pneumonia

Preferred
Clinical Setting
Laboratory
Organism Complications Antimicrobial
Studies
Therapy
Streptococcus Chronic cardio Gram stain of Bacteremia, Penicillin G
pneumoniae pulmonary disease; sputum; culture meningitis,
follows URT of blood, sputum, endocarditis,
infections pleural fluid empyema
Haemophilus Chronic cardio Culture of Empyema, Ampicillin
influenzae pulmonary disease; sputum, blood, endocarditis or amoxicillin;
follows URT pleural fluid Cefotaxime
infections or ceftriaxone
Staphylococcus Influenza Culture of Empyema, Nafcillin
aureus epidemics; sputum, blood, cavitation
nosocomial pleural fluid
Klebsiella Alcohol abuse, DM; Culture of Empyema, Cephalosporin
pneumoniae nosocomial sputum, blood, cavitation + gentamicin
pleural fluid or tobramycin
Escherichia Nosocomial; Culture of Empyema Third generation
coli rarely community sputum, blood, cephalospori
acquired pleural fluid
300 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Preferred
Organism Clinical Setting Laboratory Complications Antimicrobial
Studies
Therapy
Pseudomonas Nosocomial; cystic Culture of Cavitation Anti
aeruginosa fibrosis sputum, blood pseudomonal
penicillin +
tobramycin
Mycoplasma Young adults Complement Skin rashes, Erythromycin,
pneumoniae fixation test hemolytic anemia azithromycin or
clarithromycin
Legionella sp. Exposure to Culture of Empyema, Erythromycin,
contaminated sputum or tissue cavitation, azithromycin or
construction site, endocarditis, clarithromycin,
water source, pericarditis with or without
air conditioner; rifampin
community
acquired or
nosocomial
Adapted from Jawetz et al., Medical Microbiology 26th edition, 2014

Streptococcus pneumoniae
Also called pneumococci, streptococcus pneumoniae are gram positive, encapsulated, lancet
shaped diplococci. They are alpha hemolytic (cause partial hemolysis in culture) and are normal
inhabitants of the upper respiratory tract of 5%–40% of humans and are transient flora of the
nasopharynx. They produce disease through their ability to multiply in the tissues. The main
virulence factor is the capsule which is anti phagocytic.

Figure 17.3 Gram stain of S. pneumonia exhibiting

typical lancet shaped diplococci, with surrounding
"halos" representing capsules which demonstrate the
Neufeld Quellung reaction

Mode of Transmission
Pneumococci is mainly transmitted through droplet respiratory secretions. Nasopharyngeal
carriers serve as source of infection in 10% of cases
 Infections of the Respiratory Tract 30

Clinical Findings
The disease begins with abrupt onset of fever and chills, cough, and pleuritic chest pain.
The sputum is red or brown (“rusty”) in color. From the respiratory tract, the organism may
reach other sites. The middle ear and sinuses are the most frequently involved causing sinusitis
and otitis media. The infection may also spread from the mastoid to the meninges. The disease
is terminated promptly if antimicrobial therapy is given early.

Laboratory Diagnosis
1. Gram stain and microscopic examination of sputum
2. Blood and sputum culture
3. Capsular swelling test (Quellung reaction)
4. Optochin sensitivity
Treatment and Prevention
The recommended drug is penicillin G, alternative drugs are ceftizoxime and vancomycin.
Primary prevention consists of administration of vaccine, especially to high risk individuals
such as the elderly and those who do not have a spleen. Other preventive measures include
avoidance of risk factors (e.g., upper respiratory tract infection, alcohol or drug intoxication,
malnutrition), establishment of early diagnosis, and early administration of antimicrobial agents.

Haemophilus influenzae
The term “haemophilus” means blood loving and is attributed to the organism’s
requirement for enriched media, usually containing blood for isolation. H. influenzae is found
on the mucous membranes of the upper respiratory tract in humans. The most virulent and
invasive strain is the encapsulated strain (H. influenzae type b). Most infections occur in
children between the ages of 6 months to 6 years.

Mode of Transmission
The organism enters the body through the upper respiratory tract. Humans are the
only reservoirs.

Clinical Findings
1. Sinusitis and otitis media – H. influenzae is second only to pneumococci as the most
common cause of bacterial sinusitis and otitis media.
302 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. Epiglottitis – an inflammation of the epiglottis which can cause severe, life threatening
disease in young children due to airway obstruction, is almost exclusively caused
by H. influenzae.

3. Meningitis – the microorganisms are usually carried to the meninges by way of the
bloodstream. Prior to the use of vaccine, H. influenzae used to be the most common cause
of bacterial meningitis in children aged 5 months to 5 years. The rapid onset of fever,
headache, and stiff neck along with drowsiness is typical.
4. Bronchitis and pneumonia – these are commonly seen in elderly adults, especially those
with chronic respiratory disease.

Laboratory Diagnosis
Diagnosis is based on microscopic examination of Gram stained specimen. Definitive
diagnosis is based on culture. Specimen used consist of nasopharyngeal swabs, pus, blood, and
spinal fluid.

Treatment and Prevention

Ampicillin is the drug of choice for strains that do not produce beta lactamase. Essentially
all strains are susceptible to the newer cephalosporins. Primary prevention is through
administration of Haemophilus influenzae type b conjugate vaccine (Hib vaccine) to children.

Mycoplasma pneumoniae
Mycoplasmas are the smallest free living organisms that can self replicate in laboratory
media. The most unique characteristic of the organism is the lack of a cell wall. Its cell
membrane contains sterol. The absence of cell wall makes it resistant to cell wall inhibitor
antibiotics like penicillins, vancomycin, and cephalosporins. They are part of the normal flora of
the mouth.

Mode of Transmission
Transmission of mycoplasma may be done person to person by means of infected
respiratory secretions.

Clinical Findings
M. pneumoniae is the most common cause of atypical pneumonia or walking pneumonia.
Infected individuals are usually exposed to asymptomatic carriers. The infection may present as
tracheobronchitis with low grade fever, pharyngitis, malaise, and nonproductive cough
 Infections of the Respiratory Tract 30

Laboratory Diagnosis
Gram stains are of no value. Diagnosis is not made by culture since it will reveal only
normal flora. Serologic testing is the mainstay of diagnosis.

Treatment and Prevention

The drug of choice is either a macrolide (erythromycin or azithromycin) or a tetracycline
(e.g., doxycycline). There is no vaccine or other specific preventive measures.

Klebsiella pneumoniae
The organism is usually an opportunistic pathogen that causes community acquired
or nosocomial infections. K. pneumoniae is frequently found in the large intestine but is also
present in soil and water. The organism has a very large capsule (anti phagocytic). Patients
who develop infection are usually elderly patients, diabetics, alcoholics, and those with chronic
respiratory tract disease.

Clinical Findings
The organism produces a primary lobar pneumonia that is characterized by production of
thick, bloody sputum (“currant jelly” sputum). Necrosis and abscess formation are common.

Laboratory Diagnosis
Culture using MacConkey’s agar or EMB will differentiate it from the other members of
the family. Biochemical tests also further lead to a diagnosis.

Treatment and Prevention

The organism is highly resistant to a lot of antimicrobial drugs. The choice of drug depends
on the results of sensitivity testing. Preventive measures include changing the site of intravenous
catheters, removing urinary catheters when no longer needed, and proper care of respiratory
therapy devices. There is no vaccine available to prevent infection.

Legionella pneumophila
Legionellae are gram positive rods that stain poorly with the standard Gram stain although
they have a gram negative type of cell wall. It causes disease both in the community and in
hospitalized immunocompromisedpatients. It can survive inside macrophages and alveolar cells
enabling it to escape immune detection. The major virulence factor is lipopolysaccharide (LPS).
304 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Mode of Transmission
The organism is associated chiefly with environmental water sources such as lakes and
streams, air conditioners, and water cooling towers. Outbreaks of pneumonia in hospitals have
been attributed to the presence of the organism in water taps, sinks, and showers.

Clinical Findings
1. Pontiac Fever – a mild flu like form of infection that does not result in pneumonia. It has
an abrupt onset but resolves completely in less than one week.
2. Legionnaire’s Disease (Legionellosis) – also considered as an atypical type of pneumonia
and is characterized by very high fever and severe pneumonia accompanied by mental
confusion and non bloody diarrhea. This can be fatal in previously healthy persons but
have higher morbidity in immunocompromisedpatients.

Laboratory Diagnosis
L. pneumophila stains poorly with Gram stain but can be demonstrated in infected tissues
stained with Dieterle silver stain. Diagnosis depends on a significant increase in antibody titer
by the indirect immunofluorescence assay.

Treatment and Prevention

L. pneumophila is susceptible to macrolides (azithromycin or erythromycin) or
fluoroquinolones. Beta lactam antibiotics are ineffective because L. pneumophila produces beta
lactamase. Prevention involves reduction of cigarette and alcohol consumption, elimination of
aerosols from water sources, and use of high temperatures and hyperchlorination of hospital
water supplies.

Staphylococcus aureus
S. aureus pneumonia can occur in post operative patients or following viral respiratory tract
infections, especially influenza. It frequently leads to empyema (lung abscess). It is the most
common cause of nosocomial pneumonia in hospital settings.

Pseudomonas aeruginosa
P. aeruginosa is another major cause of nosocomial pneumonia. Involvement of the
respiratory tract, especially from contaminated respirators, results in necrotizing pneumonia
 Infections of the Respiratory Tract 30

Pertussis (Whooping Cough)

Bordetella pertussis
The organism is a small, encapsulated, gram negative rod. It is a pathogen that only
naturally infects humans. Its virulence can be attributed to the various toxins it produces that
are responsible for tissue destruction and manifestations of pertussis.

Mode of Transmission
The organism is transmitted by airborne droplets during the severe coughing episodes.

Clinical Findings
Pertussis is a highly contagious disease. It occurs primarily in infants and young children.
Pertussis consists of three stages, namely:
1. Catarrhal stage – this is the most contagious stage and lasts 1–2 weeks. It manifests
as a mild upper respiratory tract infection with non specific signs and symptoms.
The greatest number of microorganisms is produced during this stage.
2. Paroxysmal stage – this stage is characterized by a series of 5–20 forceful, hacking coughs
accompanied by production of copious amounts of mucus that ends in a high pitched
indrawn breath that makes the “whoop” noise, hence the term whooping cough. During
paroxysms, the patient may turn cyanotic, the tongue protrudes, the eyes bulge,
and neck veins engorge. This may last for 2–10 weeks.
3. Convalescent stage – this stage is characterized by a reduction in the symptoms of the
patient leading to recovery. The patient is no longer contagious.

Complications
Pertussis, like measles, can unmask underlying tuberculosis. Convulsions may occur due to
cerebral anoxia during coughing spells. Blindness can also develop resulting from hemorrhages
into the conjunctiva during paroxysms. Pneumonia, deafness, and hernias may also develop.

Laboratory Diagnosis
Diagnosis is done through the culture of specimens from nasopharyngeal swabs taken
during the paroxysmal stage is diagnostic. The culture medium used before was Bordet Gengou
medium demonstrating the “fried egg” appearance of the colonies but this has been replaced
by Regan Lowe charcoal medium.
306 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Treatment and Prevention

The drugs of choice are the macrolides. It is given not only to the patient but also to
exposed individuals. Administration of the drug during the paroxysmal stage will not alter the
course of the disease. Supportive care such as oxygen therapy and suctioning of mucus during
the paroxysmal stage is important. An acellular vaccine helps prevent infection.

Tuberculosis
Mycobacterium tuberculosis
M. tuberculosis is the main cause of tuberculosis globally. M. bovis causes tuberculosis in
cattle and other animals as well as humans. In AIDS patients, atypical tuberculosis occurs
which is caused by M. avium intracellulare complex. M. africanum is the major cause in Africa.
M. tuberculosis is an acid fast, obligately aerobic bacillus that is stained poorly by the dyes
used in Gram stain. Its cell wall contains complex lipids, one of which is mycolic acid, which
contributes to the acid fastness of the organism.

Mode of Transmission
The major mode of transmission is by person to person spread through respiratory
aerosols generated through coughing by infected individuals. A rare mode of transmission is
through killing. M. bovis is transmitted through ingestion of contaminated cow’s milk leading
to development of gastrointestinal tuberculosis. The organism may survive in fomites such as
utensils and glassware.

Clinical Findings
1. Primary infection (Primary Complex) – represents initial infection in childhood. It may
affect any part of the lung but most commonly involves the middle and lower lobes of the
lungs. The lesion is called Ghon complex. Most patients are asymptomatic.
2. Secondary or Reactivation Pulmonary Tuberculosis – usually caused by tubercle bacilli that
have survived in the primary lesion. It almost always begins at the apex of the lung, where
the oxygen tension is highest. The classical symptoms include easy fatigability, afternoon
rises in temperature, weight loss, night sweats, loss of appetite, chronic non productive
cough with or without hemoptysis.
3. Disseminated Tuberculosis – also called extrapulmonary tuberculosis. It is characterized
by multiple disseminated lesions. The most common initial organ involved in
extrapulmonary tuberculosis is the lymph nodes. In some instances, the involved
lymph nodes may aggregate and ulcerate forming what is called as scrofula
 Infections of the Respiratory Tract 307

Tuberculous meningitis and tuberculous osteomyelitis (Pott’s disease) are important

disseminated forms. Other disseminated forms: gastrointestinal, oropharyngeal, renal,
genitourinary TB, and pericardial TB.

a b

Figure 17.4 a Chest x ray findings of patient with tuberculosis and b shows the bacillus after
staining with Ziehl Neelsen method

Laboratory Diagnosis
1. Acid fast staining of sputum: requires collecting early morning sputum with adequate
amount of inoculum and must be collected on the day of consultation with the physician
followed by another collection 1 hour after. If the patient cannot wait for the second
collection, the patient is made to come back the following day.
2. Culture using Lowenstein Jensen medium not usually done because the organism is a slow
grower.

3. Chest x ray
4. Skin test
• Tuberculin Skin Test using purified protein derivative (PPD) as antigen
• Method: Mantoux (intradermal test)
• The skin test is evaluated by measuring the diameter of the induration (thickening)
surrounding the skin test and not by simply observing for the presence of erythema.
• Aanpositive skin test result indicates previous infection by the organism or exposure to
active case but not necessarily active disease.

• Skin test is considered positive in the following:

a. induration of 15 mm or more in a person with no known risk factors
b. induration of 10 mm or more in a person with high risk factors (e.g., homeless
person, IV drug users, or nursing home residents)
c. induration of 5 mm or more in a person with deficient cell mediated immunity
(e.g., AIDS patients
308 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

• The test becomes positive 4–6 weeks after infection. Immunization with BCG may
cause a positive test, but the reactions are usually only 5–10 mm and tend to decrease
with time. People with skin test result of 15 mm or more are assumed to be infected
with M. tuberculosis even if they have received the BCG vaccine.

Treatment and Prevention

Multidrug anti Koch’s or anti TB therapy is used to prevent emergence of drug resistance.
WHO recommended a Direct Observed Treatment Short Course (DOTS) program aimed at
preventing development of drug resistance and to reduce morbidity and mortality from TB.
Preventive measures include: (1) improvement of housing conditions and nutritional
status of the population; (2) prompt identification and adequate treatment of patients;
(3) careful follow up of contacts of patients with active TB (e.g., tuberculin tests, x rays,
sputum examination); (4) use of masks and other respiratory isolation procedures to prevent
spread to medical personnel; public health education; (5) eradication of tuberculosis in cattle
and pasteurization of milk; and (6) immunization with BCG vaccine.

Pulmonary Anthrax
Pulmonary or inhalation anthrax is also called Woolsorter’s disease and is transmitted by
inhalation of spores of Bacillus anthracis into the lungs. It begins with non specific symptoms
that resemble influenza which rapidly progresses to edema, enlargement of mediastinal lymph
nodes, bloody pleural effusion, septic shock, and death. Chest x ray would show widening of
the mediastinum due to enlarged lymph nodes. Hemorrhagic meningitis and hemorrhagic
mediastinitis are severe life threatening complications. The drug of choice is ciprofloxacin with
doxycycline as an alternative drug
 Infections of the Respiratory Tract 309

CHAPTER SUMMARY

• Respiratory tract infections can be caused by bacteria, viruses, and fungi and can be
transmitted from person to person either direct or indirect contact.
by

• Upper respiratory tract infections include common cold, pharyngitis, croup, tonsillitis,
diphtheria, and otitis media and externa.

• Lower respiratory tract infections are usually more severe than URTI and include
pneumonia, pertussis, tuberculosis, and anthrax.

• Common cold is the most common respiratory tract infection worldwide and most
commonly caused by Rhinovirus.

• Infections that are toxin mediated are diphtheria, pertussis, and scarlet fever.

• Influenza is caused by Influenza virus under the family of Orthomyxoviruses. There are
3 types: Influenza A, and C. Influenza A is associated with pandemics.
B,

• Coronavirus is the second most common cause of common cold but has evolved and
caused severe acute respiratory syndrome worldwide.

• Atypical pneumonia or “walking pneumonia” is caused by other bacteria that are not
common causes of pneumonia. It cannot be stained Gram stain and does not grow in
by
cultures using common media.

• The most common cause of atypical pneumonia is Mycoplasma pneumoniae.

• Legionnaires disease is associated with environmental water sources such as lakes and
streams, air conditioners, and water cooling towers.

• examination
Viral pneumonia is characterized inflammation of the interstitial spaces and physical
by
may only reveal rales on auscultation. Among the common causes of viral
pneumonia are respiratory syncytial virus, parainfluenza viruses, influenza viruses,
adenoviruses, coronaviruses, measles virus, and varicella zoster virus.

• Infections discussed in this chapter are preventable by immunization like Pneumococcal

pneumonia, pertussis, pulmonary anthrax, diphtheria, and H. influenza.

• BCG, the vaccine for M. tuberculosis, does not confer complete protection
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 Infections of the Respiratory Tract 311

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Case: A 7 year old child was brought to the ER because of fever and erythematous, generalized
rashes. The condition started 3 days prior to admission as moderate to high grade fever with
hoarseness and pain in swallowing. One day prior to admission, the patient, still with fever,
developed the rashes. On examination of the throat, there is tonsillopharyngeal congestion.
The tongue is erythematous with prominent pale taste buds resembling a strawberry.

1. The most probable diagnosis is:

a. Acute tonsillopharyngitis c. Oral Candidiasis
b. Scarlet fever d. Herpangina
2. The most probable etiologic agent is:
a. Staphylococcus aureus c. Candida albicans
b. Streptococcus pyogenes d. Coxsackie A virus
3. The toxin responsible for the manifestations of this disease is:
a. Erythrogenic toxin c. Exfoliative toxin
b. TSST 2 d. Dermotoxi

Multiple Choice.

1. Which of the following is/are correct regarding Bacillus anthracis?

a. Gram negative bacilli c. Obligate anaerobes
b. Produce endospores d. All of the above
2. This organism, to become pathogenic, must be lysogenized by the tox+ gene:
a. Corynebacterium diphtheriae c. Mycoplasma pneumoniae
b. Haemophilus influenzae d. Mycobacterium tuberculosis
312 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

3. This organism is the most common cause of Walking Pneumonia.

a. Staphylococcus aureus c. Legionella pneumophila
b. Mycoplasma pneumoniae d. Bacillus anthracis
4. Haemophilus influenza type that is most virulent and associated with the most
severe infection.
a. Type A c. Type C
b. Type B d. Type D
5. Which of the following organisms do not have a cell wall?
a. Neisseria meningitidis c. Mycoplasma pneumoniae
b. Corynebacterium diphtheriae d. Legionella pneumophila
6. Most lesions of pulmonary tuberculosis are located in the apices of the lungs.
This can be explained by which of the following?
a. Mycobacterium tuberculosis is acid fast
b. Mycobacterium tuberculosis is a strict aerobe
c. Mycobacterium tuberculosis is transmitted through inhalation
d. All of the above
7. Which of the following is a characteristic manifestation of pertussis?
a. Rusty colored sputum
b. Enlarged mediastinal lymph nodes
c. Seal bark cough
d. Inspiratory whoop
8. Pseudomembrane and “bull neck” appearance are characteristics of:
a. Diphtheria c. Pulmonary tuberculosis
b. Anthrax d. Waterhouse Friderichsen syndrome
9. Tuberculosis can be acquired through:
a. Droplet means c. Fomite
b. Ingestion d. All of the above
10. Correct statement regarding tuberculin skin test for Mycobacterium tuberculosis.
a. It is a test for exposure
b. It is a test for pathogenicity
c. It is a test for immunity
d. It is a confirmatory test for tuberculosi
 CHAPTER
Infections of
18 Gastrointestinal Tract

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. recognize common gastrointestinal tract infections based on clinical manifestations;
2. differentiate invasive from non invasive diarrhea;
3. compare the five hepatitis infections;
4. describe the characteristics of the causative organisms of each gastrointestinal tract
infection;
5. explain how the different gastrointestinal tract infections are transmitted; and
6. discuss the appropriate laboratory diagnosis, treatment, and prevention of each infection.

Definitions of Terms
1. Gastritis – inflammation of the mucosal lining of the stomach
2. Enteritis – inflammation of the small intestines
3. Colitis – inflammation of the colon (large intestines)
4. Gastroenteritis – inflammation of the mucosal lining of the stomach and intestine
5. Hepatitis – inflammation of the liver
6. Dysentery – low volume, painful, bloody diarrhe
314 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The digestive system is inhabited by many microorganisms. From the mouth down to
the colon, these different ecosystems are occupied by site specific microbial populations.
The stomach, due to its acidity, acts as an effective sterilization chamber that limits the entry
of microorganisms to the small intestines. Infections of the digestive system range from
asymptomatic infections to life threatening loss of fluids and electrolytes, or severe ulceration
accompanied by intestinal perforation and hemorrhage. The clinical manifestations vary from
one another.

Establishmentof Infectious Disease

in the Digestive System
Several factors play important roles in the causation of infectious disease in the digestive
system. The most crucial and important impediment to infective agents is an intact mucosal
epithelium covering all parts of the digestive system. Early damage to the lining may lead to
manifestations such as nausea and vomiting. Damage to the mucosa in the form of ulcerations
allow the members of the normal flora to penetrate the deep tissue and disseminate through the
bloodstream to other organs. Alteration of the acid barrier of the stomach by disease, surgery
or drugs increases the survival of pathogens leading to infection. Changes in the composition
of the normal flora allow the growth of pathogenic organisms. Certain bacteria, viruses, and
parasites can cause disease even in the absence of predisposing factors due to their ability to
produce virulence factors (e.g., toxin production).
Signs and symptoms of infections in the gastrointestinal tract are produced in several ways.
These mechanisms include:
1. Pharmacologic action
Some bacteria produce toxins which may alter normal intestinal function without
causing lasting damage to their target cells. For example, the toxin produced by Vibrio
cholerae produces voluminous, watery diarrhea due to its ability to induce increased
electrolyte secretion into the intestines.
2. Local inflammation
Invasion of the alimentary tract by microbes can lead to inflammation. Invasion
is usually limited to the epithelial layer but may spread to the deeper tissues. In the
mouth, the gums are usually affected causing periodontitis. In the intestines, infections
can cause inflammation that can result to dysentery.
3. Deep tissue invasion
Certain organisms are able to spread to adjacent tissues and enter the bloodstream.
For example, the parasite Strongyloides is capable of burrowing through the intestinal
wall. The parasite is often colonized by gut bacteria and as a result, infection by this
worm can lead to polymicrobial septicemia
 Infections of Gastrointestinal Tract 31

4. Perforation
When the mucosal epithelium is perforated, the normal flora spills into sterile
areas and invades deep tissues, often with serious consequence. For example,
perforation of an inflamed appendix can lead to peritonitis.

Mouth
Dental Caries (Tooth Decay)
Unlike other exterior surfaces, the teeth are hard and do not shed surface cells allowing
accumulation of masses of microorganisms and their products. These accumulations are
called dental plaques and are involved in the formation of dental caries or tooth decay.
Older, calcified deposits of plaque are called dental calculus or tartar. The most important
organism that causes dental caries is Streptococcus mutans, although other microorganisms may
also be involved (e.g., Actinomyces, Lactobacilli). S. mutans favors crevices or other sites on the
teeth that are protected from the shearing action of chewing or from the flushing action of
saliva. The lactic acid produced by the bacteria is not diluted or neutralized by saliva, and this
breaks down the enamel of the teeth, leading to localized softening of the external enamel.
If the initial penetration of the enamel by caries remains untreated, bacteria can penetrate
the interior of the tooth eventually advancing into the pulp of the tooth.
Preventive measures against the development of dental caries include minimal ingestion
of sucrose, brushing, and flossing, regular dental visits to remove plaque, and the use of fluoride.
The use of mouthwash may be effective, with chlorhexidine being the most effective.

Periodontal Disease
Periodontal disease is a term used to describe conditions that are characterized by
inflammation and degeneration of structures that support the teeth. Gingivitis is the reversible
inflammation of the gingivae or gums, characterized by bleeding of the gums while brushing
the teeth. This is due to overgrowth of supra gingival plaque causing irritation to the tissues of
the gums. Organisms involved are varied and include streptococci, actinomycetes, and anaerobic
gram negative bacteria (Prevotella, Bacteroides, and Fusobacterium nucleatum).
Periodontitis is a chronic gum disease that can cause bone destruction and tooth loss.
It generally causes little discomfort. The gums are inflamed and bleed easily. As the infection
progresses, the bone and tissue that supports the teeth are destroyed, leading to loosening and
loss of teeth. Numerous bacteria may be involved in the infection.
Acute necrotizing ulcerative gingivitis or Vincent’s disease or trench mouth is also
another common serious infection of the mouth. It is characterized by pain that prevents
316 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

normal chewing and may be accompanied by bad breath or halitosis. The most common
organism involved is Prevotella intermedia. The condition is treated by adequate debridement,
oxidizing agents, and administration of metronidazole.

Oral Thrush
The organism that causes this condition is Candida albicans, a fungus that is part of the
normal flora of the skin, mucous membranes, and gastrointestinal tract. The condition consists
of white patches adherent to the oral mucosa and may occur on the tongue, lips, gums, or palate.
Factors that predispose the development of oral thrush include endocrine disturbances
(e.g., diabetes), prolonged intake of antibiotics, malnutrition, malignancy, immunosuppression,
and prolonged use of steroids. Diagnosis can usually be made by inspection and confirmed
by examination of scraped material under the microscope demonstrating the characteristic
pseudohyphae. Treatment consists primarily of correcting the predisposing factor and
avoiding unnecessary use of antibiotics. Topical antifungal agents may be used. The drug of
choice is nystatin.

Mumps (Epidemic Parotitis)

Mumps is a highly communicable disease characterized by painful inflammation of
the salivary glands. This infection is caused by the mumps virus, a member of the family
Paramyxoviridae and there is only one serotype. Humans are the natural hosts. The disease
occurs worldwide, affecting people who have not been vaccinated before the age of 15. Lifelong
immunity occurs in persons who have had the disease.

Mode of Transmission
Mumps virus is transmitted via respiratory droplets.

Clinical Findings
The virus infects salivary glands, with the parotid
glands predominantly infected. After an incubation period
of 18–21 days, the patient develops fever, malaise, and
anorexia followed by tender swelling of the parotid glands
and/or other salivary glands. Involvement can be unilateral
or bilateral. A characteristic increase in parotid pain Figure 18.1 Epidemic parotitis
develops, especially when drinking citrus juices. The disease or mumps showing unilateral
is benign and resolves spontaneously within a week. enlargement of the parotid glan
 Infections of Gastrointestinal Tract 31

Complications
There are two significant complications—orchitis and meningitis. Orchitis is inflammation
of the testis. This complication is significant if it occurs in post pubertal males and if the
involvement is bilateral. Bilateral orchitis can lead to sterility. Meningitis is usually benign
and self limited.

Laboratory Diagnosis
Diagnosis is usually based on clinical manifestations. Virus isolation from saliva, spinal
fluid, or urine can be done. Measurement of antibody titers can also be made.

Treatment and Prevention

Treatment is mainly supportive. Prevention consists of immunization with the live,
attenuated vaccine (MMR) given at 15 months of age. Administration of immune globulin
does not prevent development of orchitis.

Stomach
The major pathologies involving the stomach are inflammatory in nature and consist
of two conditions—gastritis and peptic ulcer disease. The predominant organism involved is
Helicobacter pylori, the most common cause of chronic gastritis and peptic ulcers (gastric and
duodenal). It has two major virulence factors: rapid motility and urease production. Its rapid
motility enables it to penetrate the mucus blanket lining the stomach. Urease produced
by the organism leads to production of large amounts of ammonia from urea that leads to
neutralization of gastric acid.
The natural habitat of the organism is the stomach but it may also be found in saliva.
It is likely acquired through ingestion and person to person transmission may also occur.
Infection with H. pylori is a risk factor for gastric carcinoma and MALT (mucosa associated
lymphoid tissue) B cell lymphomas.

Clinical Findings
Gastritis and peptic ulcer disease are characterized by recurrent pain in the upper
abdomen, specifically around the epigastric area. This may be complicated by bleeding into
the gastrointestinal tract. The acute symptoms may last for less than one week until about
two weeks, however, the infection can persist for years.
318 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Laboratory Diagnosis
Gastric biopsy specimens can be used for histologic examination. Culture can also be done
as well as measurement of antibody levels specific for H. pylori.

Treatment
Treatment involves triple therapy with proton pump inhibitor (omeprazole), macrolide
(clarithromycin) and amoxicillin for 7 to 10 days. Proton pump inhibitors directly inhibit
H. pylori.

Liver
Inflammation of the liver is termed hepatitis. Hepatitis can be caused by varied organisms
such as bacteria, viruses, and parasites. The most important causes of hepatitis are the Hepatitis
viruses. Cytomegalovirus, Epstein Barr virus, Herpes Simplex virus, and Rubella virus.
Enteroviruses, Dengue virus, and the Yellow Fever virus are associated with sporadic hepatitis.
The clinical findings of hepatitis are virtually the same, regardless of which hepatitis virus is
the cause. Typical signs and symptoms include fever, anorexia, nausea, vomiting, and jaundice.
Dark urine and pale feces are also observed. Most cases resolve spontaneously in 2–4 weeks.
Table 18.1 provides a summary of important clinical features of the Hepatitis viruses.

Hepatitis A Virus (HAV)

HAV causes infectious hepatitis. There is only one serotype of Hepatitis A virus. It is
transmitted mainly by the fecal oral route. Sexual transmission is possible but rare. The virus
appears in the feces two weeks before the appearance of symptoms. Children are the most
frequently infected, and outbreaks may occur. The common source of outbreaks arise from
water and food contaminated with feces.
HAV has a short incubation period (3–4 weeks) and most patients with HAV infection
are asymptomatic. Fulminating infection may occur in a small number of patients. No chronic
hepatitis or chronic carrier state occurs. There is no predisposition to hepatocellular carcinoma
or cancer of the liver.
Diagnosis is confirmed by detection of antibodies directed against the virus. An increase
in anti HAV IgM antibody signifies active infection while the presence of anti HAV IgG
antibody suggests previous infection.
Treatment is mainly supportive. Active immunization with a vaccine containing inactivated
HAV is available. Two doses are given—an initial dose followed by a booster dose 6–12 months
later. It is recommended for those travelling to developing countries, children ages 2–18 years
 Infections of Gastrointestinal Tract 31

and sexually active individuals. Passive immunization can be given to individuals exposed
to infected patients and is given 14 days after exposure. Other preventive measures include
observing proper hygiene such as proper sewage disposal and thorough hand washing after
bowel movement. The virus can be inactivated by heating food for at least 1 minute to above
85 °C and disinfecting surfaces.

Hepatitis B Virus (HBV)

HBV causes serum hepatitis. It is the only DNA virus among the hepatitis viruses.
The three main modes of transmission are through blood, during sexual intercourse, and from
mother to newborn during birth or breast feeding. The most efficient method of transmitting
the virus is through injection into the bloodstream. It is found worldwide.
Majority of patients are asymptomatic, however, present symptoms are more severe than
hepatitis A infection. Hepatitis B can also present with extrahepatic manifestations like
polyarthritis, polyarteritis nodosa, rashes and glomerulonephritis. Fulminant infection can occur
as well as chronic hepatitis which can lead to cirrhosis and death. Some patients can become
carriers, especially infants born to hepatitis positive mothers. HBV is also associated with the
development of hepatocellular carcinoma.
Diagnosis can be confirmed by means of serology. Treatment is mainly supportive although
interferon alpha can be useful for the treatment of chronic infection. Prevention involves the
use of either the vaccine or hyper immune globulin or both. All blood for transfusion should be
screened for HBV.

Hepatitis C Virus (HCV)

HCV causes parenteral hepatitis. It is the predominant cause of non A, non B (NANB)
hepatitis worldwide and the most common cause of post transfusion hepatitis among IV drug
abusers. Humans are the reservoir for the virus and is transmitted primarily via blood.
At present, injection drug use accounts for almost all new HCV infections. Sexual
transmission and transmission from mother to child may occur. In addition, HCV has been
found to occur as a co infection with HIV. Approximately 90% of patients with HIV infection
are also positive for HCV RNA.
HCV primarily infects hepatocytes and remains inside the hepatocytes throughout. As a
result, chronic and persistent hepatitis is the hallmark of infection. It resembles HBV as far as
the ensuing chronic liver disease, cirrhosis, and the predisposition to hepatocellular carcinoma
are concerned. Alcoholism greatly enhances the development of hepatocellular carcinoma
in HCV infected individuals.
320 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Diagnosis is confirmed by detecting antibodies to HCV. A combination of interferon

alpha and ribavirin is the treatment of choice for chronic hepatitis C. Preventive measures
includes proper screening of blood products prior to transfusion. Reducing or completely
avoiding consumption of alcoholic beverages also reduces the risk of developing hepatocellular
carcinoma and cirrhosis.

Hepatitis D Virus (HDV/Delta Virus)

HDV causes delta hepatitis. It is an unusual virus in that it is considered as a defective
virus for it cannot replicate on its own and can only do so in cells also infected with HBV.
As such, it is considered as a viral parasite. It uses the surface antigen of HBV as its envelope.
It is therefore transmitted by the same means as HBV.
HDV can occur as co infection and superinfection. In co infection, the patient is infected
with both HDV and HBV at the same time. In a superinfection, a person previously infected
with HBV (carrier) becomes infected with HDV. Acute hepatitis resulting from a co infection
is more severe than in those infected with HBV alone. Hepatitis in chronic carriers of HBV
who become superinfected with HDV is more severe with greater incidence of fulminating
hepatitis, chronic hepatitis, and liver failure.
Diagnosis rests on detection of either delta antigen or antibody to delta antigen in the
patient’s serum. Administration of alpha interferon may minimize some of the effects of
chronic hepatitis, however, it does not eradicate the chronic carrier state. Vaccination for
hepatitis B may prevent the occurrence of delta infection because HDV cannot replicate unless
HBV infection also occurs.

Hepatitis E Virus (HEV)

HEV is the major cause of enteric hepatitis. It is transmitted purely through the fecal oral
route. Clinical manifestations are similar to hepatitis A, with the exception of a high mortality
rate in pregnant women. There is no chronic hepatitis or a prolonged carrier state. Diagnosis
is made by excluding HAV and other causes. There is no antiviral drug or vaccine available to
prevent infection.

Hepatitis G Virus (HGV/GB Virus C)

HGV is not primarily hepatotropic. The virus replicates within monocytes then finds its
way into the liver where it can cause a chronic type of hepatitis. It is transmitted through sexual
intercourse and blood transfusion and may occur as a co infection with HIV. It has been noted
that patients co infected with HIV and HGV have lower mortality rate and have less HIV in
their blood than those infected with HIV alone. In vitro studies have shown that HGV may
interfere with the replication of HIV
 Infections of Gastrointestinal Tract 321

Table 18.1 Comparison of features of hepatitis viruses

Feature
Common
A
Hepatitis
“Infectious”
Hepatitis
“Serum”
B Hepatitis
“Non A,
C Hepatitis
“Delta
D
agent”
Hepatitis
“Enteric
E

name non B post non A,

transfusion” non B
Virus Picornavirus; Hepadnavirus; Flavivirus; Viroid like; Hepevirus;
structure capsid RNA envelope, DNA envelope, RNA envelope, Calicivirus
circular RNA like capsid
Transmission Fecal oral Parenteral, Parenteral, Parenteral, Fecal oral
sexual, sexual, sexual
insidious insidious
Incubation 15–50 45–160 14–180+ 15–64 15–50
period (days)
Severity Mild Occasionally Usually sub Co infection Normal
severe clinical; 70% or super patients,
chronicity infection with mild;
HBV pregnant
women,
severe
Chronicity/ No Yes Yes Yes No
Carrier state
Other None Primary Primary Cirrhosis, None
disease hepato cellular hepato cellular fulminant
associations carcinoma, carcinoma, hepatitis
cirrhosis cirrhosis
Laboratory Symptoms and Symptoms and Symptoms Anti HDV —
diagnosis anti HAV IgM serum levels and anti HCV ELISA
of HBsAg, ELISA
HBeAg, and
anti HBc IgM

Small and Large Intestines

Diarrhea
In most cases, diarrhea is defined as a change in the normal bowel habits of an individual,
with an increase in the frequency, fluidity, looseness, and volume of feces excreted per day in
comparison to the usual fecal output of the individual. In a normal adult, the average daily
stool weight is less than 200 grams, of which 65% to 85% is water. Diarrhea implies daily
stool production in excess of 250 grams, containing 70% to 95% water. It is the final common
pathway of intestinal responses to many inciting agents and serves as an adaptive mechanism
developed by the body to get rid of noxious material
322 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Depending on the causative agent, diarrhea may be classified as invasive or non invasive.
Non invasive diarrhea is the result of disruption of the secretory process due to the toxin
released from the bacteria. This is characterized by watery diarrhea and the absence of blood
or leukocytes in the feces. In invasive diarrhea there is direct damage to the gastrointestinal
tissues due to direct invasion by the bacteria. It is characterized by fever, dysentery (blood in
stools), and leukocytes in the feces. Table 18.2 summarizes the major differences between the
two types.

Table 18.2 Differences between invasive and non invasive types of diarrhea
Non invasive Diarrhea Invasive Diarrhea
Characteristic of stool Profuse, secretory Dysenteric (blood, mucus,
(severe, watery) white blood cells)
Fever No Yes
Systemic toxicity No Yes
Abdominal pain Mild Severe (cramping; tenesmus)
Site of infection Small intestine Colon (large intestine)
Adapted from Lecture Guide in Microbiology, Department of Microbiology, College of Medicine, Our Lady of Fatima
University, 2018

Children and the elderly are more susceptible to dehydration from diarrhea. Rehydration
or replacement of the fluid and electrolyte lost is necessary for the management of any form of
diarrhea. Fluid replacement can be done by drinking more fluids or oral rehydration solution
(ORS) or through IV depending on the severity of the dehydration. Some suggest giving of
zinc supplement reduces the severity of diarrhea. A new concept in the management of diarrhea
is the use of probiotics. Some claim that probiotics are helpful in preventing traveler’s diarrhea in
children and antibiotic associated diarrhea.
Education of the public is vital in reducing the incidence of diarrhea. Preventive measures
include having a proper waste water and sewage disposal system, maintenance of clean and safe
food sources and drinking water, and good hygienic practices. In addition, studies have shown
that breastfeeding for the first six months after birth is effective in preventing diarrhea in
newborns and infants.

Mode of Transmission
The most common mode of transmission is fecal oral transmission. This includes
(1) person to person transmission, usually in association with overcrowding and poor
personal hygiene, (2) ingestion of contaminated meat, poultry products or seafood, and
(3) contamination of food during or after cooking
 Infections of Gastrointestinal Tract 32

Viral Gastroenteritis
Acute, self limited infectious diarrhea which usually involves children, is most commonly
caused by enteric viruses. It may cause severe dehydration requiring hospitalization, especially
in infants. Table 18.3 summarizes the common gastrointestinal viruses causing gastroenteritis.

Table 18.3 Common viruses associated with acute gastroenteritis in humans

Virus Host Age Mode of Transmission Epidemiology
Rotaviruses
Group A 6–24 months Person to person; water Single most important
cause of endemic severe
diarrheal illness in infants
and young children
Group B Adults and children Person to person; water Outbreaks of diarrhea in
China
Group C Children Person to person; water Sporadic and occasional
outbreaks
Enteric Child < 2 years Person to person Second most important viral
adenoviruses of age agent of endemic diarrhea
(Group F or in infants and young
serotypes 40 children worldwide
and 41)
Caliciviruses
Norovirus Older children and Person to person, water, Causes outbreaks of
adults cold foods, raw shellfish vomiting and diarrhea in
families, communities, and
institutions
Saporovirus Infants, young Sporadic and occasional
children, elderly outbreaks
Astroviruses Infants, young Person to person, water, Sporadic and occasional
children, elderly raw shellfish outbreaks
Adapted from Jawetz, Melnick & Adelberg’s Medical Microbiology 26th ed., McGraw Hill Education, 2013, p. 547

Rotavirus is the most common viral cause of gastroenteritis in children. Infants and young
children are most commonly affected although debilitated adults may also be susceptible.
It destroys mature enterocytes leading to loss of absorptive function of the small intestine with
net secretion of water and electrolytes. Outbreaks may occur in the pediatric population in
hospitals and day care centers. After an incubation period of approximately 2 days, vomiting
and watery diarrhea will occur for several days.
324 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Norwalk virus is responsible for majority of cases of non bacterial food borne epidemic
gastroenteritis in all age groups, most especially in adults. The virus causes epidemic
gastroenteritis with watery diarrhea, abdominal pain, nausea, and vomiting. Outbreaks may
occur following exposure of multiple individuals to a common source. Majority of outbreaks can
be seen in nursing homes.
There is no specific treatment for viral gastroenteritis. Management is mainly supportive
with adequate fluid and electrolyte replacement.

Bacterial Infections
Bacterial Enterocolitis (Food Poisoning)
A classic case of food poisoning has two important features: (1) similar symptoms occur in
several members of a group who shared the same meal, and (2) onset of symptoms occurs a few
hours after food ingestion. Food poisoning may occur due to either of three mechanisms:
1. Ingestion of preformed toxin – the preformed toxin may be present in contaminated
food. Major offenders are Staphylococcus aureus, Vibrio, and Clostridium perfringens.
Symptoms develop within hours consisting of explosive diarrhea and acute abdominal
pain.
2. Infection by toxigenic organisms – the organisms proliferate in the gut lumen and
elaborate an enterotoxin. Symptoms occur within hours consisting of diarrhea
and dehydration if it involves a secretory enterotoxin, or dysentery if the primary
mechanism is a cytotoxin.
3. Infection by enteroinvasive organism – the organisms proliferate, invade, and destroy
mucosal epithelial cells, leading to dysentery.
As a rule, the incubation period is less than 12 hours after ingestion of preformed toxins.
Longer incubation period indicates ingestion of live bacteria that must first proliferate before
producing the signs and symptoms of infection. Identification of the causative agent requires
isolation of the infectious agent or detection of the toxin in contaminated food. Management is
mainly supportive and in most cases, antibiotic therapy is not required. Table 18.4 summarizes
the important clinical and epidemiologic features of the more important and common
organisms that cause food poisoning
 Infections of Gastrointestinal Tract 325

Table 18.4 Clinical and epidemiologic features of food poisoning

Incubation
Organism Clinical presentation Characteristic foods
period (hours)
Bacillus cereus 1–6 Vomiting Re warmed fried rice
6–24 Watery diarrhea Meat, poultry, vegetable
Clostridium botulinum 12–17 Neuromuscular Canned foods, smoked fish,
paralysis unpasteurized honey
Staphylococcus aureus 2–4 Vomiting, diarrhea Meats, custard, salads
Vibrio parahaemolyticus 10–24 Watery diarrhea Shellfish
Clostridium perfringens 9–15 Watery diarrhea Meat, poultry
Adapted from Lecture Guide in Microbiology, Department of Microbiology, College of Medicine, Our Lady of Fatima
University

Bacillus cereus
The organism is a gram positive aerobic rod or bacillus. Bacillus cereus is mildly pathogenic
and of low virulence hence an opportunistic pathogen. Food poisoning caused by B. cereus
has two distinct forms: the emetic type, associated with fried rice, and the diarrheal type,
associated with meat dishes and sauces. The organism produces toxins that cause disease that is
more of an intoxication than a food borne infection.

Clinical Findings
The emetic form is manifested by nausea, vomiting, abdominal cramps, and occasionally
diarrhea. It is self limited with recovery occurring within 24 hours. It begins 1–5 hours after
ingestion of contaminated rice and occasionally pasta dishes. The diarrheal form has an
incubation period of 1–24 hours and is manifested by profuse diarrhea with abdominal pain and
cramps. Vomiting may occur but is uncommon. The enterotoxin may be preformed or produced
in the intestine.

Laboratory Diagnosis
Laboratory diagnostic procedures are usually not done, although isolation of the organism
from the suspected food samples followed by culture can be performed.

Treatment and Prevention

No antibiotic therapy is required since the infection is self limiting. Preventive measures
include preventing contamination of food by soil since the organism is usually found in soil.
Rice should also not be kept warm for long periods
326 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Staphylococcus aureus
S. aureus is an important cause of food poisoning and causes food poisoning with the
shortest incubation period (30 minutes to 8 hours, average of 2 hours). Enterotoxins are
produced when the organism grows in food rich in carbohydrates and protein.

Mode of Transmission
The major mode of transmission for staphylococcal food poisoning is ingestion of the
preformed heat stable toxin in contaminated food, especially salads, custards, milk products,
and processed meat. The bacteria can grow in high salt concentration hence its association with
processed meats. The food does not taste spoiled making it difficult to detect contamination.
The bacteria can be killed by reheating the food, however, it does not destroy the toxin.
The chief sources of infection are carriers and those individuals shedding human lesions,
fomites contaminated from such lesions, and the human respiratory tract and skin.

Clinical Findings
Vomiting accompanied by nausea is more prominent than diarrhea. The emetic effect is
probably the result of stimulation of the vomiting center in the central nervous system after the
toxin acts on neural receptors in the gut. There is no fever and rapid convalescence is the rule.

Laboratory Diagnosis
Isolation of the organism from the suspected food samples followed by culture can be
performed to confirm the diagnosis. Contaminated food can also be tested for the presence of
toxin, however, this is seldom done.

Treatment and Prevention

Management is mainly supportive with the replacement of lost fluid and electrolytes as
a mainstay. No antibiotic therapy is required since the infection is self limited. Cleanliness,
hygiene, and aseptic management of lesions can control the spread of staphylococci from
skin lesions.

Clostridium perfringens
C. perfringens is a large, rectangular gram positive rod. It is anaerobic and rarely produces
spores. An enterotoxin produced by this microorganism is a common cause of food poisoning
 Infections of Gastrointestinal Tract 327

Mode of Transmission
Ingestion of preformed toxin from food contaminated with containing the
soil
microorganism’s spores such as reheated foods like meat dishes is the most common means by
which the organism is acquired.

Clinical Findings
The incubation period is 8–24 hours. The disease is characterized by watery diarrhea with
abdominal cramps. Vomiting may also occur but it is not common. The disease usually resolves
in 24 hours.

Laboratory Diagnosis
Large numbers of the organism can be isolated from food samples. There is no assay for
the toxin.

Treatment and Prevention

No antibiotic therapy is needed and management is mainly supportive. Food should be
adequately cooked before consumption to kill the organism and prevent infection.

Vibrio parahaemolyticus
V. parahaemolyticus is a marine organism. It is a curved, gram negative coccobacillus.
Virulent strains produce Kanagawa hemolysin, an enterotoxin similar to the cholera toxin.
It possesses polar flagella and pili. V. parahaemolyticus is the most common cause of bacterial
gastroenteritis associated with seafood.

Mode of Transmission
The infection is acquired through ingestion of raw or undercooked seafood, especially
shellfish such as oysters.

Clinical Findings
The manifestations vary from mild to severe watery diarrhea, nausea, vomiting, abdominal
cramps, and fever. The illness is self limited, lasting about three days.

Laboratory Diagnosis
Diagnosis can be confirmed by culture. The organism is halophilic, requiring 8% sodium
chloride (NaCl) solution for growth
328 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Treatment and Prevention

Antibiotic therapy is not necessary because the disease is relatively mild and self limited.
Disease is prevented by proper refrigeration and cooking of seafood.

Gastroenteritis (Diarrhea)
Infectious diarrhea may result from multiplication of the microorganism in the
gastrointestinal tract and the mobilization of host defenses as it attempts to eliminate the
invading organism. All diarrhea producing bacteria adhere to intestinal mucosal cells by means
of fimbriae. Once bacteria start to proliferate, they can (1) induce structural abnormalities
resulting to increased excretion of fluids and electrolytes; (2) release toxins; or (3) invade
intestinal mucosa.
Bacteria producing diarrhea may be classified into invasive and non invasive bacteria.
Non invasive bacteria produce diarrhea by producing enterotoxins or cytotoxins. Enterotoxins
stimulate adenylate cyclase causing fluid secretion, leading to a watery type of diarrhea.
Cytotoxins can cause tissue damage leading to inflammation and blood loss. Invasive bacteria
penetrate the bowel epithelium, stimulating intense inflammation. There is direct damage to
the intestinal mucosa resulting in dysenteric type of diarrhea.

Table 18.5 Features of specific types of non invasive bacterial diarrhea

Vibrio +/–Clostridium
Escherichia Bacillus Staphylococcus
cholera coli perfringens cereus aureus
Incubation (hours) 12–72 24–72 6–12 3–8 1–6

Abdominal
+ ++++ ++ ++
cramps
Vomiting ––+ + ++ ++++
Adapted from Lecture Guide in Microbiology, Department of Microbiology, College of Medicine, Our Lady of Fatima
University

Table 18.6 Features of specific types of invasive bacterial diarrhea

Yersinia
Shigella spp. Escherichia coli Salmonella spp.
enterocolitica
Incubation (hours) 24–72 24–72 8–48 1–6

Abdominal cramps ++++ ++++ + ++

Vomiting ++ ++ ++++ ++
Fever ++ ++ ++++ ++
Adapted from Lecture Guide in Microbiology, Department of Microbiology, College of Medicine, Our Lady of Fatima
University
 Infections of Gastrointestinal Tract 329

Noninvasive diarrhea is usually self limited and does not require specific antibiotic therapy.
Invasive diarrhea is usually more severe and requires aggressive therapy. Table 18.7 summarizes
the common causes of bacterial diarrhea.

Table 18.7 Major causes of bacterial diarrhea

Organism Pathogenic Mechanism Source Clinical Features
Escherichia coli
ETEC+ Cholera like toxin, Food, water Traveler’s diarrhea
no invasion Watery diarrhea
EHEC+ Shiga like toxin, Undercooked Hemorrhagic colitis,
no invasion beef products HUS*
EPEC+ Attachment, Weaning foods, water Watery diarrhea
enterocyte effacement
EIEC+ Invasion, local spread Cheese, water, Fever, pain, diarrhea,
person to person dysentery
Salmonella spp. Invasion, dissemination Milk, beef, eggs, poultry Fever, pain, diarrhea
or dysentery,
bacteremia, extra
intestinal infection
Shigella spp. Invasion, local spread Person to person, Fever, pain, diarrhea,
low inoculum dysentery, epidemic
spread
Yersinia Invasion, dissemination Milk, pork Fever, pain, diarrhea,
enterocolitica extra intestinal
infection
Vibrio cholerae Enterotoxin, no invasion Water, shellfish, Watery diarrhea,
person to person pandemic spread
Clostridium Cytotoxin, local invasion Nosocomial spread Fever, pain, bloody
difficile diarrhea, following
antibiotic use
Clostridium
perfringens
Enterotoxin, no invasion Meat, poultry,
fish
Watery diarrhea,
food sources
Mycobacterium Invasion, inflammation Contaminated Chronic abdominal
tuberculosis with necrosis and milk, swallowing pain; complications
scarring of coughed up of malabsorption,
organisms stricture, perforation,
fistulae, hemorrhage
+ ETEC – enterotoxigenic E. coli; EHEC – enterohemorrhagic E. coli; EPEC – entero pathogenic E. coli;
EIEC – enteroinvasive E. coli
* HUS – hemolytic uremic syndrom
330 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Escherichia coli
E. coli is a gram negative, motile, encapsulated rod that is a member of the family
Enterobacteriaceae and is a member of the normal intestinal flora. There are 5 pathogenic
groups of E. coli namely: (1) enterotoxigenic E. coli (ETEC), (2) enteropathogenic E. coli
(EPEC), (3) enteroaggregative/adherent E. coli (EAEC), (4) enterohemorrhagic E. coli (EHEC),
and (5) enteroinvasive E. coli (EIEC).
E. coli only produces disease when it reaches the tissues outside of their normal flora sites.
ETEC, EPEC, and EAEC are primarily associated with secretory diarrhea involving the small
intestines while EHEC and EIEC involve the large intestines. E. coli is the most common
cause of urinary tract infection and gram negative sepsis. It is the most common cause of
neonatal meningitis and is most frequently associated with “traveler’s diarrhea.” It also used as
index of fecal contamination of water.
The microorganism has several components that contribute to its ability to produce disease.
These include the presence of pili (for adherence), capsule (anti phagocytic), endotoxin,
and enterotoxins (two that cause watery diarrhea and one that causes bloody diarrhea and
hemolytic uremic syndrome).

Mode of Transmission
Infection is acquired through ingesting of food or water contaminated by human
feces. EHEC is usually associated with ingestion of undercooked meat (e.g., undercooked
hamburgers).

Enterotoxigenic E. coli (ETEC)

ETEC is the major cause of “traveler’s diarrhea” or “turista” and is an important cause
of diarrhea in infants in developing countries. Some strains produce a heat labile (LT)
exotoxin that causes intense and prolonged hypersecretion of water and chlorides and inhibits
sodium re absorption. Some produce a heat stable (ST) enterotoxin that stimulates fluid
secretion. Strains with both toxins produce severe watery diarrhea. ETEC induced diarrhea is
characterized by abrupt onset of diarrhea which is self limiting. A more serious infection may
be observed in infants.

EnteropathogenicE. coli (EPEC)

EPEC is an important cause of diarrhea in infants in developing countries. It was
previously associated with outbreaks of diarrhea in nurseries in developed countries. The
organism adheres to mucosal cells of the small intestine and causes loss of microvilli. This
leads to watery diarrhea that is usually self limited but can be chronic. Dehydration, electrolyte
imbalance, and other complications may cause death so that antibiotic therapy is necessary
 Infections of Gastrointestinal Tract 33

Enteroaggregative E. coli (EAEC)

EAEC causes acute and chronic diarrhea. These are common causes of food borne illness
in industrialized countries. EAEC produces ST like toxin and a hemolysin.

Enteroinvasive E. coli (EIEC)

EIEC causes invasion of the colonic mucosa. The disease occurs most commonly in
children and travellers in developing countries. The disease characterized by acute bloody
diarrhea with malaise, headache, high fever, and abdominal pain. It is an occasional cause of
occasional outbreaks of dysentery and sporadic infection. It is the most common cause of
urinary tract infection as well as meningitis in newborns.

Enterohemorrhagic E. coli (EHEC)

EHEC produces a shiga like toxin as that produced by Shigella. It is a verotoxin that is
cytotoxic, neurotoxic, and enterotoxic. The main source of infection is undercooked meat,
especially undercooked hamburgers in fastfood restaurants. It is associated with hemorrhagic
colitis, a severe type of diarrhea which initially presents with bloody diarrhea, vomiting,
and severe abdominal pain. It is also associated with hemolytic uremic syndrome, a disease
resulting in acute renal failure, hemolytic anemia, and thrombocytopenia(low platelet count).
E. coli serotype O157:H7 is the most common. In 2011, E. coli O104:H4 caused a serious
outbreak of food borne illness resulting in 35 deaths out of 3,000 persons in Germany.

Laboratory Diagnosis
Diagnosis can be confirmed by culture of organism from stool specimen using a differential
medium (EMB or MacConkey’s agar). On EMB agar, E. coli colonies have a characteristic
greenish metallic sheen. E. coli can ferment lactose. Biochemical tests should be done to
differentiate it from the other members of Enterobacteriaceae.

Treatment and Prevention

Antibiotic therapy is usually not indicated in E. coli diarrheal diseases. Trimethroprim
sulfamethoxazole can be given to shorten symptoms and eliminate the invasive forms of the
organism. Fluid and electrolyte replacement is essential. Traveler’s diarrhea can sometimes
be prevented by the prophylactic use of doxycycline, ciprofloxacin, or trimethoprim
sulfamethoxazole. Ingesting uncooked foods and unpurified water should be avoided while
traveling in certain countries.
332 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Salmonella spp.
Salmonellae are gram negative, encapsulated, motile rods that also belong to the family
Enterobacteriaceae. The organism has three important antigens—cell wall (somatic) O, flagellar
H, and capsular Vi (virulence) antigens. The H antigen is responsible for the invasiveness of the
organism while the Vi antigen is anti phagocytic. Gastric acid is an important host defense.
A large inoculum is needed to produce infection.
Clinically, the Salmonella species can be classified into two categories, namely:
(1) the typhoidal species (S. typhi and S. paratyphi), and (2) the non typhoidal species
(S. enteritidis and S. cholerasuis). S. cholerasuis is most commonly involved in systemic infection.

Mode of Transmission
Ingestion of food and water contaminated by human and animal wastes is the major mode
of transmission. S. typhi is transmitted only by humans. All other species have both animal
and human reservoirs. The most frequent animal sources are dairy products, poultry, and eggs,
however, inadequately cooked meat products have also been implicated.

Clinical Findings
1. Enterocolitis – characterized by invasion of the small and large intestine. It begins with
nausea and vomiting which progresses to abdominal pain and diarrhea (mild to severe,
with or without blood). The disease usually lasts a few days and is self limited. In the
U.S., S. enteritidis serotype typhimurium is the most common cause. This is the most
common manifestation of salmonella infection.
2. Typhoid or enteric fever – begins in the small intestines but few gastrointestinal
symptoms occur. Survival and growth of the organism in phagocytic cells is a striking
feature of this disease as well as the predilection to invade the gallbladder, resulting in
the establishment of a carrier state—asymptomaticcarriage of the bacteria for more
than 1 year.
Typhoid fever is most commonly caused by S. typhi but can also be caused by
S. paratyphi. The illness is characterized by slow onset with fever, bradycardia and
constipation rather than vomiting and diarrhea. After the first week, as the bacteremia
becomes sustained, high fever, delirium, tenderness in the abdomen, and splenomegaly
may occur. Rose spots which is characterized by rose colored macules on the abdomen
or chest may occur in typhoid fever. The disease begins to resolve by the 3rd week.
Complications such as intestinal hemorrhage or perforation can also occur. The carrier
state is more common in women, especially those with previous gallbladder disease
and gallstones
 Infections of Gastrointestinal Tract 333

3. Septicemia – occurs in one of two settings: a patient with an underlying disease (e.g., sickle
cell anemia) or cancer, or a child with enterocolitis. Septicemia is most commonly caused
by S. cholerasuis. Symptoms begin with fever with little or no enterocolitis then proceed
to focal symptoms. Osteomyelitis, pneumonia, and meningitis are the most common
sequelae.

Figure 18.2 The typical rose spots seen on the chest

of a patient with typhoid fever

Laboratory Diagnosis
1. Enterocolitis – stool exam, stool smear, stool culture
2. Typhoid Fever or Enteric Fever
a. Isolation and identification
Culture is the best method (EMB or MacConkey’s agar)
• Blood or bone marrow – 1st to 3rd week of illness
• Stool or rectal swab – incubation period; 2nd to 4th week of illness
• Urine – first two weeks
b. Serology – Widal Test

• Positive only in 24%–60%; positive after the first week of illness

• Measures level of antibodies against the O, H, and Vi antigens (4 fold rise in
antibody titer)

• Interpretation:
» Antibody against O Ag – acute infection
» Antibody against H Ag – recovery or previous vaccination
» Antibody against Vi Ag – carrier
c. Typhidot – detects specific IgM and IgG antibodies against Salmonella
3. Biochemical tests are done to differentiate from other Enterobacteriacea
334 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Treatment and Prevention

Enteric fevers and bacteremia require antimicrobial treatment, however, the vast majority
of cases of enterocolitis do not. Clinical symptoms and excretion of the organism may be
prolonged by antimicrobial therapy in enterocolitis. Antimicrobial therapy for invasive
salmonella is done using ampicillin, trimethoprim sulfamethoxazole, or 3rd generatio
cephalosporin. In most carriers, surgical removal of the gallbladder (cholecystectomy) must be
combined with drug therapy.
Sanitary measures must be undertaken to prevent contamination of food and water.
Thorough cooking of infected poultry, meat, and eggs must be done. Carriers must be treated
promptly and not be allowed to handle food, as well as observe strict hygienic precautions.
Vaccines are available to prevent the infection, especially in endemic areas.

Shigella spp.
Shigellae are gram negative, non motile, non encapsulated rods which are members of the
family Enterobacteriaceae. The natural habitat is limited to the intestinal tracts of humans
and other primates. There are four important species of Shigella, namely S. sonnei, S. flexneri,
S. boydii, and S. dysenteriae type 1 (Shiga bacillus). Of the four species, S. dysenteriae is the most
clinically significant as it is responsible for epidemics with high mortality. The major virulence
factor of S. dysenteriae type 1 is the shiga toxin, which is a verotoxin as in E. coli. Low inoculum
is needed to produce infection.

Mode of Transmission
Shigellosis is transmitted by the four F’s, namely “food, fingers, flies, and fomites.” It may
also be transmitted through sexual contact.

Clinical Findings
Shigellosis is characterized by a short incubation period of 1–3 days. It is characterized by
lower abdominal pain, fever, and bloody, mucoid diarrhea. Bowel movement is accompanied
by tenesmus or strained defecation. In adults, more than 50% of cases resolve spontaneously.
In children and the elderly, severe dehydration may lead to death. Complications include
(1) perforation of the colon, (2) hemolytic uremic syndrome similar to E. coli, and (3) Ekiri
syndrome, a fulminant type of encephalopathy. Like Salmonella, a carrier state may occur.

Laboratory Diagnosis
1. Stool examination revealing leukocytes (wbc) and/or red blood cells in fresh stool specimen
2. Culture of feces or rectal swab specimen (EMB or MacConkey’s agar)
 Infections of Gastrointestinal Tract 33

Treatment and Prevention

The mainstay of management is fluid and electrolyte replacement. Antimicrobial agents
such as ciprofloxacin, ampicillin, doxycycline, and trimethoprim sulfamethoxazole are effective
against the organism.
Preventive measures include (1) sanitary control of water, food, and milk; (2) proper sewage
disposal, and fly control; (3) patient isolation and disinfection of excreta; (4) detection of
carriers, especially food handlers; and (5) prompt antibiotic treatment of infected individuals.

Yersinia enterocolitica
Y. enterocolitica is also a member of the family Enterobacteriaceae and are gram negative,
urease positive rods. Urease produced by the organism neutralizes the gastric acid allowing the
organism to survive and colonize the intestines.

Mode of Transmission
Ingestion of food (meat and dairy products) or water contaminated by feces of domestic
animals is the primary mode of transmission. The organism may also be transmitted
through fomites.

Clinical Findings
The organism causes inflammation and ulceration in the tissues affected. Early symptoms
include fever, abdominal pain, and diarrhea that is watery to bloody. The terminal ileum may
be involved and if the mesenteric lymph nodes are involved, it may present itself as right lower
quadrant pain and may be misdiagnosed as acute appendicitis. One to two weeks after onset,
some patients develop arthralgia, arthritis, and erythema nodosum. The organism, in rare
instances, may cause pneumonia, meningitis, or sepsis. It is however a self limiting infection.

Laboratory Diagnosis
Y. enterocolitica can grow in most culture media and can grow best with “cold enrichment”
or at low temperature of 4 °C.

Treatment and Prevention

Most cases of diarrhea are self limited and do not require antimicrobial therapy. There are
no specific preventive measures, however, preventing contamination of food and water may be
helpful in preventing infection.
336 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Vibrio cholerae
The Vibrios are among the most common bacteria in surface waters worldwide. V. cholerae is
a comma shaped, curved, motile rod with a polar flagellum. V. cholerae serogroups O1 and O139
cause cholera epidemics. Occasionally, serogroups non O1/non O139 cause cholera like illness.
V. cholerae serogroups O1 is divided into serotypes (Inaba, Hikojima, and Ogawa) and biotypes
(classical and El tor). V. cholerae O1 biotype El tor is the most common cause of cholera
epidemics while serogroup O139 or the Bengal strain was identified as the strain that caused
the most recent (8th) epidemic of cholera and has been identified as the first non O1 strain
associated with outbreaks. V. cholerae produces an enterotoxin (choleragen or cholera toxin) that
stimulates prolonged hypersecretion of water and electrolytes. It is pathogenic only for humans.

Mode of Transmission
The disease is spread by ingestion of contaminated food and water. Person to person
transmission is rare because the infectious dose is very high.

Clinical Findings
The disease is called cholera and majority of cases are asymptomatic. There is sudden
onset of nausea and vomiting, and profuse watery diarrhea (as much as 20–30 L/day) with
abdominal cramps. The stools may resemble “rice water.” There is severe dehydration which
can lead to circulatory collapse and hypovolemic shock may result in death if the patient is not
treated promptly.

Laboratory Diagnosis
Diagnosis rests on the typical clinical presentation. Microscopic examination using
darkfield or phase contrast microscopy may be done to show the rapidly motile organism.
Culture is also diagnostic.

Treatment and Prevention

Crucial to the management of cholera is rapid fluid and electrolyte replacement. The drug
of choice is tetracycline. Preventive measures include health education and improvement of
sanitation. Isolation of patients with disinfection of their excreta should be done. A vaccine is
available, however, it provides limited protection to heavily exposed persons and is not effective
in epidemic control
 Infections of Gastrointestinal Tract 33

Clostridium perfringens
C. perfringens is a toxin producing organism that can produce invasive infection. It produces
numerous toxins and enzymes that result in a spreading infection. These toxins have lethal,
necrotizing, and hemolytic properties. Some strains produce a powerful enterotoxin, especially
when grown in meat dishes.

Mode of Transmission
Infection in humans occur after ingesting food (usually meat and gravies) contaminated by
dirt or feces.

Clinical Findings
C. perfringens can produce a disease process called enteritis necroticans, an acute
necrotizing process in the small intestines that manifests with abdominal pain and bloody
diarrhea. Severe infection can lead to peritonitis and shock.

Laboratory Diagnosis
Diagnosis is done through culture under anaerobic conditions.

Treatment and Prevention

The antimicrobial agent of choice is penicillin. Antitoxins in the form of concentrated
immune globulins can be administered. There are no specific preventive measures. Food must
be adequately cooked before consumption to kill the organism.

Clostridium difficile
C. difficile is also an anaerobic, gram positive, spore forming rod. Approximately 3% of the
general population are asymptomatic carriers of the organism in the gastrointestinal tract. It is
the most common nosocomial cause of diarrhea. The organism produces exotoxins that cause
death of enterocytes.

Mode of Transmission
C. difficile is transmitted by the fecal oral route and hospital personnel are important
intermediaries.
338 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Clinical Findings
The organism causes antibiotic associated pseudomembranouscolitis. Clindamycin is
the first antibiotic recognized as a cause of the disease but other antibiotics are now implicated.
The second and third generation cephalosporins are now considered as the most common
causes. The diarrhea may be watery or bloody and frequently accompanied by abdominal
cramps, fever, and leukocytosis.

Laboratory Diagnosis
Detection of toxins in stool specimens using ELISA or cytotoxicity test is the basis for the
diagnosis. Sigmoidoscopy may also be done to visualize the pseudomembrane.

Treatment and Prevention

Discontinuance of administration of the offending antibiotic is the treatment of choice
followed by oral administration of either metronidazole or vancomycin. Fluid replacement
is also essential. Indiscriminate use of antibiotics should be avoided. There are no preventive
vaccines or drugs.

Bacillus anthracis
Gastrointestinal anthrax is very rare and is acquired by entry of spores through the mucous
membranes or by ingestion of improperly cooked meat from infected animals. Symptoms
include vomiting, abdominal pain, and bloody diarrhea. The diagnosis can be made through
microscopic examination of specimen and culture.
The drug of choice for the treatment of anthrax is ciprofloxacin. Doxycycline is
an alternative drug. Control measures include: (1) proper disposal of animal carcasses,
(2) decontamination of animal products, and (3) active immunization of domestic animals with
live attenuated vaccine. Persons with high risk should be immunized.

Mycobacterium tuberculosis
Tuberculosis of the gastrointestinal tract can be caused by either M. tuberculosis when it is
swallowed after being coughed up from a lung lesion, or by M. bovis when it is ingested in
unpasteurized milk products. It is characterized by abdominal pain and chronic diarrhea,
accompanied by fever and weight loss. Intestinal obstruction or hemorrhage may also occur.
The most common site involved is the ileocecal region. Oropharyngeal tuberculosis typically
presents with painless ulcer accompanied by lymphadenopathy
 Infections of Gastrointestinal Tract 33

Treatment involves multidrug therapy against the organism. The BCG vaccine can be used
to induce partial resistance to tuberculosis. Pasteurization of milk and elimination of infected
cattle are important preventive measures for intestinal tuberculosis.
340 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• Dental
lactic
caries is most commonly caused by S. mutans and is associated with sucrose and
acid.

• Periodontal diseases are mixed infections caused by different groups of bacteria. The
infections involve inflammation of the gums and the progressive destruction of the deeper
tissues and alveolar bone.

• Oral thrush is most commonly caused by C. albicans and is associated with a variety of
predisposing factors like immunosuppression and intake of broad spectrum antibiotics,
among others.

• Mumps
glands.
is a highly communicable infection characterized by inflammation of the salivary

• H.common
pylori produces urease which enables it to survive the acidity of the stomach. It is
cause of gastritis, peptic ulcer, gastric carcinoma, and MALT lymphomas.
a

• The most common causes of hepatitis are viruses. Hepatitis A and E are acquired through
the fecal oral route while Hepatitis B, C, and D are acquired by sexual, parenteral, and
transplacental transmission.

• Food poisoning can be caused by S. aureus, C. perfringens, B. cereus, and C. botulinum.

aureus causes food poisoning with the shortest incubation period.
S.

• The virulent strains of V. parahemolyticus produce Kanagawa hemolysin and it is the most
common cause of bacterial gastroenteritis associated with seafood.

• There are 5 pathogenic groups of E. coli namely: (1) enterotoxigenic E.

(ETEC), (2) enteropathogenic E. coli (EPEC), (3) enteroaggregative E. coli (EAEC),
coli

(4) enterohemorrhagic E. coli (EHEC), and (5) enteroinvasive E. coli (EIEC).

• Typhoid fever is caused by both S. typhi (most common) and S. paratyphi. It is acquired
through ingestion of contaminated dairy products, poultry, and eggs.

• Shigellosis
with tenesmus.
is characterized by lower abdominal pain, fever, and bloody, mucoid diarrhea

• V.bycholerae serogroups O1 and O139 cause cholera epidemics. Cholera is characterized

profuse diarrhea (rice water stools) accompanied by vomiting resulting to severe
dehydration, and can even result in the death of the patient.

• Mycobacterium tuberculosis and M. bovis can cause intestinal tuberculosis from ingestion of
contaminated milk and its products
 Infections of Gastrointestinal Tract 341

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Case: A 50 year woman was brought to the emergency room because of profuse diarrhea and
vomiting. The stool is described as “rice water” like in appearance. The patient is a resident of
Payatas, Quezon City and works as a scavenger. P.E. reveals a severely dehydrated patient with
thready pulse and hypotension.

1. This is most probably a case of:

a. Salmonellosis c. Cholera
b. Shigellosis d. Amoebiasis
2. The severe diarrhea is due to which of the following virulence factors of the
causative agent?
a. Capsule c. Toxin
b. Teichoic acid d. Flagellum
3. The infection is most probably acquired through:
a. Ingestion of contaminated food and water
b. Ingestion of contaminated poultry products
c. Indirect contact via inanimate objects
d. Contact with asymptomatic carrier

4. If this is a case of cholera, which group of Vibrio cholera is a common cause of

epidemics of cholera?
a. V. cholerae O1 c. Bengal strain
b. V. cholerae non O1 d. All of the abov
5. This infection is best managed by:
a. Fluid and electrolyte replacement
b. Giving Tetracycline
c. Isolation of the patient
d. All of the above
342 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Multiple Choice.

1. The Shigellla species that produces shiga toxin.

a. S. flexneri c. S. sonnei

b. S. boydii d. S. dysenteriae
2. Infection with which hepatitis virus almost always leads to fulminant hepatitis?
a. Hepatitis A c. Hepatitis C
b. Hepatitis B d. Hepatitis D
3. Gastroenteritis caused by this is most frequently associated with ingestion of raw
contaminated seafood.
a. S. aureus c. V. parahemolyticus
b. S. typhi d. S. flexneri
4. Food poisoning with the shortest incubation period:
a. B. cereus c. C. perfringens
b. S. aureus d. C. botulinum

5. Antibiotic associated pseudomembranousenterocolitis is caused by:

a. C. botulinum c. C. perfringens
b. C. difficile d. B. cereus
6. It is the most common cause of traveler’s diarrhea.
a. ETEC c. EAEC
b. EPEC d. EIEC
7. It is associated with hemolytic uremic syndrome.
a. EHEC c. V. cholerae
b. S. dysenteriae d. A and B
8. It occurs as co infection or super infection with Hepatitis D virus.
a. HAV c. HCV
b. HBV d. HE
 Infections of Gastrointestinal Tract 34

9. This organism is used as an index in the fecal contamination of water.

a. V. cholerae c. E. coli
b. S. boydii d. S. typhi
10. It is the most common cause of post transfusion hepatitis.
a. HAV c. HCV
b. HBV d. HDV
This page is intentionally left blank
 CHAPTER Sexually
19 Transmitted
Infections

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. recognize common sexually transmitted infections based on clinical manifestations;
2. describe the characteristics of the causative organisms of each sexually transmitted
infection;
3. identify the individuals at risk of sexually transmitted infections;
4. determine the appropriate laboratory diagnosis and treatment of each infection; and
5. discuss the global strategy for the prevention and control of sexually transmitted
infections.

The incidence of sexually transmitted diseases has increased tremendously through the
years. The age incidence has also increased not only in the adult population but more so among
adolescents. Sexually transmitted infections (STI) are mostly caused by mixed infections
and not just single organisms. The infections can be acquired through (1) unprotected sex
(vaginal, oral, or anal), (2) skin to skin contact with the genital area, (3) blood transfusion, or
(4) perinatal transmission through transplacental transfer or during childbirth as the infant
passes through the infected birth canal. STIs are worldwide in distribution and can affect all
age groups but sexually active individuals are the most vulnerable. The most common sexually
transmitted infections are listed in Table 19.1
346 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Table 19.1 Most common sexually transmitted infections

Organism Disease
Bacterial Neisseria gonorrhea Gonorrhea
Haemophilus ducreyi Chancroid
Treponema pallidum Syphilis
Chlamydia trachomatis Lymphogranuloma venereum
Viral HIV AIDS
Herpes simplex virus Genital/Perianal herpes
Human papillomavirus Condylomata acuminata
Hepatitis viruses Hepatitis
Zika virus
Fungal Candida albicans Vulvovaginal Candidiasis
Protozoal Trichomonas vaginalis Trichomoniasis
Parasitic Phthirus pubis Pediculosis pubis

The individuals at risk of sexually transmitted infections are those who engage in
unprotected sex, those with multiple sexual partners, rape victims, and IV drug users who share
needles.

Prevention and Control

of Sexually Transmitted Infections
It has always been emphasized that sexually transmitted diseases can best be prevented
by practicing safe sex especially among travelers. The correct use of condoms can lessen the
chances of contracting STIs. Avoiding sharing of needles and razors as well as getting tattoos,
body piercings, or acupuncture treatments also lessen the risk of STI. Screening of blood donors
is also being implemented to prevent the contraction of STIs. There are also available vaccines
for the Human Papillomavirus (HPV) and Hepatitis B virus.
The World Health Organization has come up with a Global strategy for the prevention
and control of sexually transmitted infections. The strategy has two components: (1) the
technical which involves methods on the use of protective barriers, promotion of sexual
behavior, accessibility to effective care system, and improved monitoring and evaluation
of control strategies; and (2) the advocacy which addresses the need for an effective STI
advocacy campaign to raise awareness and mobilize resources at the national and international
level. These strategies involve dedication and collaboration of the health sector with other
government agencies and non governmental organizations. Education of the public on sexuall
 Sexually Transmitted Infections 34

transmitted infections, identification of infected individuals, contact tracing, and prompt

treatment of infected individuals are important in preventing the spread of such infections.

Clinical Manifestations of STIs

Skin Lesions
1. Ulcerative Lesions
a. Chancre – primary lesion of syphilis; painless and well delineated.
b. Chancroid – ulcer with ragged edges; painful
c. Genital herpes – start as a vesicle that becomes an ulcer after rupture

2. Granulomatous reactions – typical of granuloma inguinale

3. Rashes – commonly seen in secondary syphilis, gonorrhea, and candidiasis
4. Warty lesions – characteristic of condylomata acuminata and molluscum contangiosum
Discharge
1. Vaginal discharge – usually accompanied by dysuria, dyspareunia, and vulvar irritation
a. Trichomonas vaginalis – thin, foamy, foul smelling
b. Neisseria gonorrhea – greenish, purulent
c. Candida albicans – thick, cheesy exudates (milk curd like appearance)
2. Urethral discharge – in males, any urethral discharge other than ejaculation is abnormal

Common STIs
Syphilis
Syphilis ranks third among the most common sexually transmitted diseases worldwide. It
is caused by Treponema pallidum, a spirochete with fine regular coils with tapered ends. It is
a strict human pathogen. It is sensitive to oxygen. The organism cannot be grown in cell free
culture medium.

Modes of Transmission
Syphilis can be transmitted: (1) through direct sexual contact; (2) congenitally; and
(3) through blood transfusion.
348 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Clinical Findings

1. Adult Syphilis
a. Primary syphilis – a highly infectious stage with abundant organisms that can be
isolated from the ulcer. The primary lesion is called chancre which starts as a hard,
painless papule that later becomes an ulcer with smooth or well delineated borders.
Within 2 months, the ulcer heals spontaneously even without treatment but will
continue to disseminate through the blood and lymphatics and eventually progress
to secondary syphilis.
b. Secondary syphilis – presents with flu like symptoms, lymphadenopathy, and a
generalized mucocutaneous rash (including the palms and soles) which can be
macular, papular, or pustular. The characteristic lesion is called condyloma latum
(plural: condylomata lata) which is a painless, wart like lesion that is highly
contagious.
c. Latent syphilis – the stage where the patient is clinically inactive or asymptomatic.
The patient may have reactivation of secondary syphilis or may progress to tertiary
syphilis.
d. Tertiary (late) syphilis – characterized by granulomatous skin lesions (gummas) that are
also found in bones and other tissues as well as other organ involvement such as
cardiovascular syphilis (aortic aneurysm) or CNS involvement (neurosyphilis).
2. Congenital Syphilis
a. Early congenital syphilis – right after birth, the infected newborn may not present
with any clinical manifestation. Later the newborn may manifest with runny nose
(snuffles), rash, and condylomata lata as well as hepatosplenomegaly.

b. Late congenital syphilis – manifested as 8th nerve deafness with bone and teeth
deformities (e.g., saddle nose, saber shins, Hutchinson’s teeth, and Mulberry or
Moon’s molars).

Laboratory Diagnosis
1. Darkfield microscopy
2. Serology
a. Non specific treponemal test – VDRL (Venereal Disease Research Laboratory) and RPR
(Rapid Plasma Reagin)
b. Specific treponemal test – Fluorescent Treponemal Antibody Absorption (FTA ABS
 Sexually Transmitted Infections 349

Treatment and Prevention

The drug of choice is penicillin. Alternative drugs in cases of penicillin resistance or allergy
are tetracycline or doxycycline. There is no vaccine for syphilis. It can be controlled through
practice of safe sex and regular testing.

a b c

d e

Figure 19.1 a The appearance of the spiral shaped Treponema pallidum. Characteristic lesions
seen in adult syphilis include the b painless chancre of primary syphilis, c mucocutaneous rash,
d condylomata lata of secondary syphilis, and e gumma of tertiary syphilis
Source: Center for Disease Control and Prevention and DermNet NZ

Figure 19.2 a X ray of infant with

late congenital syphilis showing
a b
saber shin deformity, b Hutchinson’
teeth and mulberry molars

Gonorrhea
Gonorrhea is the second most common sexually transmitted infection worldwide. It occurs
only in humans. For it has no animal reservoir. Females are asymptomatic carriers of the
infection. The risk after single exposure is higher in females (50%) than in males (20%). It is
caused by Neisseria gonorrheae, gram negative diplococci. It is kidney bean shaped when it is
350 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

single and coffee bean shaped when in pairs. It has pili which are used for attachment to host
cell, motility, transfer of genetic materials and plays an important role in the pathogenesis.

Clinical Findings

1. Gonorrhea infection in males

Males are most often asymptomatic during the early stage of infection. The infection
is restricted to the urethra and manifests as purulent urethral discharge and dysuria.

2. Gonorrhea infection in females

The primary site of infection is the cervix. Women may manifest with purulent
vaginal discharge, dysuria, and abdominal pain. Ascending infections such as salpingitis,
abscesses involving the fallopian tubes and ovaries, and pelvic inflammatory disease
(PID) may develop if not properly treated. Stricture of the fallopian tubes may lead to
sterility. Rectal and pharyngeal infections are more commonly asymptomatic than genital
infections.
3. Disseminated infections
This occurs in 1%–3% of cases and present as fever, migratory arthralgia, suppurative
arthritis of the wrists, knees, and ankles, and pustules with erythematous base over the
extremities. Other diseases associated are perihepatitis (Fitz Hugh Curtis Syndrome) and
purulent conjunctivitis in adults. In newborns, N. gonorrheae can cause a purulent ocular
infection called ophthalmia neonatorum that is acquired upon passage through the infected
birth canal.

Figure 19.3 Typical

purulent discharge from
a patient with active
gonorrhea infection

Laboratory Diagnosis
Gram negative intracellular diplococci may be seen using microscopy. Culture using
modified Thayer Martin medium as selective medium allows the growth of Neisseria only
 Sexually Transmitted Infections 351

Treatment and Prevention

For uncomplicated gonorrhea, ceftriaxone, ciprofloxacin, cefixime, or ofloxacin are
recommended. For mixed infection with Chlamydia, any of the aforementioned drugs must be
combined with doxycycline or azithromycin.
Prevention of ophthalmia neonatorum is through the use of 1% silver nitrate or 0.5%
erythromycin or tetracycline eye ointment. Education of the public and screening of sex workers
are effective control measures for all sexually transmitted diseases.

LymphogranulomaVenereum (LGV)
LGV is caused by Chlamydia trachomatis, obligate intracellular bacteria that do not have
cell walls. The organism has a unique process of development involving two forms—the
elementary bodies which are the metabolically inactive infectious form and reticulate bodies
that are metabolically active but non infectious. Serotypes D to K are associated with non
gonococcal urethritis, cervicitis, and PID while serotypes L1, L2, and L3 are associated with
lymphogranuloma venereum.

Clinical Findings
1. Urogenital tract infections
Most are asymptomatic. If symptomatic, it may manifest as cervicitis, endometritis,
urethritis, salpingitis, bartholinitis, perihepatitis, and mucopurulent discharge.
2. Lymphogranulomavenereum
A primary lesion appears at the site of infection, either a papule or ulcer, which is
small, painless, and heals rapidly. The second stage is manifested by enlarged lymph nodes
that are painful (buboes) and ruptures to form draining fistulas.

Figure 19.4 Enlarged painful lymph

nodes (buboes) that rupture and drain
with pus are characteristic of LGV
caused by Chlamydia trachomatis
Source: Fred & van Dijk, 200
352 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Laboratory Diagnosis
The organism can be visualized using Giemsa stained specimen obtained from scrapings
from the lesion. Culture is the most specific diagnostic method.

Treatment and Prevention

Treatment of the infection involves giving of azithromycin, doxycycline, or erythromycin.

Chancroid
The etiologic agent is Haemophilus ducreyi, a gram negative coccobacillus. Haemophilus
means “blood loving” and must be grown in culture medium containing blood. It only requires
hemin (X factor) for growth which is derived from the blood in the culture medium.

Clinical Findings
Chancroid presents with a soft, painful papule with an erythematous base that develops into
an ulcer with ragged edges associated with inguinal lymphadenopathy.

Figure 19.5 Characteristic

lesion in chancroid

Laboratory Diagnosis
Definitive diagnosis is made through culture on at least two kinds of enriched media
containing vancomycin.

Treatment and Prevention

Antibiotics for treatment include cephalosporins, azithromycin, erythromycin, o
ciprofloxacin.
 Sexually Transmitted Infections 35

Genital Herpes
Genital herpes is caused by Herpes Simplex Virus (HSV). It is a DNA virus under the family
of Human Herpesviridae. There are two types of HSV, type 1 and type 2. The virus is capable of
latency in the neurons hence the occurence of recurrent infections.

Modes of Transmission
The main mode of transmission is through oral secretions or sexual contact.

Clinical Findings
Genital herpes is caused by HSV types 1 and 2, but majority of cases are caused by type 2.
Most primary infections are asymptomatic. The lesions are vesicular which later on rupture
resulting to ulcers and are painful with inguinal lymphadenopathy. The lesions are seen in
the vulva, vagina, cervix, or perianal area and are accompanied by pruritus and mucoid vaginal
discharge.
Recurrent infections are often of shorter duration and less severe than the primary
infection. A consequence of genital herpes in newborns is neonatal herpes which is acquired in
utero or upon passage through the infected birth canal during delivery.

Laboratory Diagnosis
Tzanck smear and histopathologic examination are done to demonstrate the characteristic
cytopathologic effects that includes Cowdry type A inclusions, syncytia formation, and ballooning
of infected cells. A more specific diagnostic test is PCR or immunofluorescence.

Treatment and Prevention

The drug of choice is acyclovir but it does not prevent recurrences. The prevention is the
same as with other sexually transmitted diseases. No vaccine is available.

Figure 19.6 a Herpes

genitalis due to herpes
simplex virus type 2 and
b genital warts or condyloma
acuminata caused by human
papillomavirus
a b
Source: SOA AIDS Amsterdam
354 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Table 19.2 Comparison of the major genital sore diseases

Primary Syphilis Genital Herpes Chancroid
Etiologic agent Treponema pallidum Herpes Simplex Virus Haemophilus ducreyi
Incubation period 3 weeks (10–90 days) 2–7 days 3–5 days
Usual clinical Slightly tender papule Marked pain in genital Tender papule that
presentation that ulcerates over 1 to area; papules ulcerate ulcerates
several weeks in 3–6 days; fever,
headache, malaise and
inguinal adenopathy
common
Diagnostic tests Dark field exam of Virus culture of Culture in at least
exudate from chancre; cells and fluid from two kinds of
serologic tests chancre; nucleic acid enriched media with
amplification tests vancomycin
Long term sequelae Secondary syphilis Recurrent genital Inguinal bubo
with mucocutaneous herpes
lesions; tertiary
syphilis
Treatment Benzathine penicillin Acyclovir, famciclovir, Ceftriaxone,
G; doxycycline if or valacyclovir azithromycin,
allergic to penicillin erythromycin, or
ciprofloxacin
Adapted from Jawetz, Melnick, and Adelberg’s Medical Microbiology 26th ed., McGraw Hill Education, 2013, p. 806.

Condylomata Acuminata
This is caused by the Human papillomavirus (HPV) (serotypes 6 and 11). It is a DNA
virus under the family of Papovaviruses that is transmitted through sexual contact. HPV is
capable of immortalizing or transforming an infected cell leading to malignancy (usually
types 16 and 18).

Clinical Findings
Genital warts or condylomata acuminata occur most commonly in the genital or
perianal areas. The serotypes most commonly associated with condylomata acuminata are
serotypes 6 and 11. Infection of the genital tract is associated with cervical and penile cancer.
The serotypes predominantly isolated in these cases are serotypes 16 and 18
 Sexually Transmitted Infections 355

Figure 19.7 Genital

warts or condylomata
acuminate in a a male
a b
and b female patient
Source: SOA AIDS Amsterdam

Laboratory Diagnosis
Histologic examination and Papanicolaou smear.

Treatment and Prevention

Injection of interferon is the preferred and most beneficial treatment. An HPV vaccine is
already available for individuals 11 years old and above. It is recommended for adolescents and
sexually active males and females. It is either a tetravalent vaccine containing serotypes 6, 11,
16, and 18 or a bivalent vaccine containing serotypes 16 and 18.

Acquired Immunodeficiency Syndrome (AIDS)

AIDS is caused by an RNA virus under the family of retroviruses called the Human
Immunodeficiency Virus (HIV). The virus possesses the enzyme reverse transcriptase that
allows it to integrate its genome into the host cell’s DNA. It possesses a glycoprotein known
as gp120 on its envelope that binds to the CD4+ receptor on helper T cells and macrophages.
Another envelope glycoprotein, gp41, facilitates the adsorption of the virus to the CD4+ T cells
356 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

gp41 RNA
gp120 envelope
envelope protein
protein
p17 matrix
proteins

Lipid
membrane

Reverse p24 capsule

transcriptase protein

Anatomy of the AIDS Virus

Figure 19.8 Structure of the Human Immunodeficiency Virus (HIV) causing Acquired Immune
Deficiency Syndrome (AIDS)

Modes of Transmission
There are several modes of transmission for HIV, namely: (1) sexual; (2) parenteral
(blood transfusion, tattooing, ear piercing, injections); and (3) transplacental contact. HIV is
not transmitted by kissing, coughing, sneezing, insect bites, or swimming pools. Individuals
at highest risk of developing infection include: (1) sexually active individuals especially those
with multiple sexual partners; (2) intravenous drug users (with sharing of needles); (3) patients
receiving blood and blood product transfusions like hemophiliacs; and (4) newborns of HIV
positive mothers.

Clinical Findings
The incubation period lasts from less than a year to about 10 years where the patient is
asymptomatic. Initially, patients present with flu like or infectious mononucleosis like
symptoms accompanied by chronic diarrhea and generalized lymphadenopathy.This
occurs about one month after exposure to a patient with AIDS. The symptoms will
then subside followed by a long period of latency (approximately 8 years), after which
the patient will present with evidences of opportunistic infections and malignancies.
It is during this time that the patients will have very low CD4+ T cell counts considered as
full blown AIDS cases making them more susceptible to opportunistic infections and to the
 Sexually Transmitted Infections 357

wasting syndrome (diarrhea and weight loss). Lesions in the tongue and mouth (hairy cell oral
leukoplakia) due to the Epstein Barr virus are also observed. AIDS related dementia (signs of
dementia and decreased intellectual abilities) is also observed in some patients. The hallmark
of AIDS is Kaposi’s sarcoma, a form of soft tissue cancer. Infections with Pneumocystis jiroveci,
Mycobacterium avium intracellulare, and severe Cytomegalovirus infections are indicative of
very low CD4+ T cell count. AIDS patients do not die of AIDS but because of opportunistic
infections. The most common cause of death is pneumonia due to P. jiroveci.

Laboratory Diagnosis
There are several tests available for HIV. Among the very first tests are ELISA
(Enzyme linked immunosorbent assay) and Western Blot assay which serve as screening and
confirmatory tests, respectively. An early marker of infection is p24 antigen determination.
Polymerase chain reaction (PCR) can also be used to confirm diagnosis.

Treatment and Prevention

Treatment involves the use of reverse transcriptase inhibitors like azidothymidine (AZT)
and dideoxycytidine (ddC) which may be used in combination with protease inhibitors
like indinavir or saquinavir. Today, combination of several drugs is given to prevent the
development of drug resistance. This treatment modality is known as HAART (highly active
anti retroviral treatment).
Aside from the usual preventive measures for sexually transmitted infections, other
preventive measures include: (1) education of the public regarding its mode of transmission
and control measures to prevent the spread of the infection; (2) screening of blood donors;
(3) monogamous relationships and use of condoms, (4) wearing of protective clothing (gowns,
masks, and gloves); (5) avoidance of sharing of needles, and (6) disinfection of contaminated
surfaces and laundry. No vaccine is available for HIV because the virus undergoes constant
mutation due to the antigenic drift of its gp120.

Figure 19.9 Lesions of Kaposi sarcoma in various parts of the body seen in patients with AIDS
Source: Sand et al., 2010 and OpenStax College, 201
358 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Pediculosis Pubis (Pubic lice or crabs)

Etiologic Agent
Phthirus pubis is the etiologic agent for pubic lice. The organism is tiny, about 2 millimeters
(mm) long, and visible to the naked eye. It is a parasitic insect that feeds on the blood of the
host. The lice are primarily seen attached to the pubic hair and in coarse hairs found in other
parts of the body like the chest, beard, moustache, and armpits.

Mode of Transmission
Pediculosis pubis is primarily spread through sexual contact. In rare occasions, it is spread
through inanimate objects like towels, linens, or clothes.

Clinical Findings
The infestation is highly contagious and spreads easily. It is commonly seen in jails and
sexually active individuals. They readily attach to human hair and cause intense pruritus and
red spots. Secondary bacterial infection may occur and eczematous lesions may develop.

a b

Figure 19.10 a Phthirus pubis, b infestation of the pubic hair by the lice
Source: SOH AIDS Amsterdam

Diagnosis
Identification of the parasite attached to hair.

Treatment and Prevention

Insecticidal creams, lotions, and shampoos that contain 1% malathion or permethrin
may be used. The spread of infection can be prevented by treatment of infested individual or
mass control
 Sexually Transmitted Infections 359

CHAPTER SUMMARY

• Individuals at high risk for the development of sexually transmitted infections include
those who engage in unprotected sex, those with multiple sexual partners, sex workers,
rape victims, and IV drug users.

• STIs may be caused by bacteria, viruses, fungi, protozoa, or parasites.

• STIs can be acquired through unprotected sex (vaginal, oral, or anal), skin to skin contact
of the genital area, blood transfusion, transplacental transfer or during childbirth.

• STIs can present as ulcerative, granulomatous, or warty lesions or as abnormal genital

or urethral discharges.

• Gonorrhea is the most common STI worldwide followed by syphilis.

• Inreservoir
gonorrhea, males are initially asymptomatic and infected females are the primary
of the infection. Untreated infection of gonorrhea may lead to pelvic
inflammatory disease and even sterility.

• HIV is a slow virus. It is oncogenic and capable of latency.

• The WHO Global strategy for the control of STIs includes a technical and advocacy
component. The most important aspect of prevention is education of the public. It must
be a concerted effort of the national and local governments, the community, as well
as nongovernmental organizations.

• There are available vaccines for hepatitis B and Human papillomavirus.

• Early recognition, diagnosis, and treatment are very important in preventing
complications and in the control of infection
This page is intentionally left blank
 Sexually Transmitted Infections 361

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Case: A 32 year old seaman consulted a local hospital because of a hard, painless nodule over
the inferior aspect of his penis. There is no other manifestation. The patient allegedly had
unprotected sexual contact with a sex worker while he was abroad.

1. The most probable diagnosis is:

a. Chancroid c. Genital herpes
b. Lymphogranulomavenereum d. Syphilis
2. Condyloma latum is seen in which stage of the infection?
a. Primary c. Latent
b. Secondary d. Tertiary
3. Late syphilis is associated with the following EXCEPT:
a. Snuffles c. Saber shin
b. Hutchinson’s teeth d. Mulberry molar

Multiple Choice.

4. Patients with full blown AIDS die of complications. The most common cause of
death is pneumonia due to which of the following organisms?
a. Streptococcus pneumoniae c. Staphylococcus aureus
b. Pneumocystis jiroveci d. Haemophilus influenza
5. Which of the following sexually transmitted infections is caused by gram negative
diplococci described as coffee bean shaped?
a. Chancroid c. Candidiasis
b. Gonorrhea d. Lymphogranulomavenereum
362 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. Human papillomavirus serotypes most commonly associated with malignant

transformation are:
a. Serotypes 1, 2, 3, and 4 c. Serotypes 16 and 18
b. Serotypes 6 and 11 d. B and C
7. Which of the following is correct regarding genital herpes?
a. The most common cause is Herpes simplex virus type 1.
b. It is characterized by pruritic vesicular lesions.
c. Draining lymph nodes are often seen in most patients.
d. Purulent vaginal discharge is a distinctive manifestation.
8. The following sexually transmitted diseases may present with skin rashes EXCEPT:
a. Secondary syphilis c. Candidiasis
b. Gonorrhea d. Chancroid
9. The HIV receptor that binds with the CD4 T cells of the host is
a. gp120 c. p24
b. gp41 d. p17
10. Sexually transmitted disease that is caused by a parasite:
a. Candidiasis c. Pediculosis
b. Syphilis d. Chlamydi
 CHAPTER
Infections
20 of the Urinary Tract

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. recognize common manifestations of urinary tract infections;
2. differentiate an upper urinary tract infection from a lower urinary tract infection;
3. characterize the common pathogens causing urinary tract infection;
4. discuss the appropriate laboratory diagnosis and treatment of each infection; and
5. propose measures by which urinary tract infection can be prevented.

Among the most commonly encountered infections are urinary tract infections.
Community acquired UTI is more common in women and are mostly uncomplicated. This
is due to the shorter urethra and the proximity of the anal opening to the urethral orifice in
females. In hospitalized patients, UTI usually develops as a complication of prolonged urethral
catheterization, making it harder to treat because most are resistant to various antibiotics.
The urinary tract is usually protected from pathogenic organisms by the frequent flushing
action of urination and by the constant sloughing of the epithelium. The acidity of normal
urine also inhibits the growth of many microorganisms. In most cases of UTIs, the causative
organism is derived from the flora of the colon. There are two routes by which bacteria can
reach the kidneys: (1) through the bloodstream, and (2) ascending infection from the lower
urinary tract. The most common route is by ascending infection.

Predisposing Factors to UTI

1. Gender – UTI is more common in females especially school aged girls and those above
60 years of age.
2. Mechanical factors – catheterization, sexual intercourse, kidney stones, and improper use
of tampons and douches contribute to contracting UTI
364 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

3. Metabolic disorders – increased sugar content of urine, due to diabetes for instance, is
conducive for bacterial growth.
4. Anatomic abnormalities of the urinary tract – can lead to obstruction or incomplete
voiding of urine or reflux of urine.

Etiology
A. Common etiologic agents
1. Enterobacteriaceae – Escherichia coli (50%–80% of cases); Klebsiella pneumoniae
2. Staphylococcus saprophyticus

3. Enterococci (Enterococcus faecalis)

4. Opportunistic pathogens – Pseudomonas, Proteus, Serratia
B. Less common etiologic agents
1. Bacteria – S. aureus, Corynebacterium, Lactobacilli
2. Yeast – Candida
3. Viruses – Adenovirus type 2

Escherichia coli
Escherichia coli is a gram negative bacillus that is part of the normal microbial flora of
the human body, specifically the colon hence, infections are endogenous. It is a member of the
family Enterobacteriaceae. Improper washing after defecation is a factor that promotes entry
of the organism into the urinary tract, most specially in women because of the proximity of the
urethral orifice to the anal opening. It is the most common cause of community acquired UTIs.

Proteus mirabilis
Proteus mirabilis are gram negative bacilli that are members of the family
Enterobacteriaceae. The organism produces urease which causes alkalinization of urine, making
the patient more prone to development of urinary stones. It is the second most common cause
of community acquired UTI and is a major cause of nosocomial infections.

Serratia spp.
Serratia spp. are also gram negative bacilli that belong to the family Enterobacteriaceae.
These organisms are major entities in nosocomial infections. Almost all infections caused
by these organisms are associated with underlying disease, changing physiological patterns
 Infections of the Urinary Tract 36

immunosuppressivetherapy, or mechanical manipulations of the patient. The most frequently

isolated species is Serratia marcescens. The organism produces a bright red pigment called
prodigiosin which imparts a red color to the colonies. It is also associated with outbreaks of
UTI, wound infections, pneumonia, and septicemia.

Enterococcus faecalis
Enterococci are part of the normal enteric flora, belonging to the family Enterobacteriaceae.
They grow in 6.5% NaCl and are more resistant to penicillin G. Enterococcus faecalis is the most
common among the Enterococci. These are also frequent causes of nosocomial infections,
particularly in intensive care units. Enterococci are transmitted from one patient to another
primarily from the hands of hospital personnel. In patients, the most common sites of infection
are the urinary tract, wounds, biliary tract, and blood. In urinary tract infections, enterococci are
usually cultured along with other species of bacteria.

Staphylococcus saprophyticus
Staphylococcus saprophyticus is a gram positive coccus and a common cause of urinary tract
infections in sexually active young women. It is a common colonizer of the urinary tract.

Pathogenesis
In most patients with urinary tract infection, the infecting organism is derived from the
patient’s own fecal flora. There are two routes by which bacteria can reach the kidneys:
(1) through the bloodstream, and (2) from the lower urinary tract (ascending infection).
Ascending infection is the most common cause of clinical pyelonephritis. The infection begins
with colonization of the distal urethra and introitus by the invading organism. The organism
then ascends to the urinary bladder. Instances that can cause incomplete voiding of the urine
can lead to urine stasis, allowing the bacteria to further multiply. Chronic infection leads to
ascent of the organism to the kidneys, leading to the development of an upper urinary
tract infection.
All portions of the urinary tract may be involved in the infection. Cystitis is
inflammation of the urinary bladder. It is the most common type of urinary tract infection
and is most commonly caused by E. coli. Other common causes include Proteus, Klebsiella,
Enterococcus, Pseudomonas, Enterobacter, Staphylococcus saprophyticus, Staphylococcus epidermidis,
and Candida albicans.
Inflammation of the urethra is called urethritis. The organisms involved are usually
sexually transmitted, the common causes of which are Neisseria gonorrheae and Chlamydia
trachomatis (non gonococcal urethritis or NGU). Inflammation of the kidneys, particularly of
366 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

the tubules, is called pyelonephritis. The most frequent cause is E. coli. It most often is the
result of an ascending infection, but the offending organism may also reach the kidneys through
the bloodstream.
Complications arise when prompt and adequate treatment is not instituted or when the
infection is associated with urinary tract abnormalities. These include bacteremia and septic
shock, severe renal damage, or end stage chronic pyelonephritis leading to renal failure.

Clinical Manifestations
Table 20.1 Common clinical manifestations of urinary tract infections
Clinical Condition Characteristic Symptom
Lower Urinary Tract Infection
Urethritis Dysuria, frequency, urgency
Cystitis Suprapubic pain and tenderness, frequency, occasional
hematuria
Urethrocystitis May be asymptomatic; usually malodorous urine, especially
in women; incontinence
Upper Urinary Tract Infection
Acute Pyelonephritis Flank pain, fever, and chills; hematuria; (+) kidney punch

Diagnosis

• Urinalysis
Urinalysis is one of the oldest clinical laboratory procedures. The diagnosis involves
gross observation and assessment of general appearance of urine, dipstick analysis, and
microscopic examination of formed elements in urine. It is one of the most commonly
performed laboratory tests. It is important that an appropriate specimen be collected.
Urine may be collected through the following methods:
1. Clean voided mid stream technique
2. Suprapubic aspiration
3. Diagnostic catheterization
4. From an in dwelling catheter
5. During cystoscopy, ureteral catheterization or retrograde pyelography
The usual practice is to use the clean voided mid stream technique. This technique
should be properly explained by the nurse or preferably, the physician. If possible,
the instructions should be both verbal and written. Patients should be instructed to void
the first few milliliters of urine before beginning the collection. In most cases the patient,
 Infections of the Urinary Tract 36

male or female, should gently cleanse the urethral meatus with a swab and then rinse.
Although specimens collected randomly during the day are satisfactory, the most
informative specimen is the first urine voided in the morning. Overnight urine reflects
a prolonged period without fluid intake, so formed elements are concentrated.
Freshly voided urine is clear to slightly hazy and colored yellow (or straw colored).
The intensity of the color reflects the degree of concentration. Very dilute urine is
almost colorless. Examination of the urine sediment can demonstrate the presence of
white blood cells, red blood cells, epithelial cells, casts, crystals, and infectious organisms
(bacteria, yeasts, trichomonas). Normal individuals have occasional (0–2/hpf) white cells
in their urine sediment and occasional red cells (1–3/hpf). The presence of bacteria in
the urine does not necessarily mean that the individual has urinary tract infection
because these may just represent contamination. Elevation of the white blood cells in
the urine sediment is highly suggestive of urinary tract infection. The presence of casts
in the urine, especially white cell casts, is highly suggestive of an upper urinary tract
infection (acute pyelonephritis).

• Urine Culture
Urinalysis results will only reveal the probability of urinary tract infection or not.
The best method to diagnose urinary tract infection is to do urine culture. Urine
collection follows the same principles as in doing a routine urinalysis. Immediately after
collection of the urine, the specimen should be sent to the laboratory where it should be
examined within 15 minutes. If the urine is made to stand at room temperature without
bacterial examination, in a matter of two hours it will give a false positive culture.
All urine culture reports routinely include colony count, as well as identification of
the organisms. If the colony count is 100,000/mL or more in a clean voided mid stream
specimen, there is significant bacteriuria. If there is less than 1,000 colonies/mL, this
represents contamination. If the number of colonies is between 1,000 and 100,000/mL
and there is a single microbial species, this represents possible or probable infection
and the culture should be repeated.

Treatment
Any antibiotic eliminated by the kidney and to which the organism is susceptible
can be used effectively and safely. Culture and susceptibility testing are important for
pyelonephritis and complicated cases, and when the patient is not responding to the antibiotic
therapy. For uncomplicated infection with E. coli, the recommended drug of choice is
Trimethoprim Sulfamethoxazole given 3–7 days. For infections with Proteus and Pseudomonas,
Fluoroquinolone is the antibiotic of choice. In cases of acute pyelonephritis, Fluoroquinolones
or third generation cephalosporins may be given for a period of 3–10 days. Increased water
intake is also often advised to avoid dehydration.
368 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• The urinary tract is protected by the frequent urination that flushes away pathogenic
organisms, the acidity of the urine, and the constant sloughing of the epithelium.

• Metabolic disorders like diabetes, trauma like catheterization, improper use of tampons,
and anatomic abnormalities of the urinary tract can predispose an individual to develop
urinary tract infections.

• Urinary
women.
tract infections are common worldwide, however, it is most common among

• UTIs are commonly caused by the indigenous flora of the human body and most
infections are uncomplicated.

• About 80% of community acquired UTIs are due to Escherichia coli.

• UTI may involve the urethra (urethritis), urinary bladder (cystitis), or the kidneys and its
tubules (pyelonephritis).

• The most commonly employed method of diagnosis of UTI is urinalysis. The best
specimen is early morning midstream catch urine.

• The definitive diagnosis is culture. A colony count of less than 1,000/mL indicates
contamination, 1,000/mL to 100,000/mL is possible bacteriuria and more than
100,000/mL urine indicates significant bacteriuria.
• Trimethoprim Sulfamethoxazoleis effective for uncomplicated cases of UTI
 Infections of the Urinary Tract 369

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Case: A 28 year old housewife consulted a physician because of scanty urine, increased
frequency of urination, and burning sensation at the end of urination. Urinalysis showed
numerous bacteria, white blood cells, and pus cells. Diagnosis is Urinary Tract Infection.

1. The most common cause of UTI in humans.

a. S. aureus c. E. coli
b. K. pneumoniae d. Serratia
2. Urine culture colony count of more than 100,000/mL urine is indicative of:
a. Probable infection c. Significant bacteriuria
b. Contamination d. None of the above
3. The most common method of urine collection.
a. Catheterization
b. Midstream catch early morning urine
c. Suprapubic aspiration
d. None of the above
4. Which of the following bacteria is associated with stone formation?
a. E. coli c. Serratia spp.
b. Proteus spp. d. Enterococcus faecalis
5. Which of the following common causes of UTI produces red pigment?
a. Staphylococcus aureus c. Pseudomonas aeruginosa
b. Candida albicans d. Serratia marcescen
370 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

6. Females are more prone to UTI because:

a. Women have shorter urethra
b. Close proximity of the urethra to the anus
c. Women have monthly menstruation
d. A and B
e. A, B, and C
7. The most common cause of non gonococcal urethritis.
a. E. coli c. P. vulgaris
b. C. trachomatis d. P. aeruginosa
8. The organism which is a common cause of UTI in sexually active young women.
a. E. coli c. S. saprophyticus

b. S. epidermidis d. Klebsiella
9. Fever, chills, flank pains, and positive kidney punch are suggestive of:
a. Cystitis c. Pyelonephritis
b. Urethritis d. Urethrocystitis
10. Which of the following is correct about urinary tract infections?
a. It is least likely caused by indigenous flora of the human body.
b. UTI must always be treated with antibiotics.
c. Infections are always the result of trauma to the urinary tract.
d. Organisms may reach the kidneys through the bloodstream
 CHAPTER

21 Infections of the Eyes

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. recognize manifestations of common eye infections;
2. characterize the common pathogens causing infections of the eyes;
3. discuss the appropriate laboratory diagnosis and treatment of each infection; and
4. propose measures to prevent the development of eye infections.

Infections involving the eyes may be in the form of: (1) conjunctivitis, inflammation or
infection involving the conjunctiva; (2) keratitis, inflammation or infection involving the
cornea; and (3) keratoconjunctivitis, inflammation or infection involving both the conjunctiva
and the cornea. Eye infections may be caused by bacteria or viruses.

Bacterial Infections
Bacterial Conjunctivitis
Bacterial conjunctivitis is also known as pink eye conjunctivitis and is highly contagious.
The infection can be transmitted through: (1) human to human transmission via contact with
eye and respiratory discharges; (2) contaminated fingers; and (3) fomites like clothing, facial
tissues, eye makeup, eye medications, and ophthalmic instruments. Manifestations of the
infection include: (1) eye irritation; (2) reddening of the conjunctiva; (3) swelling of the eyelids;
(4) mucopurulent discharge; and (5) sensitivity to light (photophobia)
372 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Etiologic Agents
Haemophilus influenzae biogroup aegyptius
Haemophilus influenzae biogroup aegyptius (Koch Weeks bacillus) is a gram negative rod
or coccobacillus. It is associated with epidemics of acute, purulent conjunctivitis that commonly
occur during the summer months. Its virulence is due to the pili which function for attachment.
Mechanical transmission through gnats has also been suspected as a mode of transmission.

Streptococcus pneumoniae
Streptococcus pneumoniae are gram positive diplococci, arranged in pairs or short chains and
are encapsulated. The organism is alpha hemolytic when grown aerobically and beta hemolytic
when grown anaerobically. One side of the bacteria is slightly pointed assuming a “lancet
shape” appearance. The virulence can be attributed to adhesins on its surface, capsule, toxin
pneumolysin, and IgA protease.

Staphylococcus aureus and Pseudomonas aeruginosa

(see chapter 16)
Chlamydia trachomatis
Chlamydia trachomatis resembles gram negative bacteria, however, it does not have
peptidoglycan in its outer wall. The cell membrane has very high lipid content. The bacteria
do not have the components to produce its own energy or ATP and are dependent on the host
hence they are classified as obligate intracellular parasites. C. trachomatis have round vacuolar
inclusions filled with glycogen and are susceptible to sulfonamides. It is associated with three
types of eye infections—conjunctivitis, inclusion conjunctivitis, and trachoma.
Conjunctivitis caused by C. trachomatis is usually acquired through swimming in
non chlorinated or poorly chlorinated swimming pool waters, hence, it is sometimes referred to
as “swimming pool conjunctivitis.” In adults, it may be a result of the transfer of the organisms
from the genitals to the eyes and often occur concurrently with non gonococcal urethritis or
cervicitis. It manifests as mucopurulent eye discharge and occurs concurrently with chlamydial
nasopharyngitis or pneumonia.
Infection in newborns is referred to as inclusion conjunctivitis that can be acquired
upon passage through the infected birth canal. In adults, it is usually associated with genital
infections. Conjunctions is caused by serotypes D to K and is manifested by swelling of the
eyelids with mucopurulent discharge, keratitis, corneal infiltrates, and corneal vascularization.
Trachoma is a chronic keratoconjunctivitis caused by serotypes A, B, Ba, and C.
It is a leading cause of preventable blindness in developing countries. It can be transmitted
eye to eye by droplet, fomites, and by eye seeking flies. It can also be transmitted through the
feces and respiratory droplets. It is endemic in areas characterized by poor living conditions
 Infections of the Eyes 373

The infection initially presents as follicular conjunctivitis with diffuse inflammation involving
the entire conjunctiva which may progress to conjunctival scarring producing in turned eyelids.
The in turned eyelids cause constant abrasion of the cornea leading to ulceration, scarring,
invasion of vessels into the cornea, pannus formation, and eventually loss of vision.

Figure 21.1 Trachoma caused by Chlamydia trachomatis serotypes

Source: OMICS International

Neisseria gonorrheae
Neisseria gonorrheae, also known as gonococcus, is a common cause of sexually transmitted
diseases. It can cause a neonatal infection known as ophthalmia neonatorum, which is acquired
upon passage through the infected birth canal. In adults, it is transmitted through finger to eye
contact with infectious genital secretions. The infection is manifested by redness and swelling of
the conjunctiva with purulent eye discharge. If untreated, the infection may progress to corneal
ulceration, perforation, and eventually blindness. Ophthalmia neonatorum can be prevented by
instilling 1% silver nitrate immediately after delivery (Crede’s prophylaxis) or 1% tetracycline
eye ointments or 0.5% erythromycin eye ointments.

Viral Infections
Eye infections due to viruses may also take the form of conjunctivitis, keratitis, or
keratoconjunctivitis. The infection is highly contagious and can spread through airborne means
like sneezing and coughing. The infection is self limited. Clinically, viral conjunctivitis differs
from bacterial conjunctivitis in that there is no purulent eye discharge.

Etiology
Adenoviruses
Adenoviruses are double stranded DNA viruses. A unique characteristic of these viruses is
the fiber that projects from each penton bases. The fiber functions for attachment and acts as
hemagglutinin. Adenoviruses are latent in the adenoids and tonsillar tissues and have affinity to
mucous epithelium of the conjunctivae. The virus is resistant to mild chlorination
374 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Eye infections caused by adenoviruses can range from mild to severe infections. The mild
eye infection is called “swimming pool” conjunctivitis because the source of infection can be
traced to poorly chlorinated swimming pool waters or ponds. It is most commonly caused by
serotypes 3 and 7. Sometimes the conjunctivitis is accompanied by pharyngitis hence called
pharyngoconjunctival fever.
A more severe eye infection caused by Adenoviruses is epidemic keratoconjunctivitis.
Adenovirus types 8, 19, and 23 are the most common causes of epidemic keratoconjunctivitis.
This infection is more common in adults and considered an occupational hazard following
exposure to dusts and other eye irritants. It may present as acute conjunctivitis, keratitis and
later leave residual sub epithelial opacities in the cornea.

Figure 21.2 Epidemic keratoconjunctivitis

caused by adenovirus. Note the absence
of purulent eye discharge.
Source: OMICS International

Enterovirus 70 and Coxsackie A24 virus

Enterovirus 70 and Coxsackie A24 virus cause acute hemorrhagic conjunctivitis, a highly
contagious eye infection. The infection is characterized by conjunctival congestion, vascular
dilatation, and onset of edema. There is no available treatment. Management is purely
symptomatic unless a secondary bacterial infection occurs which will necessitate antimicrobial
and steroid treatment. Most of the time, the infection resolves spontaneously.

Herpes simplex virus

Herpes simplex virus type 1 causes severe keratoconjunctivitis and recurrences are common,
which may present as dendritic keratitis and corneal ulcers which may lead to blindness. HSV
type 1 is the second leading cause of blindness in the United States.

Measles virus
Conjunctivitis is only one of the classical manifestations of Rubeola observed in children.
It is also associated with photophobia or sensitivity to light
 Infections of the Eyes 375

CHAPTER SUMMARY

• Infections involving the eyes may be classified as conjunctivitis, keratitis, or

keratoconjunctivitis. These infections may be caused by bacteria or viruses.

• Among the bacterial causes are Staphyloccusaureus, Strepcoccus

aegyptius, Neisseria gonorrheae, and Pseudomonas aeruginosa.
pneumoniae, Haemophilus

• Viral
measles
causes are Enterovirus 70, Coxsackie A24, Herpes simplex virus type 1, and
virus.

• The infections are highly contagious and can be transmitted through: contact with eye
and respiratory discharges, contaminated fingers or fomites like clothing, facial tissues,
eye makeup, eye medications, and ophthalmic instruments.

• The common manifestations of eye infections are eye irritation, reddening of the
conjunctiva, swelling of the eyelids, watery to mucopurulent discharge, and sensitivity to
light (photophobia).

• Chlamydia trachomatis resembles gram negative bacteria but do not have peptidoglycan
in its outer cell wall.

• Trachoma is a form of severe keratoconjunctivits that may lead to blindness.

• Neisseria gonorrhea causes ophthalmia neonatorum, acquired by the newborn upon
passage through the infected birth canal. The prophylaxis for this infection is called
Crede’s prophylaxis and involves instilling 1% silver nitrate, erythromycin or tetracycline
to both eyes of the newborn.
• Adenoviruses cause “swimming pool” conjunctivitis and the source is poorly chlorinated
swimming pool water or contaminated ponds.

• Adenovirus is also associated with epidemic keratoconjunctivitis that may lead to residual
corneal opacities.

• Herpes simplex virus type 1 can cause severe keratitis which can also lead to blindness.
• Enterovirus 70 and Coxsackie A virus cause a highly contagious eye infection, acute
hemorrhagic conjunctivitis
 Infections of the Eyes 377

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Case: A 10 year old boy scout is experiencing a fever, sore throat, and redness of the
eyes after participating in a 3 day camping activity. He was diagnosed with acute
pharyngoconjunctival fever.

1. The most common cause of this eye infection is:

a. Herpes simplex virus c. Coxsackie A24
b. Enterovirus 70 d. Adenovirus
2. Which of the following is correct regarding the causative agent for the above case?
a. The causative agent is a single stranded DNA virus.
b. It possesses fibers attached to penton bases.
c. It is easily destroyed by chlorination.
d. It is latent in the neurons.
3. Which of the following is correct regarding epidemic keratoconjunctivitis due to
adenovirus?

a. The most common causes are serotypes 6 and 11.

b. It is associated with dendritic keratitis.
c. It leaves residual corneal opacities.
d. It is the most common cause of blindness.
4. Acute hemorrhagic conjunctivitis is caused by which of the following viruses?
a. Enterovirus 70 d. A and B
b. Coxsackie A24 e. A, B, and
c. Adenoviruses
378 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

5. A common cause of purulent conjunctivitis that may be transmitted through gnats.

a. Chlamydia trachomatis c. Streptococcus pneumonia
b. Haemophilus aegyptius d. Neisseria gonorrheae
6. The mode of transmission of ophthalmia neonatorum.
a. Through traumatic inoculation
b. Upon passage through infected birth canal
c. Through fomites
d. From aerosolized organism
7. Which of the following is correct regarding C. trachomatis?
a. It has no peptidoglycan in its outer wall.
b. It is sensitive to sulfonamides.
c. It is an obligate intracellular parasite.
d. A and B
e. A, B, and C
8. The second most common cause of blindness in the US which is characterized by
dendritic keratitis and corneal ulcerations.
a. C. trachomatis c. Adenovirus
b. HSV type 1 d. N. gonorrheae
9. Which of the following is NOT a manifestation of viral conjunctivitis?
a. Redness c. Purulent discharge
b. Swelling d. Photophobia
10. Which of the following organisms possesses glycogen filled vacuoles?
a. Chlamydia trachomatis c. Haemophilus aegypticus
b. Neisseria gonorrheae d. Streptococcus pneumoni
 CHAPTER
Infections of the
22 Nervous System

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. recognize infections of the nervous system based on clinical manifestations;
2. differentiate the various infections involving the nervous system;
3. describe the characteristics of the causative organisms of each infection of the nervous
system;

4. compare the important arbovirus diseases as to their etiology and vector;

5. discuss the appropriate laboratory diagnosis and treatment of each infection; and
6. propose measures for the prevention and control of infections involving the nervous
system.

Definition of Terms
• Encephalitis – inflammation or infection involving the brain parenchyma
• Encephalomyelitis – inflammation or infection involving the brain and the spinal cord
• Meningitis – inflammation or infection involving the leptomeninges (pia mater and
arachnoid mater)

• Meningoencephalitis – inflammation involving the brain and the meninges

• Myelitis – inflammation of the spinal cord
380 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The central nervous system (CNS) is impenetrable to infectious agents because it is

surrounded by cerebrospinal fluid and meninges and enclosed by bones. The blood brain barrier
is another structure that protects the CNS from large molecules and microorganisms. Because
of these protective barriers and structures, the brain has no indigenous flora. Microorganisms
gain access to the CNS through the bloodstream and the lymphatics and through trauma.
Infections of the nervous system may present with non specific manifestations of fever and
headache that most would consider benign. Clinical clues that will suggest infections of the
nervous system include seizures, altered consciousness, or localizing signs.

Bacterial Meningitis
Acute Bacterial Meningitis
Bacterial meningitis is a suppurative infection of the meninges and subarachnoid space
with associated inflammation of the CNS. Groups that are at a high risk for development of
bacterial meningitis are children between six to twelve months of age. The most common cause
of this disease in newborns is Escherichia coli. Other common causes are Group B Streptococci
(Streptococcus agalactiae) and Listeria monocytogenes, the incidence of which is increasing in
the elderly and immunocompromisedindividuals. In older infants and children, majority of
infections were due to Haemophilus influenza type b, the incidence of which has been greatly
reduced due to the availability of the vaccine against the organism. Streptococcus pneumoniae
is currently the most common organism that causes community acquired meningitis in both
children over 1 month of age and adults followed by Neisseria meningitidis.
Meningitis is manifested by the classic clinical triad of fever, headache, and nuchal rigidity
(stiff neck) with associated nausea, vomiting, irritability, and back pain. Positive Kernig’s and
Brudzinski’s sign can be elicited. In infants, symptoms may be non specific and may include
irritability, restlessness, or poor feeding. The headache, nausea, and vomiting are signs of
increased intracranial pressure. For infants, an additional physical examination finding is a
bulging anterior fontanelle. Cerebrospinal fluid (CSF) examination is usually requested to give
a preliminary diagnosis if the meningitis is bacterial, viral, or fungal in nature. Table 22.1 shows
the characteristic CSF findings in bacterial meningitis
 Infections of the Nervous System 381

Table 22.1 Cerebrospinal fluid findings in bacterial, viral, and fungal meningitis
Normal Bacterial Viral Fungal
Appearance Watery and clear Turbid Clear Slightly turbi
Pressure 5–20 > 30 Normal to
(cm H2 O) slightly increased
Glucose 2.5–3.5 mmol/L < 2.2 Normal 1.6–2.5
(50–80 mg/100
mL)
Protein 0.18–0.45 g/L >1 <1 0.1–0.5
(g/L) (15–60 mg/mL)
WBC 0–5 (all > 500 (mostly < 1000 (mostly 100–500 (mostly
mononuclear) polymorphonuclears) monocytes) monocytes)

Neisseria meningitidis
This microorganism is also known as Meningococcus. It is a gram negative, coffee bean
shaped (or kidney bean shaped) diplococcus that is a transient flora of the nasopharynx. The
encapsulated types are virulent.

Mode of Transmission
Inhalation of respiratory droplets among contacts is the main mode of transmission of
meningococcus. Carriers can also transmit the infection through respiratory aerosols.

Clinical Findings
Neisseria meningitidis begins as throat infection. The microorganism will enter
the bloodstream causing bacteremia and go into the meninges causing meningitis.
Meningococcemia (overwhelming sepsis) with or without meningitis is a life threatening
infection. Thrombosis of small blood vessels and multi organ involvement are characteristic.
Petechiae or purpuric skin lesions over the trunk and the lower extremities is an important
presumptive sign of meningococcal infection. The disease may progress to massive
disseminated intravascular coagulopathy with destruction of the adrenal glands called the
Waterhouse Friderichsen syndrome.
382 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Laboratory Diagnosis
Blood and the CSF are the most useful specimens for culture of N. meningitidis.
Gram stain used as preliminary examination would show gram negative, coffee bean shaped
(kidney bean shaped) diplococci inside polymorphonuclear cells (Figure 22.1). Counter
immunoelectrophoresis,agglutination, or latex particles coated with specific antibodies can also
be used to detect polysaccharide antigen.

Treatment and Prevention

Penicillin is the drug of choice but resistance to this drug is significantly increasing.
Alternative antibiotics are broad spectrum cephalosporins and chloramphenicol. For the
treatment of carriers, minocycline and rifampicin are recommended. For contacts, the
prophylaxis includes sulfonamides and rifampicin for sulfonamide resistant strains.

Figure 22.1 Gram stain of

N. meningitidis in CSF with
associated PMNs
Source: OMICS International

Listeria monocytogenes
L. monocytogenes are short, motile, gram positive bacilli that appear individually, in pairs
or chains. The bacteria are cold loving (capable of growth at 1 °C) but are also capable of
growth at 45 °C and in high salt concentration. In healthy adults, disease is usually mild or
asymptomatic. Listeria monocytogenes mainly infects immunocompromisedindividuals.

Mode of Transmission
The primary source of infection is ingestion of contaminated food products but
transplacental transmission is also common during pregnancy or at birth. It has a special affinity
for growth in the CNS and the placenta. Infection during pregnancy may lead to spontaneous
abortion or stillbirth
 Infections of the Nervous System 38

Clinical Findings

In newborns, it may present as early onset listeriosis when acquired transplacentally

or late onset listeriosis when acquired during or right after delivery. A severe form of early
onset listeriosis which present with granuloma and abscess formation in several organs, is
granulomatosis infantiseptica. Late onset infection presents as meningitis or a combination of
meningitis and encephalitis with septicemia.

Laboratory Diagnosis
Diagnosis is through culture of blood, spinal fluid, or the placenta in selective media with
cold enrichment. Observation of tumbling end to end motility in liquid or semi solid media is
also useful in initial identification.

Treatment and Prevention

Penicillin or ampicillin either singly or combined with gentamicin is the treatment
of choice for listeriosis. Prevention is by avoiding eating contaminated food products and
thorough washing of raw vegetables.

Granulomatous Meningitis
Granulomatous meningitis is characterized by the formation of granulomas. It is a chronic
type of meningitis commonly caused by Mycobacterium tuberculosis and Cryptococcus neoformans.
It is characterized by remissions and relapses.

Tuberculous Meningitis
Tuberculous meningitis most commonly affects children younger than 6 years old, however,
it is rarely seen in less than 4 months of age. It usually appears 3 6 months after initial infection
and accompanies miliary tuberculosis in 50% of cases. Unrelenting headache, stiff neck, fever,
fatigue and night sweats are characteristics of tuberculous meningitis. These manifestations
together with the CSF picture are suggestive of the infection.
Aids in the diagnosis include a history of contact with an adult with tuberculosis, a positive
tuberculin skin test (including siblings), and a CSF examination to include acid fast staining of
the CSF. Treatment involves giving of quadruple anti TB regimen.
384 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Fungal Meningitis
Cryptococcus neoformans and Cryptococcus gatti are the most common causes of fungal
meningitis. Other causes are Histoplasma capsulatum and Coccidiodes immitis.
C. neoformans is an encapsulated yeast, a common saprophyte of the soil particularly
soil enriched with pigeon droppings. It is monomorphic (exists only as yeast) and of low
virulence. It is distributed worldwide. The fungus is opportunistic and only cause infection in
immunocompromisedindividuals and infections tend to be severe and fatal. Its virulence is
mainly due to its capsule and its ability to produce melanin, both of which protect C. neoformans
from phagocytosis. It is able to grow at 37 °C.

Mode of Transmission
Cryptococcal meningitis is acquired by inhalation of the fungus from the environment.
Infection spreads from the lungs into the blood, affecting the CNS.

Clinical Findings
The patients may experience headaches, nausea, vomiting, loss of vision, and other focal
neurologic findings of several weeks’ duration. The classical manifestation of meningismus is
usually absent in cryptococcosis.

Diagnosis
Diagnosis is made by direct examination of the CSF fluid stained with India ink or
Nigrosin to demonstrate the capsule of the fungus (Figure 22.2). CSF examination will show
high opening pressure, mononuclear cell pleocytosis, increased protein concentration and low
glucose concentration.

Figure 22.2 India ink preparation

demonstrating the capsule of
Cryptococcus neoformans

Treatment and Prevention

Treatment consists of Amphotericin B and Flucytosine for 4–8 weeks followed by
fluconazole. Lifelong maintenance therapy with fluconazole is required
 Infections of the Nervous System 38

Acute Viral Aseptic Meningitis or Encephalitis

Viral meningitis is generally self limiting. Encephalitis is a more serious disease causing
significant morbidity and mortality. Cerebral dysfunction is a prominent feature of viral
encephalitis. The most common cause of viral encephalitis is the Enteroviruses—Poliovirus
and Coxsackie virus. Other common causes include Mumps virus, Herpes simplex virus,
Epstein Barr virus, and Arboviruses.
Viral meningitis may initially present flu like manifestations such as fever, malaise, sore
throat, and myalgia followed several days later by signs of meningeal irritation (headache and
stiff neck). For viral encephalitis, the characteristic feature is profound cerebral dysfunction.
Changes in the level of consciousness, abnormal behavior, seizures, and deterioration in
cognitive function are more common symptoms presented by the patient.
There are several factors that may increase the risk for viral encephalitis namely (1) age, as
some types of viral encephalitis are commonly seen in certain age groups, (2) geographical areas,
as arthropod or mosquito borne infections are common in certain regions like Southeast Asia,
(3) season of the year, and (4) weakened immune state.
Inflammation in the brain can cause damage which may lead to coma or death. Other
complications can be paralysis, lack of muscle coordination or muscle weakness, speech
impairment, and visual or hearing defect, and memory problems.
Diagnosis is based on detailed history, clinical presentation, and the characteristic
CSF findings. Treatment is basically supportive and symptomatic. Prevention includes
vaccination (if there is an available vaccine for the causative agent), good personal hygiene,
maintenance of clean surroundings to eliminate breeding grounds for mosquitoes, use of insect
repellents or insecticides, and wearing thick clothing.

Enteroviruses Tick borne virus

Poliovirus Powassan virus
Coxsackievirus Others
Herpesviruses Measles virus
HSV type 1 and type 2 Rubella virus
Epstein Barr Virus Mumps virus
Varicella Zoster Virus Rabies virus
Mosquito borne viruses
Togaviruses
Flaviviruses

Box 22.1 Common causes of viral encephalitis

386 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Other Infections of the Nervous System

Tetanus
Tetanus is caused by C. tetani, a gram positive bacilli that are strict anaerobes (hence
difficult to grow in culture) and capable of producing endospores. The spore is located at the
terminal end of the bacillus giving rise to a “drumstick” or “tennis racket” appearance. It is
a common contaminant of the soil and are transient colonizers of the gastrointestinal tract
of animals and humans. The virulence of C. tetani is due to the neurotoxin produced by the
organism known as tetanospasmin that inhibits the release of inhibitory neurotransmitters
(gamma aminobutyric acid [GABA] or glycine) that leads to unregulated excitatory synaptic
activity resulting to spastic paralysis.

Clinical findings
Tetanus may present as generalized tetanus or localized tetanus. The most common form is
generalized tetanus and manifests as trismus or lockjaw (due to involvement of the masseter).
Other manifestations are risus sardonicus (sardonic smile due to sustained contraction of
the facial muscles), and persistent spasms of the back and neck muscles (opisthotonus).
In localized tetanus the infection is confined to the muscle at the primary site of infection.
Tetanus neonatorum, tetanus in the newborns, had a high mortality rate in underdeveloped
and developing countries before the Expanded Program of Immunization (EPI) by WHO.
The primary focus of infection is the umbilical stump.

a b c

Figure 22.3 a Micrograph showing the characteristic “drumstick” appearance

of Clostridium tetani. Characteristic spastic paralysis: b opisthotonus and
c risus sardonicus

Laboratory Diagnosis
The diagnosis of tetanus is based mainly on the clinical presentation. Culture is useful
however it only yields positive cultures 30% of the time as the bacteria are easily destroyed after
exposure to air. Another test is the tetanus antitoxin neutralization test in mice
 Infections of the Nervous System 387

Treatment and Prevention

The treatment for tetanus is thorough debridement and cleansing of the wound, antibiotics
(penicillin), passive immunization with tetanus immunoglobulin, and vaccination with tetanus
toxoid. Prevention is done through vaccination with tetanus toxoid given in three doses and
booster doses every ten years. Pregnant women are given the tetanus toxoid during the last
trimester of pregnancy.

Botulism
The causative agent of botulism is C. botulinum, a gram positive, anaerobic bacillus
capable of producing spores. The bacteria produce seven toxins collectively called botulinum
toxin that prevents the release of the neurotransmitter acetylcholine leading to a flaccid
paralysis. Regeneration of the nerve endings is required for the recovery of the function of the
involved muscle.

Clinical Findings
There are three forms of botulism that have been identified so far: classical or food borne,
infant, and wound botulism. The classical or food borne botulism is associated with ingestion
of canned food that is improperly canned and cooked, as well as improperly smoked fish. Infant
botulism is usually associated with ingestion of unpasteurized honey.
In the food borne type, symptoms develop one to two days after consumption of the
contaminated food. These symptoms include dizziness and weakness, accompanied by dry
mouth, blurred vision with dilated pupils, constipation, and abdominal pain. During the
entire course of the disease, the patient’s sensorium remains clear. Flaccid paralysis is seen in
progressive disease leading to bilateral descending weakness involving the peripheral muscles.
Respiratory paralysis may also develop, which may result in death.
Infants experience flaccid paralysis, seen as loss of muscle tone, hence the description
floppy baby (Figure 22.4). Ingestion of food (such as unpasteurized honey) contaminated

Figure 22.4 Loss of muscle tone in infant botulis

388 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

with botulinum spores from soil or dust is the most common mode of transmission for the
disease. C. botulinum has also been implicated in the causation of Sudden Infant Death
Syndrome (SIDS) or crib death. In both cases, death is due to respiratory paralysis.

Laboratory Diagnosis
Diagnosis is confirmed by culture of the patient’s feces or the suspected food sample. Toxin
activity can be tested by mouse bioassay.

Treatment and Prevention

Management involves an antitoxin that must be promptly administered intravenously.
Trivalent antitoxin must be given to bind with the toxins in the bloodstream. The drug of
choice is penicillin. Supportive measures include maintenance of adequate ventilation. Gastric
lavage must also be done to remove the organism from the gastrointestinal tract.
Prevention includes adequate sterilization of canned foods. Boiling food for more than
20 minutes before consumption may destroy the toxin. In order to prevent possible infection
honey should not be given to children below 1 year of age.

Leprosy (Hansen’s Disease)

The etiologic agent is Mycobacterium leprae, formerly known as Hansen’s bacillus, an
acid fast bacillus that has high lipid in its cell wall. The lipid rich cell wall accounts for its
acid fastness. The organism is located intracellularly and found inside foam cells or lepra
cells (endothelial cells of blood vessels). It is probably the only bacterium that grows in the
peripheral nervous system. The organism has preference to the cooler areas of the body like the
skin, superficial nerves, nose, pharynx, larynx, eyes, and testicles.

Mode of Transmission
The organism is spread through inhalation of respiratory aerosols (most common) and skin
contact with the lesion or wound exudates. Prolonged contact is necessary for transmission
to occur.

Clinical Findings
Leprosy affects both the skin and the peripheral nerves. There are two forms of
leprosy: tuberculoid or lepromatous leprosy, although a borderline form of the disease is
also recognized. The infection is insidious in onset and manifestations develop slowly, as long as
20 years after contracting the infection depending on the patient’s immune response
 Infections of the Nervous System 389

a b c d

Figure 22.5 Clinical features of leprosy: a skin path characteristics of paucibacillary leprosy;
b typical leonine facies, saddle nose deformity, and eye changes; c skin lesions; and d typical
hand deformity
Source: Marcos et al., 2013; Mayrabem, 2014; and Jones, 2008

The skin lesions of leprosy may present as hypopigmented, anesthetic macular lesions about
1–10 cm in diameter, or discrete erythematous, infiltrated nodules 1–5 cm in diameter or as
diffuse skin infiltration. If the infection remains untreated, it will progress to nerve infiltration,
trophic ulcers, anesthesia (focal or diffused), bone resorption that may result to shortening of
digits, “saddle nose” deformity, and leonine facies (Figure 22.5). The differences between the
two types of leprosy is summarized in Table 22.2.

Table 22.2 Clinical differences between tuberculoid and lepromatous leprosy

Tuberculoid Form (neural form) Lepromatous Form (cutaneous form)
Skin Lesions Hypopigmented lesions Nodular lesions (diffuse)
Amount of bacilli in Paucibacillary (very few) Multibacillary (abundant)
lesions
Immune response Strong CMI reaction Strong antibody response; defective CMI
Sensory loss Complete Patchy
Disfigurement None Leonine facies, clawed hands, saddle
nose deformity
Lepromin test Positive Negative
Infectivity Low High
Erythema nodosum Absent Usually present
leprosum

Laboratory Diagnosis
Acid fast staining of the wound exudates or respiratory aerosol is usually done. Microscopy
is only sensitive for the lepromatous type but not the tuberculoid type. The organism cannot be
cultured in artificial laboratory media. The bacteria can only be grown in the footpads of mice,
armadillos, and chimpanzees. The lepromin test is diagnostic for tuberculoid type but not for
lepromatous type
390 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Treatment and Prevention

It has been recommended by WHO that tuberculoid form be treated with dapsone
combined with rifampicin for a minimum of 6 months. The lepromatous form should be
treated with dapsone, clofazimine, and rifampicin for 12 months. Lifelong treatment with
dapsone prevents relapse of the infection. The administration of BCG vaccine can help prevent
development of disease.

Poliomyelitis
Poliovirus is classified under the family of Picornavirus. It is a single stranded RNA
virus. The virus is “naked,” it has no envelope but its outer covering, the capsid, is resistant to
adverse environmental conditions. There are three poliovirus types—type 1, type 2, and type 3.
The most common cause of poliomyelitis is type 1. The virus is shed from the feces months
after infection.

Mode of Transmission
The disease is transmitted through ingestion of food and water contaminated with feces
containing the virus. It can also be acquired through contact with nasal secretions or mouth
droplets from untreated patients.

Clinical Findings

1. Inapparent, asymptomatic infection – accounts for 90% of poliomyelitis cases.

The infection is confined to the oropharynx and the gastrointestinal tract.
2. Abortive poliomyelitis – also referred to as minor illness. It presents with mild febrile illness
manifesting as headache, sore throat, nausea, and vomiting.
3. Non paralytic poliomyelitis – initially presents as minor illness and later manifests with
back pains and stiff neck.
4. Paralytic poliomyelitis – also referred to as major illness. It is biphasic, initially presenting
as minor illness and later developing asymmetrical flaccid paralysis without sensory
loss. This is due to the involvement of the motor neurons of the anterior horn cells of
the spinal cord. It may also involve the motor cortex of the brain. Involvement of the
medullary respiratory center is referred to as bulbar poliomyelitis. One or all four
extremities may be affected in spinal paralysis. Bulbar poliomyelitis may involve the
muscles of the vocal cords, pharynx, and respiratory muscles resulting to the death
of the patient
 Infections of the Nervous System 39

5. Post poliomyelitis syndrome – occurs years after the initial polio infection and involves
the previously affected muscle, showing marked deterioration of the residual function of
the muscle.

Laboratory Diagnosis
Diagnosis is based mainly on the clinical manifestations. Isolation of the virus from feces,
CSF, and throat secretions with cell culture techniques are helpful in the diagnosis however
they are not usually done in developing countries.

Treatment and Prevention

Treatment for poliomyelitis is mainly symptomatic and supportive. Pleconaril is a new
antiviral drug that inhibits penetration of the virus into the host cell. Its availability, however, is
limited and must be administered in the early stage of the infection.
There are 2 types of polio vaccine—live attenuated polio vaccine (OPV or Sabin vaccine)
and inactivated polio vaccine (IPV or Salk vaccine). Both vaccines contain the 3 types of
poliovirus. The Salk vaccine is considered safer than the Sabin vaccine because there is no
reversion to virulence, however, the Sabin vaccine is safer and easier to administer (oral), it also
costs less and provides longer immunity.

Rabies
The rabies virus is an RNA virus that belongs to the family Rhabdoviridae, a bullet shaped
virus. Rabies is primarily a disease involving warm blooded animals. It is most prevalent in
dogs worldwide, however, it is more prevalent in cats in the United States. The virus replicates
initially at the site of bite, travels along the peripheral nerves, reaches the CNS and is shed
through the saliva. Once the virus reaches the peripheral nerves it is sequestered from the cells
of the immune system.

Mode of Transmission
1. Bite of a rabid animal – the most common mode of transmission
2. Non bite:
a. licking open skin, scratching, patting or petting of the animal
b. inhalation of aerosolized virus in bat droppings
c. transplanted infected tissue (e.g., corneal transplant tissue)
392 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Envelope
(Membrane) Matrix protein
Glycoprotein

Ribonucleoprotein
Figure 22.6 Diagrammatic representation of the structure of the rabies virus

Clinical Findings
Rabies is a fatal disease and once the clinical disease is apparent, death is imminent.
The incubation period is variable and may range from 6 days to 6 months. The duration of the
incubation period depends on the following: (1) size of the inoculum; (2) location of the wound
(a major bite is closer to the CNS and a minor bite is farther from the CNS); (3) severity of
the wound; (4) host’s immune status; (5) condition of the animal prior to the bite (provoked or
not), and (6) age of the patient.
The initial site of multiplication of the virus is the muscle at the site of the bite, where the
virus remains for days to months before traveling to the peripheral nerves. From the peripheral
nerves, the virus gains access to the spinal cord, followed by rapid infection of the brain.
The prodromal stage is manifested by fever, pain or paresthesia at the bite site,
headache, fatigue, and anorexia. Two to ten days later, neurological manifestations appear.
The most characteristic symptom is hydrophobia (fear of water). Seizures, hallucinations,
and disorientation may also occur during this phase of the disease. Some patients may
exhibit paralysis as the only manifestation. Following the neurological phase, the patient
becomes comatose. Death occurs later due to neurologic and respiratory complications.
In some cases, rabies may present as paralytic polio (dumb type) with flaccid paralysis as
the only manifestation.

Laboratory Diagnosis
The most specific diagnostic tool for rabies is histopathologic demonstrationof
Negri bodies from the infected neurons. Antigen detection by immunofluorescence is also
widely used in the diagnosis of rabies
 Infections of the Nervous System 393

Treatment and Prevention

Treatment for rabies is only supportive once the symptoms have appeared. Pre exposure
prophylaxis is Human Diploid Cell Vaccine (HDCV) for individuals at risk like veterinarians,
animal handlers, and laboratory workers. The vaccine is given in three doses and provides
protection for two years.
Post exposure prophylaxis includes: (1) thorough washing of the wound with soap and
water; (2) immediate infiltration of Rabies Immunoglobulin (RIG) around the bite site;
and (3) rabies vaccine (HDCV) given in five doses (days 0, 3, 7, 14, and 28) intramuscularly
at different sites. The World Health Organization (WHO) recommends a sixth dose as an
additional margin of safety. Vaccination of domestic animals and removal of stray animals are
also important in the prevention and control of rabies.

Arboviral (Arthropod Borne) Encephalitis

Encephalitis caused by arbovirus infection. Most arboviral encephalitis is transmitted by
mosquitoes.

Japanese B Encephalitis
It is a common infection in the Far East. The etiologic agent is Flavivirus and Encephalitis
the vector is a mosquito (Culex mosquito). Common hosts are pigs and birds. The infection
initially presents with flu like symptoms like fever, chills, and body aches followed by
manifestations of encephalitis.
Prevention includes elimination of the vector and its breeding places, and avoidance of
mosquito bites by applying insect repellent, wearing thick clothing, and using mosquito nets.
Table 22.3 shows the important arbovirus infections.

Table 22.3 Important arbovirus infections

Disease Etiology Vector
Japanese B encephalitis Flavivirus Aedes mosquito
Eastern Equine encephalitis Togavirus Culiseta mosquito
Western Equine encephalitis Togavirus Culex mosquito
St. Louis encephalitis Flavivirus Culex mosquito
California encephalitis Bunyavirus Culex mosquit
394 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• Most infections in the central nervous system (CNS) involve the meninges and the
subarachnoid space (meningitis) or the brain (encephalitis). It may be caused by bacteria,
viruses, or fungi.

• The most common

by Streptococcus
cause of bacterial meningitis in newborns is Escherichia coli followed
agalactiae and Listeria monocytogenes.

• Haemophilus
while
influenza
Streptococcus
type B is the most common cause of bacterial meningitis in infants
pneumoniae and Neisseria meningitidis are common causes in adults.

• The classic clinical triad of meningitis are fever, headache, and nuchal rigidity (stiff neck).
Kernig’s and Brudzinski’s sign may not always be present.

• Cerebrospinal fluid examination through lumbar tap is the most commonly used method
for preliminary identification of the probable etiologic agent.

• Neisseria meningitides infections are acquired from infected individuals or carriers and
may present as meningitis, meningococcemia,or Waterhouse Friederichsen syndrome.

• Granulomatous meningitis is caused by Mycobacterium tuberculosis, Cryptococcus

neoformans, the most common cause of fungal meningitis. It is an encapsulated yeast with
low virulence.

• Viral meningitis is generally self limiting while encephalitis is a more serious disease
associated with high morbidity and mortality. Cerebral dysfunction is a prominent feature
of viral encephalitis.

• The manifestations of tetanus and botulism are both due to neurotoxins, tetanospasmin
and botulinum toxins respectively.

• Leprosy may be tuberculoid type or lepromatous type. It is associated with nerve

infiltration causing anesthesia and bone resorption resulting to shortening of digits,
saddle nose, and leonine facies.

• Poliomyelitis is caused by Poliovirus. 90% of cases are asymptomatic. The infection

may involve the respiratory center causing bulbar poliomyelitis. It is preventable
by vaccination.

• Rabies is a zoonotic infection transmitted to humans by warm blooded animals, most

commonly dogs. The pathognomonic manifestation is hydrophobia. Negri bodies are seen
in histopathologic examination of the brain.

• Most arboviral encephalitis is transmitted by mosquitoes. Japanese B encephalitis is

common in the Far East
 Infections of the Nervous System 395

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Case: A 10 year child with leukemia died because of meningitis. Further interview with the
family revealed that the older brother of the patient is raising pigeons as a hobby.

1. The most probable causative agent for this infection is:

a. Haemophilus influenzae c. Cryptococcus neoformans
b. Enterovirus d. Naegleria fowleri
2. The infection in the above case is acquired through:
a. Direct person to person contact for prolonged period
b. Inhalation of aerosolized spores
c. Ingestion of pigeon droppings
d. Blood transfusion
3. The causative agent is encapsulated which can be best demonstrated by:
a. Quellung capsular swelling c. Gram staining
b. India ink staining d. H & E staining

Multiple Choice.

4. The most common cause of meningitis in infants.

a. Haemophilus influenzae c. Listeria monocytogenes
b. Escherichia coli d. Streptococcus agalactia
396 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

5. The mechanism of action of tetanospasmin:

a. It inhibits the release of neurotransmitters for inhibitory synapses
b. It prevents the release of the neurotransmitter acetylcholine
c. It destroys the motor neurons in the anterior horn of the spinal cord
d. It destroys the motor cortex of the brain
6. It is commonly associated with home made canned foods and smoked fish.
a. Clostridium tetani c. Mycobacterium leprae
b. Clostridium botulinum d. Listeria monocytogenes

7. Which of the following bacteria can only be grown in the foot pads of
experimental animals?
a. Streptococcus pneumoniae c. Mycobacterium leprae
b. Streptococcus agalactiae d. Listeria monocytogenes
8. Lock jaw and risus sardonicus are manifestations of which of the following
diseases?

a. Botulism c. Meningococcemia
b. Rabies d. Tetanus
9. Which of the following is a correct statement regarding Sabin polio vaccine.
a. It is a killed vaccine
b. It is not associated with reversion to virulence
c. It provides longer protection
d. It is safer to use
10. Mode of transmission for poliomyelitis.
a. Fecal oral c. Parenteral
b. Sexual contact d. Transplacenta
 CHAPTER

23 Viral Exanthems

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. recognize the common viral exanthems based on clinical manifestations;
2. compare and contrast the viral exanthems based on etiology, mode of transmission, signs
and symptoms, and prevention;
3. describe the characteristics of the causative organisms of each viral exanthem;
4. discuss the appropriate laboratory diagnosis and treatment of each infection; and
5. propose measures for the prevention and control of the common exanthems.

A number of viruses and bacteria produce infections that have skin manifestations. These
skin manifestations may be a part of the disease and are referred to as exanthems. The most
common causes are viruses.
Skin lesions may take several forms. These may take the form of an alteration in skin color
that cannot be palpated (macule). Some are palpable solid lesions smaller than 0.5–1.0 cm
called papules. Nodules are palpable lesions that are larger than a papule. In some infections,
the lesions may take the form of vesicles, which are raised, fluid filled lesions less than 0.5 cm
in diameter. Larger forms of vesicles are called bullae. Pustules are similar to vesicles but
contain purulent material instead
398 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Infections Associated
with MaculopapularExanthem
Measles (Rubeola)
Measles is a common and highly contagious childhood exanthem associated with serious
complications. The infection is seen worldwide. It is common among children and young
adults but the incidence decreased dramatically since a vaccine has been developed and used
worldwide. The infection is limited to humans and there is no animal reservoir or host.

Etiologic Agent
The etiologic agent for measles is the Rubeola virus or the measles virus that belongs to
the family of Paramyxoviruses.There is only one stable serotype. The virus is a single stranded
RNA virus with envelope. On the envelope are two antigens—hemagglutinin (H antigen) and
fusion protein (F protein). Hemagglutinin is the viral attachment protein and the target of
neutralizing antibodies. The fusion of the viral protein with the host membrane is mediated
by the fusion protein resulting in the formation of multinucleated giant cells known as
syncytia formation.

Mode of Transmission
Measles is transmitted through inhalation of respiratory droplets. The infection is
contagious even before the onset of symptoms but most contagious during the prodromal
period.

Clinical Findings
During the initial stage of measles, the patient develops high grade fever with the 3 C’s
of measles—cough, coryza (common cold or runny nose), and conjunctivitis with photophobia.
This stage is highly infectious. The pathognomonic enanthem, Koplik’s spots, develops after
two days of prodrome. It is described as appearing like “grains of salt” over the inner cheek
opposite the second molar that lasts for only 24 to 48 hours. The Koplik’s spots may also
appear in other mucous membranes like the conjunctivae and vagina. This is followed by the
appearance of maculopapular rashes that undergo branny desquamation. Fever persists as the
temperature continues to increase as the rashes appear, and the child is sickest at this point.
The fever subsides once all the rashes have appeared throughout the body
 Viral Exanthems 399

Complications
Pneumonia is the most common and serious complication of measles, associated with
very high mortality of 60%, especially in immunocompromisedindividuals. There can also be
superimposed bacterial pneumonia on top of measles pneumonia. Otitis media is the second
most common complication. Post infectious encephalitis is a rare complication occurring in
less than 1% of cases but associated with about 15% mortality.
Subacute sclerosing panencephalitis (SSPE) is a very late and serious neurologic sequela
of measles. It occurs approximately 7 years after the initial measles infection and common in
children who had measles earlier than 2 years old. This occurs when wild type measles virus
persist in the brain and behave like a slow virus. This is manifested by changes in behavior and
personality, spasticity, myoclonic jerks, and blindness.

Figure 23.1 a Koplik’s spots: the

pathognomonic enanthem of measles
described as “grains of salt” opposite
the molar teeth. b Typical maculopapular
rashes of measles undergoing branny
a b
desquamation

Laboratory Diagnosis
Diagnosis of measles is based primarily on the clinical manifestations.

Treatment and Prevention

Treatment is symptomatic and supportive. Prevention is done through administration of
a live attenuated vaccine given in combination with Mumps and Rubella (MMR) at 2 years
of age. Immune globulin may be given to exposed susceptible individuals like those who are
immunocompromised.

Rubella (German Measles)

Rubella is one of the common viral exanthems in childhood together with measles,
chickenpox, Fifth disease, and roseola. It is a benign infection in children, however, infections
in adults can be more severe especially when acquired during the first two weeks of pregnancy,
when organ development occurs
400 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Etiologic Agent
The Rubella virus is a single stranded RNA virus under the genus Rubivirus and is
a member of the Togavirus family. There is only one stable serotype and humans are the
only hosts.

Mode of Transmission
The virus is mainly spread through inhaling respiratory droplets. However, transplacental
transmission can also occur when a seronegative mother becomes infected during pregnancy.

Clinical Findings
The rubella virus causes German measles, also known as the “three day measles.”
It manifests with fever, followed by the appearance of maculopapular rashes that lasts for
three days. The rashes are pruritic and unlike measles due to Rubeola virus, do not undergo
desquamation. It is associated with conjunctivitis without photophobia, post auricular
or occipital lymphadenopathy,and arthralgia. Pearly white dot like lesions, known as
Forschemer spots can be present in the palate. Comparison between rubella and rubeola
is listed in Table 23.1. The fever usually disappears as the rashes appear. Natural infection leads
to lifetime immunity.

Table 23.1 Comparison between Rubeola and Rubella

Features Rubeola Rubella
Common name Measles German measles
Etiology Paramyxovirus Togavirus
Mode/s of transmission Inhalation of droplets Inhalation, transplacental
Fever Present Present
3 C’s (coryza, cough, Present; conjunctivitis with Cough & coryza absent;
conjunctivitis) photophobia conjunctivitis without photophobia
Exanthem Maculopapular Maculopapular
Branny desquamation Yes No
Lymphadenopathy None Present
Arthralgia None Present
Congenital infection No Yes
Vaccine Yes Ye
 Viral Exanthems 401

Congenital rubella is the most serious outcome. The most common manifestations are
microcephaly, mental retardation, intrauterine growth retardation, cataracts and other ocular
defects, deafness, failure to thrive, and congenital heart disease. This is associated with high
mortality for the infected baby during pregnancy and during the first year after birth.

a b

Figure 23.2 a Characteristic rashes seen in patients with

Rubella infection or German measles. b Infant born with
Congenital Rubella with distinct rashes (“blueberry muffin”).

Laboratory Diagnosis
Diagnosis of Rubella is based primarily on the clinical manifestations. Diagnosis is
confirmed by presence of anti Rubella specific IgM.

Treatment and Prevention

Treatment is mainly symptomatic and supportive. Prevention involves administration
of vaccine (MMR vaccine) at 2 years of age as part of the immunization program for
infants. The primary goal of vaccination is to reduce the number of seronegative women
hence, lowering the incidence of congenital infection. The vaccine is contraindicated in
pregnant women.

Roseola Infantum (Exanthem Subitum or Sixth Disease)

Etiologic Agent
Sixth Disease is caused by Human Herpes Virus 6 (HHV6) that belongs to the family of
Herpesviridae. It primarily infects lymphocytes particularly CD4+ T cells. The virus is latent in
T cells and monocytes
402 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Mode of Transmission
The mode of transmission is still unknown but respiratory transmission and oral secretions
are most likely because the virus replicates in the salivary glands.

Clinical Findings
Roseola is manifested by sudden onset of high grade fever followed by a generalized rash
that lasts for two days. However, it may also cause a spectrum of illness including: fever without
rash, rash without fever, encephalitis, hepatitis, and more serious infections. Roseola is the most
common cause of febrile seizures in children.

Laboratory Diagnosis
Diagnosis of roseola is based on clinical manifestations.

Treatment and Prevention

Treatment for roseola is symptomatic. No vaccine is available for HHV6.

Erythema Infectiosum (Fifth Disease)

Etiologic Agent
Fifth Disease is caused by Parvovirus B 19, a single stranded DNA virus that belongs
to family Parvoviridae, the smallest among the DNA viruses. They are dependent on rapidly
replicating host cells or other viruses. The target of this virus is the erythroid progenitor cells,
causing lysis of these cells. The virus is associated with viremia and can cross the placenta and
infect the fetus.

Mode of Transmission
Fifth disease is transmitted by respiratory droplets and oral secretions. It can also be
transmitted by blood transfusions and vertical transmission from an infected mother.

Clinical Findings
Fifth disease is common in early school age children and less common in adults. It is a
biphasic infection consisting of the lytic stage and the immunologic stage. The initial or
lytic stage is manifested by mild signs and symptoms of upper respiratory tract infections.
Although the manifestations are mild during this stage, it is also the most contagious stag
 Viral Exanthems 403

of the infection. This is followed by the immunologic stage characterized by a generalized

lace like rash most prominent over the face (“slapped cheek” appearance) and arthralgia.
B19 infection in adults leads to polyarthritis involving the wrists, knees, and ankles.
The most serious complication is aplastic crisis in patients with chronic hemolytic anemia.
In pregnant women, it is associated with high risk of fetal death due to congestive heart
failure (hydrops fetalis).

Laboratory Diagnosis
Diagnosis of the fifth disease is based on the clinical presentation of the patient. Definitive
diagnosis can also be accomplished through ELISA and polymerase chain reaction (PCR).

Treatment and Prevention

There is currently no specific antiviral treatment or vaccine available for fifth disease.

Figure 23.3 Characteristic “slapped

cheek” appearance of infant with
erythema infectiosum

Infections Associated with Vesicular Exanthem

Varicella (Chickenpox)
Varicella is a benign, self–limiting, and highly communicable infection in children but
associated with severe infections in adults.

Etiologic Agent
The causative agent is the Varicella Zoster Virus (VZV), a double stranded, enveloped
DNA virus that belongs to the Herpesvirus family of viruses. It infects mucoepithelial cells and
establishes latency in nerve ganglia. Because of the latency, the virus persists in the infected
host for an indefinite period and produces recurrent infections (zoster or shingles) especially in
elderly and immunocompromisedpersons
404 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Mode of Transmission
The disease is most commonly transmitted by inhalation of respiratory droplets but may
also be transmitted by direct contact with the lesions.

Clinical Findings
Varicella is characterized by fever and vesicular eruptions on the skin and mucous
membranes. The rashes are initially maculopapular which later becomes vesicular with
associated intense pruritus. The vesicles rupture and ulcerate and later leads to scab formation
(crusts). The lesions appear in crops of different stages and all the stages of the lesions
(macule, papules, vesicles, ulcers, crust) appear simultaneously. The vesicles are described as
“teardrop on a pink base” or “dew drop on a rose petal.” The lesions are superficial and do not leave
permanent scars. Complications include pneumonia (in adults) and encephalitis (in children).

Figure 23.4 The typical rashes of chickenpox showing

the lesions at various stages at a given area ranging from
macules to vesicles to papules to pustules.
Source: F malan, 2010

Laboratory Diagnosis
Diagnosis is based on clinical manifestations and a Tzanck smear of skin scrapings or swab
from the vesicle to demonstrate the Cowdry type A inclusions and multinucleated giant cells.

Treatment and Prevention

The treatment is mainly symptomatic. The drug acyclovir has been shown to be effective
in reducing the course of the disease but it does not prevent latency and recurrent infections.
Prevention is by administration of Varicella Zoster Virus vaccine, a live attenuated vaccine
 Viral Exanthems 405

Herpes Zoster (Shingles)

Zoster or shingles is a disease that occurs in adults or immunocompromisedindividuals and
is caused by the same virus that causes chickenpox. It is caused by the reactivation of a latent
chickenpox infection. The first manifestation is severe radicular pain over the skin supplied
by sensory ganglia. The most commonly involved dermatome is the thoracic dermatome.
The disease may also involve other cranial nerves like the trigeminal, facial, or auditory
nerves. The rashes are similar in appearance to that of chickenpox but differ from chickenpox
in two aspects, namely: (1) its limited distribution to the skin innervated by a single sensory
ganglion, and (2) the rashes are painful rather than pruritic. The most common complication is
post herpetic neuralgia, a form of prolonged pain that may persist for months.

a b

Figure 23.5 a Characteristic vesiculopustular lesions of chickenpox

in a child. b Reactivation of latent chickenpox infection showing the
dermatomal distribution of the lesion
Source: Government of South Australia, Department of Health and Wellbeing

Variola (Smallpox)
Variola or smallpox is a contagious infection responsible for very high fatality rate
worldwide before the 18th century. For centuries, smallpox was controlled through the process
known as variolation, which involved inoculation of high risk individuals with live virulent
virus. The process was relatively dangerous but greatly helped reduce the rate of outbreaks
and epidemics. It was Edward Jenner who developed a live vaccine from cowpox in the
17th century. The last reported case was reported in Somalia in 1977. In 1980, smallpox was
declared totally eradicated through vaccination
406 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

The success of vaccination is attributed to several factors, including: (1) there is only
one, stable serotype, (2) there is no animal reservoir and humans are the only hosts, (3) there
is no subclinical state, and (4) it is easily clinically recognizable. Smallpox is listed among
the Category A bioterrorism biowarfare agents by the Center for Disease Control and
Prevention of the United States.

Etiologic Agent
The etiologic agent is the Variola virus, a member of the human Poxviruses. Poxviruses are
the largest among the DNA viruses. It shares antigenic determinants with animal poxviruses
and because of this, the Cowpox virus has been successfully used in the development of vaccines
for smallpox.

Mode of Transmission
The primary mode of transmission is through inhalation. It can also be transmitted
by direct contact with the lesions, dried virus, or contaminated materials like clothing.

Clinical Findings
There are two variants of smallpox—smallpox minor (1% mortality) and smallpox major
(up to 40% mortality). The disease initially presents with fever and malaise, followed by the
appearance of rashes that are macular that then become papular, later becoming vesicular, and
eventually pustular. Unlike chickenpox, the lesions of smallpox appear one stage at a time.
In addition, the lesions are deep seated, leaving permanent scars. In severe cases, the rashes may
become hemorrhagic. The comparison of varicella and variola is shown in Table 23.2.

Table 23.2 Comparison between chickenpox and smallpox

Features Varicella Variol
Common name Chickenpox Smallpox
Mode of transmission Inhalation, direct contact Inhalation, direct contact
Exanthem Vesicular Vesicular
Eruption of lesions Crops of different stages Lesions appear one stage at a time
Scar formation Rare Common
Latency Yes No
Severity Children: mild, self limited Generally severe and fatal
Adults: severe
Vaccine Yes Yes
 Viral Exanthems 407

Figure 23.6 Smallpox with lesions appearing one stage

at a time

Laboratory Diagnosis
The disease is easy to recognize based on the symptoms. Virus isolation can be done by
growing of the virus in chorioallantoic membrane of embryonated eggs where the characteristic
pocks develop. Antibody assays can confirm the diagnosis.

Treatment and Prevention

Methisazone is effective as prophylaxis but not for therapeutic purposes. The vaccine is a
live, attenuated vaccine. Smallpox has been totally eradicated since 1980 because of the success
of vaccination
408 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• The five most common childhood exanthems are measles, chickenpox, German measles,
roseola, and fifth disease.

• All five exanthems are caused by viruses, worldwide in distribution, and highly
contagious.

• Measles, Rubella, and chickenpox are preventable vaccination.

by

• Rubeola or measles is characterized by fever, a prodrome consisting of the 3 C’s (coryza,

cough, conjunctivitis with photophobia), Koplik’s spots, and maculopapular rash
with desquamation.

• Rubella is a common cause of congenital viral infection. It is manifested by fever,

lymphadenopathy, joint pains, and maculopapular rash.

• Roseola is the most common cause of febrile seizures in children.

• Chickenpox is a benign self limiting infection in children but severe in adults and
characterized by vesicular lesions.

• Herpes zoster or shingles present with severe pain over the path of sensory nerve
distribution followed appearance of vesicular lesions. The thoracic dermatome is most
by
commonly affected.

• Fifth disease generalized erythematous rash but it is most prominent over the face and is
described as “slapped cheek” appearance.

• Smallpox
by
is a highly contagious viral infection. It was totally eradicated in 1980
vaccination
 Viral Exanthems 409

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Case: A 33 year old female was brought to the out patient department of a government
hospital because of generalized maculopapular rashes. The condition started 2 days prior to
admission as fever, conjunctivitis, and arthralgia. On examination, lymph nodes are palpable
over the occipital area. The patient has been given OPV and BCG immunization during the
first year of life.

Physical examination: Temp: 38 °C RR: 18/min PR: 90 beats/min

Skin: (+) maculopapular rashes over the face, trunk, and extremities
Eyes: Reddish conjunctivae with tearing and non purulent discharge
Nose: No flaring of alae nasi
Chest and Lungs: No intercostal or subcostal retractions on inspection. On auscultation, there
are no rales heard on both lung fields. No wheezes.

1. The most probable diagnosis is:

a. Rubella c. Roseola infantum
b. Rubeola d. Fifth disease
2. Congenital infection in the newborn associated with this infection presents as:
a. Microcephaly c. Deafness
b. Mental retardation d. All of the above
3. The vaccine used to prevent this infection is a:
a. Live, attenuated viral vaccine c. Killed viral vaccine
b. Inactivated viral vaccine d. Subunit vaccin
410 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Multiple Choice.

4. “Slapped cheek” appearance is characteristic of which of the following exanthems?

a. Roseola infantum c. Erythema infectiosum
b. Rubella d. Rubeola
5. “Teardrop on a pink base” appearance of the lesions is a characteristic feature of
which of the following?
a. Varicella c. Exanthem subitum
b. Variola d. Fifth disease
6. Koplik spots is the pathognomonic exanthem of:
a. Fifth disease c. Rubella
b. Rubeola d. Roseola infantum
7. The late sequela of Rubeola.
a. Otitis media c. Subacute sclerosing panencephalitis
b. Pneumonia d. Congenital infection
8. Which of the following infections was successfully eradicated through
vaccination?

a. Varicella c. Exanthem subitum

b. Variola d. Fifth disease
9. Which of the following infections is the most common cause of febrile seizures in
children?

a. Rubeola c. Roseola
b. Rubella d. Fifth disease
10. The following statements are correct regarding shingles EXCEPT:
a. Shingles most commonly involves the trunk.
b. It is common in the elderly and immunocompromised.
c. It is the primary infection caused by Varicella zoster virus.
d. It is associated with congenital infections
 CHAPTER
Other Systemic
24 Infections

LEARNING OBJECTIVES

At the end of this chapter, the student should be able to:

1. recognize the common systemic infections based on clinical manifestations;
2. compare and contrast dengue fever and chikungunya based on etiology, mode of
transmission, signs and symptoms, and prevention;
3. describe the characteristics of the causative organisms of each systemic infection;
4. discuss the appropriate laboratory diagnosis and treatment of each infection; and
5. discuss the prevention and control of common systemic infections.

Dengue Fever
Dengue fever is an arthropod borne infection and is common in the Far East. The
incidence is highest during the rainy season because the vector, Aedes aegypti which is a
household mosquito, lays its eggs in clean stagnant water. Humans are reservoir hosts for
Dengue virus.

Etiologic Agent
Dengue fever is caused by Dengue virus under the family Flaviviridae (historically classified
as Arboviruses). It is a single stranded, enveloped RNA virus, and there are four strains of
the virus
412 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Mode of Transmission
Dengue fever is transmitted through the bite of a female mosquito known as Aedes aegypti
which is the more effective vector in urban areas or cities. In rural areas the vector is Aedes
albopictus that breeds in vegetations as well as banana and abaca plantations. The mosquito is
also known as Asian tiger mosquito because of the dots on its body and stripes on its legs. It is
a low flying day biting mosquito with short range flights. Hence the most common sites of
bites are the lower extremities. The peak of biting is 2 to 3 hours after daylight and a few hours
before nighttime. Humans are the only known hosts.

Figure 24.1 The Aedes aegypti mosquito during

a blood meal. Take note of the characteristic
white stripes on the legs of the mosquito, earning
for itself the name “Asian tiger mosquito.”
It is the female mosquito that usually bites.

Clinical Findings

• Classical Dengue Fever

This is manifested by abrupt onset of high
grade fever for 3 to 6 days which then subsides
and reappears after 2 to 3 days. This pattern is
described as “camel back” or “saddle back”
pattern of fever. The fever is accompanied by
generalized and excruciating muscle and bone
pains, hence the term “break bone fever.”
Patients may also manifest severe headache
and retro orbital pain. This is also associated
Figure 24.2 Punctuate rash of dengue
with transient punctuate rash over the knees fever known as Herman’s sign
and elbows referred to as Herman’s sign (Figure
24.2). On the 3rd to 5th day, the rash becomes morbilliform or scarlatiniform over the
trunk, face, and extremities. There is also generalized lymphadenopathy and leucopenia.

• Dengue Hemorrhagic Fever (DHF) or Shock Syndrome (DSS)

This occurs when a patient who had previous infection with one serotype of the virus
becomes infected by a different serotype. There is a tendency for bleeding and shock due
to increased amounts of cytokines that causes increased vascular permeability and plasm
 Other Systemic Infections 41

leakage. It has been observed that the risk for DSS or DHF is more likely in cases of
secondary infection with serotype 2 after a previous infection with serotype 1 of the virus.
The third and fourth infections are usually associated with a milder clinical course.
During the early phase of the infection, the patient presents with classical dengue
fever. Two to five days later, the infection worsens with manifestations of prostration,
restlessness, facial flushing, abdominal pain, and dehydration. Signs and symptoms
of bleeding are also present like appearance of petechiae, epistaxis, hematemesis or
melena, purpura, or ecchymosis. Hepatomegaly may also be present indicating possible
development of hepatitis. Development of DHF usually occurs if the patient is bitten
again by a mosquito that carries a different strain of the virus than that which caused the
first infection. The bleeding manifestations can be attributed to the decrease in platelet
count (thrombocytopenia) due to type III hypersensitivity reaction elicited by the virus.
As bleeding or third space loss continues, the patient may manifest signs and symptoms
of circulatory collapse (DSS) such as cold extremities and tachycardia.
The World Health Organization case definition of DHF includes: (1) fever,
(2) hemorrhagic manifestations, (3) thrombocytopenia(platelet count < 100,000/ cu mm,
and (4) hemoconcentration (increase in hematocrit).

Laboratory Diagnosis
The diagnosis is mainly based on the clinical manifestations and blood picture of the
patient. Culture and identification using living cells like suckling mice and mosquito cell
lines are done but not usually requested. Serology (MAC ELISA, complement fixation,
hemagglutinin inhibition) can also be done for the isolation and identification of the virus.
Detection of viral nucleic acid and antigens can be achieved with the use of PCR.

Treatment
There is no specific treatment for dengue. Management of the infection is supportive care.

Prevention and Control

A vaccine against dengue (Dengvaxia) has been developed and became commercially
available in the Philippines, Mexico, Brazil, Peru, Indonesia, Singapore, Thailand, Guatemala,
El Salvador, Costa Rica, and Paraguay which are all endemic for dengue. The vaccine
(Dengvaxia or also known as CYD TDV) was developed by Sanofi Pasteur. It is a live
recombinant tetravalent dengue vaccine from yellow fever 17D vaccine strain. The vaccine
should be given in a series of 3 doses every 6 months. It is recommended to be given to
seropositive individuals (those who had previous dengue infection).
414 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Prevention is mainly focused on education of the public, active surveillance of cases, and
mosquito control. Mosquito control by larval source reduction (destroying the breeding places
of the mosquito) is the best way of preventing the disease. Secondary preventive measures
include prevention of mosquito bites by applying mosquito repellants, wearing thick clothing,
and screening windows and doors. Fogging is no longer recommended as it does not really
destroy the mosquitoes but merely drives them away.

Chikungunya
Chikungunya is a re emergent infection caused by alphavirus (Simliki Forest virus) that
belongs to the family Flaviviridae. It is transmitted through bite of the mosquito Aedes aegypti.
It is similar to dengue fever based on benign clinical syndrome of break bone fever, but without
retro orbital pain and only mild headache. Unlike dengue, it presents with more severe muscle
and joint pains that the patient literally folds up. Sequelae of chikungunya are crippling joint
pain and hemorrhagic fever.

Table 24.1 Comparison between dengue fever and chikungunya

Features Dengue Fever Chikungunya
Etiology Dengue Virus - Flavivirus Alphavirus - Flavivirus
Vector Aedes aegypti (rural) Aedes aegypti (rural)
Aedes albopictus (urban) Aedes albopictus (urban)
Incubation Period 2–7 days 2–4 days
Age Group All ages All ages
% Symptomatic 20%–60% 70%
Symptoms
Fever Present Present
Headache Severe Mild
Rash Present Present
Retroorbital pain Present Absent
Joint pain Present More severe
Sequelae DHF or DSS Crippling arthritis
Vaccine Yes Non
 Other Systemic Infections 415

Zika
Zika virus infections are already in existence in Southeast Asia, Africa, and the Pacific
Islands. For a long time there had never been any reported case of Zika infection until 2015
when an outbreak was confirmed in Brazil, associated with microcephaly in newborns
(Figure 24.3). From then on, outbreaks have been reported in many countries.

Etiology
Zika is caused by the Zika virus under the family Flaviviridae. It is single stranded RNA
virus with an envelope.

Figure 24.3 Microcephaly in congenital Zika infection

Modes of Transmission
1. Bite of mosquito – Aedes aegypti and Aedes albopictus are two species of mosquito that can
carry the virus.
2. Mother to fetus – Zika virus can be passed to the fetus from the pregnant mother during
pregnancy. The Zika virus has been proven to cause microcephaly and other severe fetal
brain defects.
3. Sexual contact – Zika virus can be spread by an infected man to his sexual partner even
before the appearance of symptoms. Studies have shown that the virus is present in semen
longer than in blood.
4. Blood transfusion – the virus can also be transmitted by blood transfusion, requiring blood
from donors to be tested for the virus. A significant number of blood donors tested
positive for Zika in Brazil and Polynesian countries
416 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Clinical Findings
Zika does not cause any symptoms or may cause only mild symptoms that may last for
several days to a week. If symptomatic, the common manifestations are fever, headache, joint
pain, muscle pain, conjunctivitis, or a rash. Severe disease is uncommon. An infected person is
protected from future infections after recovery.

Treatment and Prevention

There is no treatment for Zika. The treatment is supportive like rest and plenty of fluids.
Prevention includes vector control, practicing safe sex, and screening of blood donors.

Infectious Mononucleosis
Etiology
The causative agent is Epstein Barr virus (EBV), a double stranded, enveloped DNA
virus under the family of Herpesviridae. The B lymphocytes are the major targets of EBV.
The virus can cause latent infection of the B cells. This agent is oncogenic and capable of
immortalizing and transforming infected cells. It is strongly associated with transformation
to nasopharyngeal carcinoma, Burkitt’s lymphoma, and other B cell lymphomas. The virus
causes an opportunistic infection in the tongue and mouth (hairy cell oral leukoplakia) in
AIDS patients. EBV stimulates cell growth as B cell mitogen and prevents apoptosis causing
immortalization of B cells.

Mode of Transmission
EBV is primarily transmitted through the exchange of saliva, hence, the infection is also
known as “kissing disease.”

Clinical Findings
The incidence is highest among adolescents and young adults. The infection is manifested
by clinical triad of sore throat, lymphadenopathy, and splenomegaly with associated fever,
anorexia, and lethargy. Hepatitis is also common (hepatosplenomegaly)
 Other Systemic Infections 41

Diagnosis
Diagnosis can be obtained by hematologic examination which shows lymphocytosis with
atypical lymphocytes known as Downey cells. Detection of antibodies against the viral capsid
antigen is an important diagnostic tool. Serologic tests like a positive heterophil antibody test is
also useful for early detection.

Treatment and Prevention

There is no specific antiviral drug or vaccine for EBV.

Cytomegalovirus Infection
Cytomegalovirus (CMV) is a double stranded DNA virus under the Herpesviridae
family and the largest among the Herpesviruses. It causes enlargement of the infected cells
(cytomegaly). Infections with this virus are common, primarily affecting newborns, normal
healthy adults, and immunocompromisedindividuals. CMV establishes latency in monocytes,
myeloid stem cells, lymphocytes, macrophages, and other cells. It can be isolated in the blood,
saliva, stool, tears, throat, semen, vaginal and cervical secretions, and amniotic fluids and tissues.

Modes of Transmission
Since CMV can be isolated or is present in body fluids and tissues, the virus can be
transmitted through the oral route, sexual contact, tissue transplantation, and blood transfusion.
The virus may also spread through congenital transmission.

Clinical Syndromes

• Congenital and Neonatal Infections

Cytomegalovirus is the most common viral cause of congenital infections and mental
retardation. Approximately 90% of cases are asymptomatic. The remaining 10% present
with congenital abnormalities such as microcephaly, mental retardation, visual defects like
cataract and glaucoma, deafness, hepatosplenomegaly,rash, and congenital heart defect.
Neonatal CMV infections are acquired during delivery as the infant passes through
the birth canal, from the mother’s milk, or through blood transfusion. Infected newborns
continue to excrete the virus through the feces for months after infection.
418 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

• Mononucleosis like Syndrome

Mononucleosis like syndrome may be asymptomatic or may present as mild
mononucleosis syndrome similar to Epstein Barr virus (fever, mild pharyngitis,
and lymphadenopathy)but unlike EBV, CMV is negative for heterophil antibodies.
A comparison between EBV and CMV mononucleosis syndrome is summarized
in Table 24.2.

Table 24.2 Comparison between CMV and EBV mononucleosis

Features Mononucleosis like Infectious

Syndrome (CMV) Mononucleosis (EBV)
Etiology Cytomegalovirus Epstein Barr Virus
Mode of transmission Oral secretions Oral secretions
Fever + lymphadenopathy Present but milder Present
Sore throat Present but milder Present
Hepatosplenomegaly Present Present
Lymphocytosis Present Present
Atypical lymphocytes Present Present
Heterophil antibody test Negative Positive

Infection in ImmunocompromisedPatients
CMV commonly causes chorioretinitis (common in AIDS patients), encephalitis,
pneumonia, and esophagitis. CMV is a common pathogen in bone marrow transplant patients.

Laboratory Diagnosis
Diagnosis of the disease can be done by means of the following:
1. Histological examination of tissues and urine. Specific intracellular inclusion bodies
called “owl’s eye” inclusions are histologic hallmarks of CMV infection.
2. Culture in fibroblast cells.
3. Serological detection of IgM and IgG antibodies to CMV antigens.

Treatment and Prevention

The drug recommended for CMV is ganciclovir. An alternative drug is foscarnet. CMV
infections can be prevented by using mechanical barriers like condoms. Avoidance of unusual
sexual practices such as unprotected anal sex is also important since sexual contact is a common
route of transmission. Blood and organ donors should also be screened for CMV. There is an
available vaccine for CMV which is a live, attenuated vaccine
 Other Systemic Infections 41

Rickettsial Infections
Rickettsial infections are transmitted by the bite of arthropods like ticks, mites, lice, and
fleas except for Q fever, which is transmitted by inhalation of aerosols. Rickettsial infections are
zoonotic (with animal reservoirs) except for Epidemic typhus which occurs only in humans.
Rickettsial infections are divided into six groups, namely: (1) Typhus Group – Epidemic
typhus, Murine typhus (Endemic typhus), and Scrub typhus; (2) Spotted Fever Group – Rocky
Mountain Spotted Fever; (3) Traditional group – Rickettsialpox; (4) Q Fever; (5) Trench fever;
and (6) Ehrlichiosis.
The Spotted Fever group is characterized by rashes that appear first on the extremities,
with involvement of the palms and soles. The Typhus group is also characterized by
maculopapular rashes that are prominent in the trunk and extremities with sparing of the
palms and soles.

General Characteristics
1. Very small size (0.3 × 1–2 um)
2. Have gram negative cell wall composed of peptidoglycan, muramic acid, and
diaminopimelic acid
3. Stain poorly with Gram stain but stain well using Giemsa or Gimenez stain
4. Pleomorphic – cocci or short bacilli
5. Obligate intracellular parasites
6. Transmitted by arthropod vector except Q fever
7. Easily destroyed by heating, dyeing, and bactericidal agents like tetracycline
8. Growth enhanced by sulfonamides
Table 24.3 Clinical diseases associated with Rickettsiae
Disease Etiology Vector
Rocky Mountain Spotted Fever Rickettsia rickettsii Tick
Rickettsialpox Rickettsia akari Mite
Epidemic typhus Rickettsia prowazekii Louse
Murine typhus Rickettsia typhi Flea
Scrub typhus Orientia tsutsugamushi Mite
Ehrlichiosis
Human monocyte ehrlichiosis Ehrlichia chaffeensis Tick
Human granulocyte ehrlichiosis Anaplasma phagocytophilum Tick
Ewingii ehrlichiosis Ehrlichia ewingii Tick
Q fever Coxiella burnetii None
420 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Disease Organism Vector Reservoir

Rocky Mountain R. rickettsii Tick borne Ticks, wild rodents
spotted fever
Ehrlichiosis E. chaffeensis Ticks

Rickettsialpox R. akari Mile borne Miles, wild rodents

Scrub typhus R. tsutsugamushi Miles (chiggers), wild rodents

Epidemic typhus R. prowazekii Louse borne Humans, squirrel fleas, flying

squirrels
Trench fever R. quintana Humans

Murine typhus R. typhi Flea borne Wild rodents

Q fever C. burnetii None* Cattle, sheep, goats, cats

*Tick vectors may be responsible for animal to animal transmission.

Figure 24.4 Vectors for the various Rickettsial infections

Spotted Fever Group

Rocky Mountain Spotted Fever
The causative agent is Rickettsia rickettsii and is transmitted through the bite of ticks. This
is common in the mountainous areas of the United States. The early manifestations are similar
to Rickettsialpox. Maculopapular rashes appear on the hands and feet, then later in the trunk in
the following 2–6 days.

Figure 24.5 Typical lesions of Rocky

Mountain Spotted Feve
 Other Systemic Infections 421

Typhus Group
Epidemic Typhus (Louse borne Typhus)
The etiologic agent is Rickettsia prowazekii and is transmitted through the bite of lice.
The manifestations are similar to the other rickettsial infections, with the same non specific
symptoms and maculopapular rashes, although there is sparing of the palms and soles. It
also presents with more severe systemic infection and prostration, and is more fatal. This is
associated with a recrudescent infection known as Brill Zinsser Disease.

Endemic Typhus (Murine Typhus)

The etiologic agent is Rickettsia typhi which is transmitted by the bite of a flea. This
presents with similar features as Epidemic typhus however it is milder and rarely fatal except
in the elderly.

Scrub Typhus
The causative organism is Orientia tsutsugamushi (formerly known as Rickettsia
tsutsugamushi). This is transmitted through the bite of mites. This infection resembles
Epidemic typhus clinically except for the eschar (punched out ulcer covered with blackened
scab that indicates the site of the mite bite) with associated generalized lymphadenopathy and
lymphocytosis. The disease may also involve severe cardiac and cerebral complications.

Traditional Group
Rickettsialpox
The etiologic agent is Rickettsia akari and is transmitted through the bite of mites. It is
a mild disease resembling Varicella. The infection is manifested by fever, headache, chills,
myalgia, and the appearance of a firm red macule at the bite site which later develops into a
deep seated vesicle that ruptures and presents with a blackened scab known as eschar.

Q Fever (Query Fever)

The etiologic agent Coxiella burnetti, transmitted by inhalation of dust containing the
organism or aerosols. The infection resembles influenza and non bacterial pneumonia, hepatitis,
or encephalopathy. The infection does not present any rash or local lesion
422 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Ehrlichiosis
The disease is caused by Ehrlichia sennetsu for Sennetsu Fever and Ehrlichia chaffeensis for
Human Ehrlichiosis. These organisms parasitize lymphocytes, neutrophils, and monocytes,
and manifest non specific symptoms with thrombocytopenia.

Leptospirosis
Etiologic Agent
Leptospirosis is caused by a spirochete Leptospira interrogans. This spirochete is
tightly coiled with a hook on one or both ends and is highly motile. The reservoir hosts are
rats and other rodents (most common reservoir), household pets, and livestock (accidental
hosts). Leptospira is excreted in the urine of reservoir hosts and contaminate soil and water.
The infection is worldwide in distribution.

Figure 24.6 Leptospira interrogans, causative agent of

leptospirosis with its characteristic hooked end

Mode of Transmission
Since leptospira excreted with urine can contaminate water and soil, it is commonly
acquired when the organism enters through breaks in the skin or mucous membrane by wading
or swimming in contaminated water. It can also be transmitted through ingesting contaminated
water and food. Individuals at risk of the infection are sewage workers, farmers, and miners
 Other Systemic Infections 423

Clinical Syndrome
Leptospirosis is a biphasic infection. It initially presents flu like symptoms of fever, severe
headache, myalgia, and chills. These symptoms will recede for a short period. This will then be
followed by the immune period and manifest with signs and symptoms of meningitis. In severe
cases, the meningitis is associated with impaired renal function and liver damage (Weil’s disease
or infective jaundice). Patients who survive the infection may recover from the renal failure and
hepatic damage.

Laboratory Diagnosis
Leptospira cannot be stained with dyes but can be visualized using darkfield microscopy.
Aside from the clinical findings, diagnosis is confirmed by an increase in agglutinating
antibodies.

Treatment, Prevention, and Control

The recommended drug is penicillin. There has been no reported development of resistance
to the drug to date. The effective prophylaxis for exposed individuals is doxycycline. The disease
can be prevented by avoiding wading in contaminated water and avoiding contact with
contaminated soil. Rodent control is also important in the control and prevention of the spread
of infection.

Lyme Disease (Lyme Borreliosis)

Etiologic Agent
Lyme disease is caused by Borrelia burgdorferi, a flexible spirochete with coarse, irregular
coils. It is equipped with endoflagella and is highly motile.

a b

Figure 24.7 a Characteristic morphology of Borrelia; and

b Erythema chronicum migrans, the distinctive reaction
at the site of the bite of the vector
Source: Garrison, 200
424 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Mode of Transmission
Lyme disease is an arthropod borne infection transmitted through the bite of a tick
(Ixodes). Reservoir of Borrelia burgdorferi is the wood rat and the obligatory hosts are mammals,
particularly deer where the tick completes its life cycle.

Clinical Findings
Lyme disease is a progressing disease divided into three stages. During the first stage,
a painless, circular red rash known as erythema chronicum migrans that is spread with a clear
center at the site of the bite. This is the characteristic finding accompanied by arthralgia.
This may or may not be accompanied by non specific symptoms of fever, headache, chills and
fatigue. After a few weeks or months, the second stage sets in and manifests as myocarditis or
pericarditis, aseptic meningitis, Bell’s palsy, and neuropathies. This is then followed by a latent
period lasting several weeks and months. The third stage is manifested by arthritis involving the
large joints like the knees and a progressive chronic involvement of the central nervous system.

Laboratory Diagnosis
Borrelia burgdorferi can be stained with Giemsa or silver stains and can be visualized by
darkfield microscopy. Culture is rarely positive. Serological tests like ELISA or indirect
immunofluorescence are valuable in the diagnosis. Confirmatory test is Western Blot Assay.
Polymerase chain reaction (PCR) is also valuable in detecting Borrelia burgdorferi DNA.

Treatment and Prevention

For mild infections, effective treatment involves amoxicillin or doxycycline. For late stage
infections, the more effective drug is penicillin G or ceftriaxone. No resistance to the
aforementioned drugs has been reported so far.
Prevention is focused on preventing tick bites by wearing thick clothing and application of
insect repellants.

Relapsing Fever
Etiologic Agent
Borrelia recurrentis is the major etiologic agent for Relapsing Fever. Other Borreliae like
B. hermsii can also cause the infection. The organism is very flexible and highly motile (motility
is rotatory and twitching). The organism can survive low temperature (4 °C) in blood or culture
for months
 Other Systemic Infections 42

Mode of Transmission
Relapsing fever is transmitted from one person to another through the bite of the human
body louse (Pediculus humanus). The main reservoirs are rodents and other small animals. The
infection is transmitted from these reservoirs through bite of ticks (Ornithodorus).

Clinical Findings
During the bite, the vector introduces the organism into the skin and multiplies in the
tissues. The infection initially manifests as fever, headache, and chills. The fever lasts for a few
days and resolves but recurs after a week with associated multi organ dysfunction. There are
around 3–10 recurrences, with each recurrence the manifestations become less severe.

Laboratory Diagnosis
Examination of the peripheral blood smear using Giemsa or Wright stain will demonstrate
the spirochetes. The best time for collecting specimen is during the height of the fever where
the spirochete is always present. Culture using special media is also useful in the diagnosis.
Serological tests however are not useful in the diagnosis.

Treatment and Prevention

An effective drug in the treatment of early infection and prevention of relapses is
tetracycline. Avoidance of areas infested by arthropod vectors and protection from bites are
important in preventing infection.
426 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

CHAPTER SUMMARY

• Dengue fever, chikungunya, and Zika fever are all caused by Flaviviruses and all are
arthropod borne infections acquired through bite of mosquitoes—Aedes aegypti and
Aedes albopictus.

• Dengue fever and chikungunya are both manifested by joint pains (break bone fever)
but the joint pain is more severe in chikungunya.

• Secondary infection with another strain or serotype of dengue virus can lead to dengue
hemorrhagic fever or dengue shock syndrome which can be fatal.

• Zika is a re emerging infection. Most cases are asymptomatic. Aside from mosquito bite,
it can also be transmitted from the infected mother to the fetus causing microcephaly and
other brain defects.
• Ain vaccine for dengue fever was made available but it has encountered much controversy
the Philippines.
• EBV infectious mononucleosis and CMV mononucleosis like syndrome are very much
alike. Both are associated with lymphocytosis and formation of atypical lymphocytes.
Diagnosis can be established by heterophil antibody test where EBV would yield
a positive result.

• Cytomegalovirus is the most common viral cause of congenital infection. It is detrimental

when acquired during the first trimester of pregnancy where the various body organs are
undergoing development. The congenital infection is manifested by microcephaly, mental
retardation, cataract, deafness, and congenital heart defect.
• Rickettsial infections are also arthropod borne infections acquired through bites of ticks,
mites, lice, or fleas except Qfever which is transmitted through the respiratory route.
All infections present with maculopapular rashes except rickettsialpox which is vesicular
with eschar formation.
• Among the rickettsial infections, only epidemic typhus caused by Rickettsia prowazekii
is associated with recrudescent infection, Brill Zinsser disease.

• Both rickettsiapox and scrub typhus are manifested by eschar.

• Leptospirosis is caused by Leptospira interrogans, a spirochete with fine regular coils and
hook on one or both ends. The infection is associated with urine of rodents and other
animals and acquired through breaks in the skin and mucous membrane.
• Lyme disease and relapsing fever are both arthropod borne infections caused
Borrelia burgdorferi and Borrelia recurrentis respectively.
by

• Both Leptospira and Borrelia are highly flexible and highly motile organisms.

• The best control measures for arthropod borne infections is prevention and protection
from arthropod bites and vector control
 Other Systemic Infections 427

SELF ASSESSMENT QUESTIONS

Name: Score:
Section: Date:

Case: A 7 year old boy was brought to the ER because of nose bleeding and vomiting of blood.
The patient was apparently well until 4 days prior to admission when the patient developed
moderate grade fever, headache, and muscle pains over the lower extremities. Two days prior to
admission, the maculopapular rashes were noted over the trunk and extremities. Few minutes
prior to admission, the patient had epistaxis and two bouts of hematemesis. He was diagnosed
as a case of dengue hemorrhagic fever.

1. Which of the following best differentiates dengue fever from chikungunya?

a. More severe joint pains in chikungunya
b. Presence of punctuate rash in dengue fever
c. More severe retro orbital pain in chikungunya
d. Microcephaly in the newborn
2. The mode of transmission for this infection.
a. Bite of mosquito c. Bite of ticks
b. Inhalation of droplets d. All of the above
3. The manifestations of Classical dengue fever include the following EXCEPT:
a. Break bone fever c. Headache
b. Rash d. Bleeding
4. Which of the following is correct regarding dengue?
a. Lifelong immunity develops after the primary infection.
b. Severe infection is more likely after primary infection.
c. The dengue vaccine is ineffective in seropositive individuals.
d. Blood transfusion is a mainstay in management of dengue shock syndrome
428 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Multiple Choice.

5. A maculopapular rash involving the palms and soles.

a. Endemic typhus c. Epidemic typhus
b. RMSF d. Q fever
6. A disease transmitted by mites.
a. Scrub typhus c. Murine typhus
b. RMSF d. Q fever
7. Chickenpox like lesions is commonly seen in infection caused by:
a. R. akari c. R. prowazekii
b. R. rickettsii d. R. typhi
8. Which of the following is/are correct statement/s regarding Zika?
a. Most cases are asymptomatic
b. It can be transmitted by sexual contact
c. Screening of blood donors is important for infection control
d. A and B
e. A, B, and C

9. The most common viral cause of congenital infections.

a. EBV c. CMV
b. Rubella d. HSV
10. Which of the following is/are similarities between EBV infectious mononucleosis
and CMV mononucleosis like syndrome?
a. Both are associated with lymphocytosis
b. Both produce atypical lymphocytes
c. Both produce heterophil antibodies
d. A and B
e. A, B, and
 P art II
LABORATORY
EXERCISE
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 Laboratory Exercises 43

Rules of Conduct
in a Microbiology Laboratory
The rules enumerated below shall be strictly implemented. The main objective of these
rules is to avoid the dangers of infection which may arise from the neglect of necessary
precautions. Failure to follow these rules puts not only a student at risk, but also puts others at
risk of infection.
1. Each student is required to wear a laboratory gown, coat, or smock gown while working
in the laboratory. This will be used only in the microbiology laboratory. These should
not be laid on the worktables. When dirty, these should be wrapped properly before
bringing home where boiling or soaking in a disinfectant solution should be done
before washing.
2. Students are advised not to wear any item of jewelry while working in the laboratory.
Other items of clothing and personal belonging should not be placed on the
worktables to prevent contamination with microorganisms.
3. Eating is absolutely forbidden within the laboratory. If one needs to drink or eat, he
or she may leave the laboratory for a short time with the permission of the teacher.
Hands must be properly and thoroughly washed before going out to eat or drink.
Leave your laboratory gown behind. One should never go into an eating place wearing
his or her laboratory gown.
4. All accidents such as burns, abrasions, cuts, as well as spillages of cultures and breakage
or loss of equipment should be reported immediately to the instructor in charge or to
the technician on duty if the instructor is not around.
5. Each group must have a large towel which they will dip in disinfectant solution. The
towel should be wrung slightly so that it is not dripping wet. The towel should be
spread out on the worktable and all work should be done on this towel. Contaminated
surfaces must be cleaned immediately with a disinfectant solution.
6. All non infectious solid wastes like paper, cotton, matchsticks, etc., should be placed
in pails or waste boxes provided for that purpose. These are not to be discarded on
tabletops, sinks, or on the floor. The laboratory should be kept neat and clean at all
times.
7. All laboratory equipment used should be cleaned with a disinfectant solution before
returning them to the laboratory technician.
8. Cultures should not be left on tabletops nor thrown into the sinks. They should be
returned immediately to the technician for proper disposal.
9. Loitering, making unnecessary noise, and borrowing equipment from other groups or
students are not allowed. Each student or group must have their own set of equipment
as well as their own colored pencils if needed.
432 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

10. At the end of the laboratory period, all equipment and apparatus used must be returned
to the technician. All working areas must be thoroughly cleaned with disinfectant.
Hands must be thoroughly washed with soap and water
 EXERCISE NO.

1 The Microscope

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. identify the different parts of a microscope;
2. discuss the function of each part of the microscope; and
3. name the different kinds of microscopes and their uses.

I. Label the parts of the microscope.

a.
F
b.
A
c.

B D d.
C e.
G
f.
H
K g.

E I h.
i.
J
j.

k
434 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

II. Give the functions of the following parts of the microscope.

1. Mirror

2. Condenser

3. Diaphragm

4. Coarse adjustment knob

5. Fine adjustment knob

6. Body tube

7. Objective lenses

8. Low power objective

9. High power objective

10. Oil immersion

11. Eyepiec
 The Microscope 43

III. Enumerate the different types of microscopes and their uses.

This page is intentionally left blank
 EXERCISE NO.

2 The Cell

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. differentiate between prokaryotic cells and eukaryotic cells;
2. identify the different parts of the cell and the function/s of each part; and
3. tabulate the similarities and differences among medically important organisms.

I. Differentiate eukaryotic cell from prokaryotic cell.

Features Eukaryotic Cell Prokaryotic Cell

Nucleus with nuclear

membrane

DNA associated histones

Membrane bound organelles

Cell wall

Reproductio
438 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

II. Label the parts:

1. Prokaryotic cell

2. Eukaryotic cel
 The Cell 439

III. Indicate the function/s of each of the following parts of the cell.
Cell Part Function
Nucleus

Nucleolus

Cell wall

Cell membrane

Mitochondria

Ribosomes

Endoplasmic reticulum

Golgi apparatus

Lysosomes

IV. Differentiate the medically important organisms.

Characteristic Bacteria Fungi Viruses Protozoa Alga

Type of nucleus

Outer covering

Nucleic acid
present

Ribosome

Mitochondria

Type of
reproduction
This page is intentionally left blank
 EXERCISE NO.

3 Gram staining

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. perform the Gram staining procedure;
2. identify the Gram staining reaction; and
3. distinguish the fundamental shapes of bacteria.

INTRODUCTION

Gram staining is one of the most basic staining procedures done in the study of
microorganisms. It is a differential stain that groups organisms into gram positive and gram
negative organisms and can aid in the basic identification of organisms. It also demonstrates the
basic shape of the organism being studied. The following rules will help the student remember
the Gram staining reaction of the more important bacteria:
1. All bacilli are gram negative except Corynebacterium, Mycobacterium, aerobic spore
formers (Bacillus), and anaerobic spore formers (Clostridium).
2. All cocci are gram positive except Neisseria, Veillonella, and Branhamella
442 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

MATERIALS AND EQUIPMENT

• Glass slide
• Alcohol lamp
• Microscope with oil immersion objective
• Inoculation loop
• Reagents for Gram staining
PROCEDURE

Preparation of bacterial smear

1. Take a clean glass slide and gently heat one side to remove any grease that may be present.
The slide should always be held along the edges to prevent recontamination with grease
from the fingertips. Lay the slide on the table with the flamed side up.
2. Sterilize the wire loop with the flame from the alcohol lamp until it is red hot.
3. If the source of the specimen is growing on solid media, pick a small colony and emulsify
in a drop of distilled water in the center of the slide. If the source of the specimen is from
liquid media, take one or two loopfuls of the culture and spread over the center of the
slide over an area of 1 inch by 1⁄2inch.
4. Fix the smear with heat by passing the slide (with the smear side up) over the flame five or
six times. This will cause the preparation to adhere to the slide so that it will not be easily
washed off during the staining process.
5. Stain the preparation with the desired staining method.
Gram staining
1. Make a thin smear of the specimen.
2. Cover the whole smear with a few drops of crystal violet for 1 minute.
3. Gently wash off the excess stain with water until no more stain comes off.
4. Put a few drops of Gram’s iodine over the smear and let stand for 1 minute.
5. Wash off iodine with water
 Gram staining 44

6. Flood the smear with 95% alcohol and let it stand for 15 to 30 seconds. Repeat this step
until no more color comes off with the alcohol.
7. Wash with water.
8. Counter stain with safranin for 30 seconds.
9. Air dry and examine in the microscope using oil immersion objective.
Illustrated Gram staining procedure

KEY
Crystal violet
Iodine
Alcohol
Safranin

Gram positive

Gram negative

1 Application of 2 Application 3 Alcohol wash 4 Application

crystal violet of iodine (decolorization) of safranin
(purple dye) (mordant) (counterstain)

I. Give the Gram staining reaction and morphology (shape) of the following bacteria.
Gram stain reaction:

Morphology/Shape:

Gram stain reaction:

Morphology/Shape:

Gram stain reaction:

Morphology/Shape:
444 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

II. Indicate the function of the different reagents used in Gram staining.
Expected Result
Reagent Function
Gram positive Gram negative

Crystal violet

Gram’s iodine

95% Alcohol

Safranin

III. Differentiate gram positive cell wall from gram negative cell wall.
Features Gram positive Cell Wall Gram negative Cell Wall

Peptidoglycan

Complexity

Teichoic acid

Lipopolysaccharide
complexes

Endotoxi
 EXERCISE NO.

4 Acid fast Staining

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. perform the acid fast staining procedure;
2. identify the acid fast staining reaction; and
3. differentiate the methods used for acid fast staining.

INTRODUCTION

The acid fast staining procedure is another basic staining procedure which is utilized
in microbiology. It can be done using the Ziehl Neelsen or the Kinyoun method (refer to
Chapter 3 regarding the differences between the two methods). It is also a differential type of
staining method that distinguishes acid fast from non acid fast organisms. All Mycobacteria are
acid fast. Nocardia is partially acid fast.

MATERIALS

• Glass slide • Alcohol lamp

• Inoculation loop • Reagents for acid fast stainin
• Microscope with oil immersion objective
446 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

PROCEDURE

Kinyoun method
1. Make a smear of the specimen and fix it by gentle heating over flame.
2. Stain with Kinyoun’s carbol fuchsin for 3 minutes. Do not heat.
3. Gently wash with running water.
4. Decolorize with acid alcohol for about 2 minutes until no more color appears in the
washing.
5. Wash with water.
6. Add a few drops of malachite green and let it stay for 30 seconds.
7. Wash with water.
8. Air dry.

I. Look at the picture below and identify the following:

Acid fast reaction:

Morphology/Shape:

Method used:

Acid fast reaction:

Morphology/Shape:

Method used
 Acid fast Staining 447

II. What is responsible for the acid fastness of these organisms?

III. Why is the Ziehl Neelsen method called the “hot method” while the Kinyoun method
the “cold method?” Explain briefly.

IV. Differentiate the two methods of acid fast staining.

Expected Result
Reagent Function
Ziehl Neelsen Kinyoun

Carbol fuchsin

Acid alcohol

Safrani
This page is intentionally left blank
 Sterilization
EXERCISE NO.

5 and Disinfection
Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. interpret the result of each method of sterilization;
2. differentiate bacteriostatic from bactericidal agents; and
3. discuss the functions and uses of commonly used physical methods of sterilization and
commonly used chemical agents.

INTRODUCTION

There are several methods that can be employed to kill organisms or inhibit their growth.
These can be classified into physical and chemical methods (refer to Chapter 7). Of the
physical methods, heating is the most reliable and whenever possible, it should be the method
of choice. It is also the method that is readily accessible and universally accepted.
Chemical agents can also be used to achieve sterilization and disinfection. Disinfection
is important in infection control, not only in hospitals but also at home. A wide variety of
chemical agents belonging to several groups (e.g., detergents, heavy metals, alkylating agents,
etc.) can be used. Different modes of action have been ascribed to them. These chemical agents
may interfere with the functions of the cell membrane, denature proteins, or destroy or modify
the functional groups of proteins. In this exercise, different physical methods of sterilization, as
well as chemical agents for disinfection, will be evaluated
450 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

PROCEDURE

Physical Methods of Inactivation of Organisms

Materials:

• 4 broth cultures of Bacillus subtilis

• 4 broth cultures of Escherichia coli

• 8 tubes of nutrient broth

1. Treat the four culture tubes of each organism as follows:
• First tube – autoclave at 15 pounds per square inch (psi) for 15 minutes
• Second tube – stand in a pan of boiling water for 30 minutes
• Third tube – stand in a pan of water at 60 °C for one hour
• Fourth tube – control tube
2. Transfer a loopful of the suspension of the microorganism from each tube to another
tube of nutrient broth and incubate at 35 °C for 24 hours.
3. Record the growth with sub cultured tubes (no growth, minimal growth, moderate
growth, or heavy growth) in the table below.
Growth Obtained
Exposure to Heat
Bacillus subtilis Escherichia col
Autoclave – 15 psi for 15 minutes
Boiling water – 30 minutes
Heating – 60 °C for one hour

Chemical Method of Inactivation of Organisms

Materials:

• One broth culture with light suspension of Staphylococcus aureus

• Four 1 mL water blanks

• Nutrient agar plate
Chemical agents:

• 1:1000 Zephiran
• 70% alcohol
• Povidone iodine
 Sterilization and Disinfection 45

1. Using a glass pencil, divide the nutrient agar plate into four quadrants by marking
the bottom of the dish. Label the quadrants 1, 2, 3, and 4, and the name of the
disinfectant to be used.
2. Transfer a loopful of the S. aureus suspension to each of the water blanks. Label each
test tube as follows:

• Test tube no. 1 – control (no chemical agent added)

• Test tube no. 2 – 0.3 mL of 1:1000 Zephiran added
• Test tube no. 3 – 0.3 mL 70% alcohol added
• Test tube no. 4 – 0.3 mL povidone iodide added
3. Let stand for three minutes after addition of the different chemical agents.
4. After three minutes, transfer a loopful of the suspension from the control test tube and
streak on quadrant number 1 of the nutrient agar plate. From test tube number 2,
get another loopful and streak on quadrant number 2. From test tube number
3, get a loopful and streak on quadrant number 3. Finally, from test tube number
4, get a loopful and streak on quadrant number 4. Remember to sterilize the wire
loops in between each step to avoid contamination.
e. Incubate at 35 °C for 24 hours.
f. Observe growth and record results as follows: (+) – with growth; (–) – without growth
of organisms.
Chemical Agent Growth Observed
Control
1:1000 Zephiran
70% alcohol
Povidone iodide

Answer the following:

1. Differentiate sterilization from disinfection.
452 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. Enumerate the factors that may influence the efficiency of chemical agents.

3. What is thermal death time?

4. Define the following terms:

a. Bactericidal

b. Bacteriostatic

c. Antisepsi
 Sterilization and Disinfection 453

5. Identify the method of sterilization/chemical agent described. Write your answers on the
space provided.

a. A method of sterilization that involves the physical separation of microorganisms from

the fluid.

b. This method is utilized to deprive the organisms of moisture.

c. This method is used to destroy disease producing microorganisms in milk, milk

products, food, and beverages.

d. At 15 psi, the temperature reaches 121 °C requiring only 15 to 20 minutes to sterilize

the material.

e. The material to be sterilized is exposed to live steam for 30 minutes for 3 consecutive
days
454 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

f. This chemical agent is used as a standard for evaluating new chemical agents.

g. This chemical agent is used as water disinfectant.

h. A phenol derivative that is less toxic and more potent than phenol.

i. Considered the best antiseptic

j. Used as prophylaxis for ophthalmia neonatoru

 EXERCISE NO.

6 Bacterial Structures

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. identify the different structures of a bacterium;
2. discuss the importance and functions of each bacterial structure; and
3. name the method used to demonstrate each bacterial structure.

INTRODUCTION

Bacteria are equipped with specialized structures that enable them to establish infection and
produce disease. These structures are not readily visualized using the standard Gram stain and
acid fast stain. However, there are special stains that can be used to visualize each specialized
structure. This exercise aims to demonstrate these special structures and stains used to visualize
them.

MATERIALS

• Microscope
• Demonstration slides: (a) capsule, (b) spore, and (c) flagell
456 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

I. Identify the bacterial structure shown and give the special stain used to demonstrate this
structure:

Structure:

Stain used:

Structure:

Stain used:

Structure:

Stain used
 Bacterial Structures 457

II. Identify the type of flagella

1.

2.

3.

4.

III. Give the functions of the following bacterial structures.

1. Cell wall

2. Cell membrane

3. LPS

4. Ribosome
458 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

5. Nucleoid

6. Granules

7. Pili

8. Flagella

9. Endospores

10. Capsul
 EXERCISE NO. Antimicrobial
7 Susceptibility
Testing
Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. discuss the importance of antimicrobial susceptibility testing;
2. interpret results of antimicrobial susceptibility testing;
3. classify antibiotics based on mechanism of action;
4. describe the characteristics of an ideal antibiotic; and
5. discuss the mechanisms of drug resistance.

INTRODUCTION

Antibiotics are given to treat infectious diseases. The physician faces the problem of
deciding which antibiotic to use for a given infectious disease. To make sure that the antibiotic
to be given is suited for a specific organism, an antimicrobial susceptibility test must be
requested. This test will tell the physician if the organism involved in the disease process is
susceptible to or resistant to a particular antibiotic, thereby saving the patient from spending
money on a drug that will not work on the particular organism involved in the first place.
Susceptibility testing is most often indicated when the etiologic agent involved is known to be
capable of developing resistance to commonly used antimicrobial agents. It is rarely done if the
organism is not known to develop resistance against a given antibiotic
460 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

There are two methods used in susceptibility testing—the disc diffusion method and the
test tube method. The test tube method is a serial dilution method that is tedious and time
consuming. It is used to determine the bacteriostatic and bactericidal concentration of the
antibiotic. The method routinely used in most laboratories is the disc diffusion method. In this
method, paper discs are impregnated with known concentration of different antibiotics. These
are then placed on agar plates where the microorganism has been inoculated. It is relatively
simple to do and interpret. Interpretation is done by measuring the zone of inhibition around
the colonies. However, there may be variations in results which may be due to several factors,
namely: (1) size of inoculum, (2) size of antibiotic molecule, and (3) length of incubation.
The disc method can be used to determine bacteriostasis only. It is applicable to fast growing
aerobes and facultative microorganisms.

MATERIALS

• Prepared cultures of aureus and E.

Staphylococcus coli

• Commercially prepared antibiotic discs (Penicillin, Chloramphenicol, Tetracycline,

Methicillin, Erythromycin). Other antibiotic discs may be used if available.

PROCEDURE

1. On the culture plates, place equidistant from each other and in a circular fashion, one disc
each of the commercially prepared antibiotic discs.
2. Incubate at 35 °C for 16–18 hours.
3. After incubation, measure with a ruler the widest diameter of the zones of inhibition of
each antibiotic (expressed in millimeters).
4. Compare the measurements obtained with the reference table. Record your results with the
interpretation (susceptible, intermediate susceptibility, resistant) on the table on the next
page.

Measurement of zone of inhibitio

 Antimicrobial Susceptibility Testing 461

INTERPRETATION OF RESULTS

Antibiotic Zone Size Interpretatio

462 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Answer the following:

1. What are the characteristics of an ideal antimicrobial agent?

2. List down the different antibiotics used for the exercise and give the mechanism of action.

3. What is meant by resistance? Give the mechanisms by which organisms develop resistance
 EXERCISE NO.

8 Bacteria and Disease

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. differentiate
a. disease and infection,
b. pollution and contamination, and
c. virulence and pathogenicity;
2. describe the different types of host pathogen relationships;
3. discuss the mechanisms of disease production; and
4. tabulate the differences between endotoxin and exotoxin.

INTRODUCTION

Different organisms establish different types of relationships. These relationships may

be beneficial only for the organism or may cause harm to other organisms. The relationship
between bacteria and humans is also similar. There are some bacteria that enter the human
body without producing disease (e.g., normal flora) while there are a great number of them that
produce harm to humans by causing disease. The concept on bacteria and disease has been fully
discussed in the textbook and in the classroom. This exercise serves as an assessment of the
students’ understanding regarding the concepts discussed in class
464 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

I. Answer the following:

1. Differentiate:
a. disease from infection

b. virulence from pathogenicity

c. mutualism from commensalism and parasitism

2. What are the different mechanisms of disease production?

3. Enumerate the different stages of an infectious disease and what occurs during
each stage?

II. Compare exotoxin from endotoxin by filling out the table below.
Feature Exotoxin Endotoxi
Relation to cell

Toxicity

Stability

Antigenicity

Conversion to toxoid
 Bacteria and Disease 465

III. Define the following types of infection:

1. Endemic

2. Epidemic

3. Sporadic

4. Exotic

IV. Match the following terms with their respective definitions:

1. Carrier a. Insects that transmit infection

2. Fomite b. Presence of bacteria in the blood
3. Vector
c. Person harboring the organism without
signs and symptoms of the disease
4. Vehicle d. Hospital acquired infections
e. Infection confined to a limited area
5. Septicemia
f. Inanimate object which can carry
6. Fulminant infection microorganisms
7. Nosocomial infection g. Multiplying bacteria in the bloodstream
8. Local infection h. Biological substances that can carry
organisms
9. Bacteremia i. Presence of toxin in the bloodstream
10. Toxemia j. Infection that always leads to deat
This page is intentionally left blank
 9
EXERCISE NO.

Immunology

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. differentiate
a. innate from adaptive immunity,
b. antigen from immunogen, and
c. cell mediated from humoral immunity;
2. give examples of primary and secondary lymphoid organs;
3. compare the different types of hypersensitivity reactions; and
4. describe the five types of immunoglobulins.

INTRODUCTION

Several factors play a role in the occurrence of infection. One of these factors, and probably
the most important of them all, is the defensive powers of the host. Each human being is
equipped with an arsenal of responses that aids him or her in fighting disease producing
organisms. The human body’s first line of defense (e.g., skin, sweat, sebaceous secretions)
helps prevent the entry of organisms into our body. If the organisms gain entry into the body,
the second line of defense (inflammation) inhibits their growth and multiplication. Finally,
organisms that escape the second line of defense are dealt with by the third line of defense, the
immune response. This exercise is meant to assess the understanding of the students on selected
concepts in immunology
468 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

I. Answer the following:

1. Differentiate the following:
a. innate from adaptive immunity

b. antigen from immunogen

c. cell mediated from humoral immunity

2. Enumerate the primary and secondary lymphoid organs

II. Enumerate the different hypersensitivity reactions and give examples for each reaction.
Hypersensitivity
Mechanism Mediator/s Example/s
Reaction
Type I

Type II

Type III

Type IV

III. Give the function/s of the different immunoglobulins.

Immunoglobulin Function/
IgA

IgD

IgE

IgG

IgM
 EXERCISE NO.

10 Protozoans

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. compare the different protozoal infections based on
a. source of infection,
b. mode of transmission,
c. characteristic manifestation, and
d. vector; and
2. characterize the different malarial infections based on
a. hypnozoites produced,
b. type of RBC infected,
c. relapse, and
d. CNS involvement.

INTRODUCTION

Protozoa refer to unicellular, eukaryotic organisms that are the most primitive among
the parasites. Like bacteria, protozoa divide by binary fission. The classification is based on
the mode of locomotion of the different members. Sporozoans are those that hardly exhibit
any movement. A member of this group is Plasmodium, which is responsible for malaria
470 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Members of Phylum Sarcodina, which include the etiologic agent for amoebiasis, move
by means of pseudopodia (“false feet”). The Mastigophora group consist of the flagellates,
which move by means of flagella. Important members of this group are Giardia lamblia and
Trichomonas vaginalis. Balantidium coli is the sole member of Phylum Ciliophora that is
pathogenic to humans. Motility is by means of cilia.

MATERIALS

• Microscope
• Demonstration slides or freshly mounted specimens of the different protozoans
I. Compare the different Plasmodium species.
Feature P. falciparum P. vivax P. ovale P. malariae

Type of RBC
infected

Production of
hypnozoites

Occurrence
of relapse

Timing of
paroxysms

Blackwater
fever

Cerebral
malari
 Protozoans 471

II. Compare the important protozoans.

Parasite Source Mode Characteristic
of Infection of Transmission Manifestation

Entamoeba
histolytica

Naegleria sp.

Acanthamoeba
sp.

Giardia
lamblia

Trichomonas
vaginalis

Trypanosoma
cruzi

Trypanosoma
brucei

Leishmania
donovani

Toxoplasma
gondii

Plasmodium
falciparu
472 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

III. The following protozoans are transmitted by bite of a vector. Indicate the vector for each
of these parasites.

1. Trypanosoma cruzi

2. Trypanosoma spp.

3. Leishmania spp.

4. Plasmodium spp
 EXERCISE NO.

11 Cestodes

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. compare the different cestode infections based on
a. source of infection,
b. mode of transmission, and
c. characteristic manifestation;
2. discuss the infective stage and intermediate hosts of common Cestodes;
3. discuss cysticercosis and its management; and
4. describe hydatid cyst disease and its complications.

INTRODUCTION

Cestodes are worms that belong to the Phylum Platyhelminthes or flatworms. Cestodes are
commonly called tapeworms because their bodies are divided into segments which are called
proglottids. These are hermaphroditic worms, with each proglottid containing both male and
female reproductive organs. Hence, each proglottid is capable of laying eggs. These worms
also have an organ of attachment called a scolex, which helps differentiate one from the other.
The tapeworms can be divided into two major groups—intestinal and extraintestinal
474 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

Intestinal tapeworms are characterized by having an animal intermediate host (cattle, pig,
fish, beetle). Humans serve as the definitive host. On the other hand, the definitive host for
the extraintestinal tapeworm Echinococcus granulosus is an animal (dog) while humans serve as
accidental or dead end hosts.

MATERIALS

• Microscope
• Demonstration slides as well as preserved specimens of the different tapeworms
I. Fill out the table below with appropriate answers.
Source Mode Characteristic
Parasite
of Infection of Transmission Manifestation

Taenia
saginata

Taenia
solium

Diphyllobothrium
latum

Hymenolepis
nana

Echinococcus
granulosu
 Cestodes 475

II. Answer the following:

1. What is cysticercosis? How is it treated?

2. What is hydatid cyst disease? What complication/s can occur

476 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

III. Fill out the table below with the necessary information.
Parasite Common Name Infective Form Intermediate Host

Taenia
solium

Taenia
saginata

Diphyllobothrium
latum

Echinococcus
granulosus

Hymenolepis
nan
 EXERCISE NO.

12 Trematodes

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to:
1. compare the different trematode infections based on
a. source of infection,
b mode of transmission, and
c. characteristic manifestation; and
2. discuss the intermediate hosts, site of infections, and complications of common
trematode infections.

INTRODUCTION

Trematodes or flukes are also flatworms like cestodes but unlike cestodes, their bodies are
not divided into segments. They are also more developed than cestodes because they possess
a primitive nervous system. All flukes are hermaphroditic and have two intermediate hosts
except for blood flukes. For blood flukes, the intermediate host is a freshwater snail. Freshwater
snail also serves as the first intermediate host of other flukes. Those with two intermediate
hosts differ only in their second intermediate hosts. The usual mode of transmission for the
trematodes is ingestion of improperly cooked or raw second intermediate host. Again, the
exception is blood flukes, which are transmitted by skin penetration of the infective larvae
478 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

MATERIALS

• Microscope
• Demonstration slides as well as preserved specimens of the different trematodes
I. Fill out the table below with appropriate answers.
Parasite Source of Infection Mode of Transmission Characteristic
Manifestation

Schistosoma
spp.

Clonorchis
sinensis

Paragonimus
westermani

Fasciolopsis
buski

Fasciola
hepatic
 Trematodes 479

II. Indicate the first and second intermediate hosts of the different trematodes.
Parasite First Intermediate Host Second Intermediate Host

Schistosoma
spp.

Clonorchis
sinensis

Paragonimus
westermani

Fasciolopsis
buski

Fasciola
hepatica

III. Differentiate blood flukes.

Parasite Site of Infection Associated Complication

Schistosoma
japonicum

Schistosoma
mansoni

Schistosoma
haematobiu
This page is intentionally left blank
 EXERCISE NO.

13 Nematodes

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to differentiate the various
nematode infections based on their:
a. primary site of infection,
b. infective stage,
c. source of infection,
d. mode of transmission,
e. characteristic manifestations, and
f. complications.

INTRODUCTION

Nematodes or roundworms are the most developed among the various existent parasites.
The body of nematodes is cylindrical. These worms possess muscles that enable them
to move. They have a complete digestive tract as well as a highly developed nervous system
consisting of nerve bundles, ganglia, and special sensory organs. Roundworms are divided
into intestinal roundworms, and the blood and tissue roundworms. The most common
among the intestinal roundworms is the giant intestinal roundworm Ascaris lumbricoides.
All roundworms are non hermaphroditic. The female worm is usually larger than the male.
The eggs are usually excreted with the feces. There are three major modes of transmission of
the roundworms—ingestion, skin penetration by the infective larvae, and bite of a vector
482 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

MATERIALS

• Microscope
• Demonstration slides of the ova of the different roundworms
• Preserved specimens of the different roundworms
I. Fill out the table below comparing the various nematodes.
Characteristic
Parasite Source of Infection Mode of Transmission Manifestation

Ascaris
lumbricoides

Trichuris
trichiura

Strongyloides
stercoralis

Hookworms

Capillaria
philippinensis

Trichinella
spiralis

Wuchereria
bancrofti

Brugia malay
 Nematodes 483

II. Answer the following:

1. What is Loeffler’s syndrome? This condition is associated with which nematode?

2. Which among the nematodes are transmitted by skin penetration?

3. Which of the nematodes have a lung phase in their life cycle?

4. Differentiate Wuchereria bancrofti and Brugia malayi infection clinically

This page is intentionally left blank
 Infections
EXERCISE NO.

14 of the Skin
Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to develop the skills in
analyzing cases pertaining to skin infections.

I. Case Study
1. A 6 year old boy was brought to a physician because of skin lesions with “honey
colored crusts” over the lower extremities. The condition started a week prior to
consultation as vesicular lesions which later ruptured and formed crusts.

a. What is the most probable diagnosis?

b. What is the most probable causative agent for this condition

486 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

c. What are the distinctive characteristics of the causative agent?

d. What diagnostic tests would you request to establish the diagnosis?

e. What advice will you give to the parents of the child?

f. How can the infection be prevented?

2. A 15 year old girl consulted a physician because of hypopigmentedmacules over

the right cheek. On physical examination, the lesions appear scaly and dry, with a
“chalky” appearance. There are no other accompanying signs and symptoms.

a. What is the probable diagnosis

 Infections of the Skin 487

b. What is the most likely etiologic agent?

c. What tests or procedures should be requested to establish the diagnosis?

II. Identify the dermatophytic fungal infections affecting the following sites:
1. Scalp
2. Groin
3. Hands
4. Bearded area
5. Nails
6. Feet
7. Body or trun
This page is intentionally left blank
 Infections of the
EXERCISE NO.

15 Respiratory Tract

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to develop the skills in
analyzing cases pertaining to infections of the respiratory tract.

I. Case Study
1. A 7 year old child was brought to the emergency room because of fever and a
prolonged episode of forceful, dry hacking cough with a distinct inspiratory whoop.
The patient has no history of immunization.
a. What is the most probable diagnosis?

b. What is the most probable causative agent and its characteristics

490 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

c. What specimen is used for culturing the organism? What culture medium of
choice is used?

d. What are the stages of this infection and the corresponding manifestations of
each stage?

e. How is the infection prevented? Which vaccine should be given, how is it given,
and what is the schedule for giving the vaccine?

2. A 35 year old male construction worker complains of blood streaked sputum. The
condition started one month prior to consultation as dry, non productive cough of
two weeks duration associated with rise in body temperature in the afternoon and
night sweats. The wife noticed that since the condition started, the patient had loss
of appetite and weight.
a. What is the most probable diagnosis
 Infections of the Respiratory Tract 491

b. What is the most probable causative agent?

c. What tests should you request for the diagnosis of this infection?

d. What is the proper way of collecting specimen for sputum examination?

e. Give the characteristics of M. tuberculosis based on the following:

i. Staining reaction
ii. Cell wall components
iii. Growth characteristics
iv. Virulence factors

f. Discuss ways by which the disease can be prevented and controlled

This page is intentionally left blank
 EXERCISE NO. Infections of the
16 Gastrointestinal
Tract
Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to develop the skills in
analyzing cases pertaining to infections of the gastrointestinal tract.

I. Case Study
1. A 50 year old woman was brought to the emergency room because of prolonged
fever of two weeks duration and crampy abdominal pain. The condition started two
weeks prior to consultation as moderate grade fever with loose bowel movement
that was later replaced by constipation.
a. What is the most probable diagnosis?

b. What are the common sources of this infection

494 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

c. Give the characteristics of the probable causative agent.

d. How is this condition managed?

e. What are ways by which the condition can be prevented?

2. A 25 year old male was brought to the hospital because of fever and yellowish
discoloration of the skin and sclerae. On physical examination, the liver is enlarged
and tender. Laboratory results reveal (+) HBs Ag. Gram stain was negative for
bacteria.

a. What is the probable condition involved in this case? What is the most likely
etiologic agent?

b. Based on your answer on no. 1, what are the ways by which the most likely
etiologic agent is transmitted
 Infections of the Gastrointestinal Tract 495

c. Which of the hepatitis viruses is/are associated with the following?

i. Fulminant infection:
ii. Most common cause of post transfusion hepatitis:
iii. Vaccine preventable hepatitis:
iv. Occur as co infection or super infection with HBV:
v. Caused by a DNA virus:

II. Answer the following:

1. Define diarrhea.

2. What are some ways by which diarrhea can be prevented?

3. What is the mainstay in the management of diarrhea

This page is intentionally left blank
 EXERCISE NO. UTI and
17 Sexually transmitted
Infections
Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to develop the skills in
analyzing cases pertaining to infections of the genitourinary tract.

I. Case Study
1. A 28 year old seaman consulted a physician because of a solitary nodule on the shaft
of his penis that is hard and painless accompanied by painless enlargement of his
inguinal lymph nodes. The nodule later formed an ulcer with smooth edges.

a. What is the most probable diagnosis and the most likely etiologic agent?

b. Give two other conditions that can present with a lesion like what is presented by
the patient. How are they different from the case presented
498 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

c. Differentiate syphilis from gonorrhea by answering yes or no on the table below.

Syphilis Gonorrhea
Congenital transmission
Neonatal transmission
Systemic spread
Presence of skin lesions
Purulent discharge

d. In a patient with HIV infection, what is the specific target of the virus and what
will be the effect on the infected person?

e. What is reverse transcriptase and what is its importance in the management of

HIV infection?

2. A 25 year old sexually active female sought consultation because of a burning

sensation during urination. She also complained of frequency and urgency in
urination. The attending physician is thinking of a probable urinary tract infection.
a. Enumerate and explain briefly the factors that can predispose the development
of UTI
 UTI and Sexually transmitted Infections 499

b. Differentiate lower UTI from upper UTI as to clinical manifestations. How do

bacteria reach the kidneys?

c. Why is urinary tract infection more common in females than in males?

d. What is the proper way of collecting urine specimen? What instructions should
be given to the patient when collecting a urine sample?

e. What possible complications can arise in persons with untreated and repeated
urinary tract infection
This page is intentionally left blank
 EXERCISE NO. Infections of the
18 Eyes and Central
Nervous System
Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to develop the skills in
analyzing cases pertaining to infections of the eyes and the central nervous system.

I. Case Study
1. A 30 year old male was attacked by a stray dog on his way home. He went to the
hospital where you are working as an emergency nurse.

a. How will you manage the patient?

b. What factors will affect the length of the incubation period for the development
of rabies infection
502 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

c. Other than animal bite, what are the other modes of transmission of rabies?

d. What is the most distinctive manifestation of rabies?

e. What is the recommended management of rabies?

2. A 6 year old child was brought to the hospital because of seizures. This was
accompanied by fever, nausea, vomiting, and stiff neck.

a. What is the difference between encephalitis and meningitis?

b. How do you elicit Kernig’s and Brudzinski’s signs? What is the expected positive
result for each test
 Infections of the Eyes and Central Nervous System 503

c. What are the most common causes of bacterial meningitis and the corresponding
age groups most commonly affected?

3. A 30 year old male sought consultation because of “eye redness” accompanied by

purulent discharge.

a. What are the common causes of conjunctivitis?

b. What are the ways by which conjunctivitis can be prevented?

c. How would you differentiate bacterial conjunctivitis from viral conjunctivitis

clinically
This page is intentionally left blank
 EXERCISE NO.

19 Viral Exanthems

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to develop the skills in
analyzing cases pertaining to viral exanthems.

I. Case Study
A 3 year old child was brought to the emergency room because of difficulty in
breathing. The condition started four days prior to admission as fever, colds, cough,
and conjunctivitis with associated photophobia. Three days prior to admission,
maculopapular rashes were noted over the face and trunk of the patient which later spread
to the extremities. Immunization history: the patient has been given OPV and BCG
immunization during the first year of life.

• Physical examination: Temp: 38.7 °C; RR: 30/min; PR: 90 beats/min

• Skin: Maculopapular rashes over the face, trunk, and extremities
• Eyes: Reddish conjunctivae with tearing and non purulent discharge
• Nose: (+) Flaring of alae nasi
• Chest and lungs: Intercostal and subcostal retractions on inspection. On
auscultation, (+) crepitant rales on both lung fields. No wheezes
506 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

1. What is the most probable diagnosis? How is the disease transmitted?

2. What do you call the pathognomonicenanthem associated with this disease?

Where is it located and how is it described?

3. What are the most common complications associated with this infectious disease?

4. How is this disease prevented

 Viral Exanthems 507

II. Differentiate rubeola from rubella.

Features Rubeola Rubella

Etiology

Common name

Mode of
transmission

Enanthem

Exanthem

Conjunctivitis

Post auricular
lymphadenopathy
Arthralgia

Congenital viral
infection

Vaccine

III. Differentiate varicella from variola.

Features Varicella Variol
Etiology

Common
name
Mode/s of
transmission

Lesion

Distribution
of lesions

Severity

Vaccine
This page is intentionally left blank
 Other Systemic
20
EXERCISE NO.

Infections

Name: Score:
Section: Date:

LEARNING OBJECTIVES

At the end of the laboratory period, the student should be able to develop the skills in
analyzing cases related to selected systemic infections.

I. Case Study
A 7 year old boy was brought to the emergency room because of nose bleeding
(epistaxis) and vomiting of blood (hematemesis). The patient was apparently well until
7 days prior to admission when the patient developed moderate to high grade fever,
headache, and muscle pains over the lower extremities. Two days prior to admission,
maculopapular rashes were noted over the trunk and extremities. Few minutes prior
to admission, the patient had epistaxis and two bouts of hematemesis.

1. What is the most probable diagnosis and etiologic agent for this case
510 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

2. What is the mode of transmission of this infection?

3. If this is dengue fever, what are the manifestations of classical dengue fever?

4. What is the WHO definition for Dengue Shock Syndrome?

5. What measures should be done to prevent this disease

 Other Systemic Infections 511

II. Answer the following:

1. What are arthropod borne infections? Give examples.

2. How can you prevent arthropod borne infections?

3. What are the congenital abnormalities associated with CMV infection

512 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences

4. Give the similarities and differences between infectious mononucleosis syndrome due
to Epstein Barr virus and Mononucleosis like syndrome due to cytomegalovirus.

5. Tabulate the different Rickettsial infections, their corresponding causative agents,

and vectors
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Index

A Ancylostoma duodenale, 249–251

Animalcules, 4
Abortive poliomyelitis, 390
Anthrax, kinds of, 276
Acanthamoeba, 180
cutaneous, 276
Acidophiles, 44
gastrointestinal, 276, 338
Acquired Immunodeficiency
pulmonary, 276, 308
Syndrome (AIDS), 355
Anthropophilic, 278
Actinomycetes, 279
Antibiotic associated pseudomembranous
Acute bacterial meningitis, 380
colitis, 338
Acute bacterial pneumonia, 299
Antibiotics, 91
Acute necrotizing ulcerative gingivitis, 315
classification of, 92–97
Acute viral aseptic meningitis, 385
Antibodies, 110
Acyclovir, 404
structure of, 110
Adenovirus, 289, 290, 373
Antibody dependent cellular cytotoxicity, 118
Adhesins, 140, 273 Antibody mediated hypersensitivity, 117–119
Aedes aegypti, 411–412 Antigen presenting cells, 103–104
Aedes albopictus, 412 Antigenic variations, types of, 293
Aerobes, 5, 43 antigenic drift, 293
African Sleeping Sickness, 191–193 antigenic shift, 293
AIDS Antigens, 101
See Acquired Immunodeficiency Syndrome properties of, 102
Airborne transmission, 138 Antimicrobial, 91
Alcohols, 86 Antisepsis, 77
Algae, 23 Antiseptic, 62
Alkalophiles, 44 Arboviral encephalitis, 393–394
Alkylating agents, 87 Ascariasis, 243–244
Allergic phenomena, 156 Ascaris lumbricoides, 241–244
Allergic reaction, 115 Asepsis, 62–63
Alpha hemolysis, 14 Asian Flu, 294
Amantadine, 295 Asymptomatic infection, 182, 226
Amoebiasis, 172–173 Autoclave, 80
Anaerobes, 5, 43 Autotrophs, 42
Anaerobic media, 15 Avian flu, 293
Anaphylactic hypersensitivity, 115–117 Axial filaments, 3
520 Index

B C

Bacillus anthracis, 275–276, 308, 338 Candida albicans, 316, 347

Bacillus cereus, forms of, 325 Capillaria philippinensis, 255–256
diarrheal form, 325 Capnophiles, 43
emetic form, 325 Carbuncle, 270
Bacitracin test, 273 Cell wall, 30
Bacteremia, 134 Cell mediated immunity, 113
Bacteria, 22, 29–35 Cellulitis, 272
Bacterial conjunctivitis, 371 Central lymphoid organs, 102
Bacterial enterocolitis, 324–327 Central nervous system, 380
Bacterial growth, 41–46 Cervical cancer, 354
nutritional requirements, 41–42 Cestodes, 207–218
physical requirements, 43–44 extra intestinal, 216
Bacterial infections, 324–339 intestinal, 208–216
Bacterial meningitis, 380–385 Chagas disease, 189–191
Bacterial morphology, 29–36 Chancre, 347
Bactericidal agent, 77 Chancroid, 347, 352
Bacteriology, 3 Chemical disinfectants, classification of, 85
Bacteriophages, 22 Chickenpox, 403–404
Bacteriostatic agent, 77 Chikungunya, 414
Balantidiasis, 179–180 Chlamydia trachomatis, 351, 372
Balantidium coli, 178–179 Chocolate agar, 14
Basophils, 104 Cholera, 336
Bengal strain, 336 Chromoblastomycosis, 279
Beta hemolysis, 14 Chronic bronchitis, 297
Beta hemolytic, 272 Chronic disease, 143
Binnig, Gerd, 10 Classical dengue fever, 412
Blood agar, 14 Clindamycin, 94
Boil, 270 Clostridium botulinum, 387–388
Boiling, 79 Clostridium difficile, 337–338
Bordet Gengou medium, 305 Clostridium perfringens, 274–275, 326, 337
Bordetella pertussis, 305 Clostridium tetani, 386
Borrelia burgdorferi, 423 CMV
Borrelia recurrentis, 424 See Cytomegalovirus
Botulinum toxin, 387 Coccidiodes immitis, 384
Botulism, 387 Colitis, 313
Brill Zinsser Disease, 421 Commensalism, 133, 151
Bronchiolitis, 297 Communicable disease, 142
Bronchitis, 290 Complement system, 113
Bronchopneumonia, 298 pathways, 113–114
Brugia malayi, 258–259 Condyloma acuminatum, 280
Bulbar poliomyelitis, 390 Condyloma latum, 348
Bullae, 269, 397 Condylomata acuminata, 35
 Index 521

Conjugation, 95
Diatoms, 24
Conjunctivitis, 371–372
Contagious disease, 142 Differential media, 15
Contamination, 62, 134 Digenea, 223
Coronavirus, 289 Dinoflagellate, 24
Corynebacterium diphtheriae, 295 Diphtheria, 295
Coxiella burnetti, 421
Diphtheria/Tetanus toxoid and acellular
pertussis (DTaP), 297
Croup, 293
Diphtheria toxin, 295
Cryptococcal meningitis, 384
Diphyllobothriasis, 214
Cryptococcus gatti, 384
Diphyllobothrium latum, 212–213
Cryptococcus neoformans, 138, 384
Diplococci, 29
Culture media, 13–15
Discrete erythematous, 389
classification according to chemical
composition, 13 Disinfection, 62
classification according to functional Dorner stain, 276
type, 14–15 Downey cells, 417
classification according to physical state, 13 Drug modification, 95
Cutaneous mycoses, 278–279 Drug resistance, mechanism of, 94
Cysticercosis, 211 acquired, 94
Cystitis, 365 intrinsic, 94
Cytomegalovirus (CMV), 417 Dry heat, methods of, 81
Cytomegalovirus infections, 417–418 DTaP
Cytotoxins, 328 See Diphtheria/Tetanus toxoid
and acellular pertussis vaccine
Dysentery, 313
D
Dyspnea, 296
Death phase, 46
Decontamination, 62 E
Definitive hosts, 152
Delta hepatitis, 320 Early congenital syphilis, 348
Delta virus EBV
See Hepatitis D virus See Epstein Barr virus

Denaturing agents, 86 Echinococcosis, 218

Dengue fever, 411 Echinococcus granulosus, 216–217
Dengue hemorrhagic fever, 412–413 Ectoparasites, 152
Dengue shock syndrome Efflux pump, 95
See Dengue hemorrhagic fever Ehrlich, Paul, 5
Dental calculus, 315 Ehrlichia chaffeensis, 422
Dental caries, 315 Ehrlichia sennetsu, 422
Dental plaques, 315 Ehrlichiosis, 424
Dermatophytes, 278 ELISA
Dessication, 82 See Enzyme linked immunosorbent assay
Diarrhea, 321–322 Encephalitis, 379
invasive, 322 Encephalomyelitis, 379
non invasive, 322 Encystation, 16
522 Index

Endemic typhus, 421 Exogenous, 143

Endoflagella, 33 Exotoxins, 141
Endogenous, 143 Eye infections, 371
Endoparasites, 152 bacterial, 371–373
Endospores, 35 viral, 373–374
Endotoxins, 141
Enrichment media, 14 F
Entamoeba histolytica, 170
Facultative organisms, 43
Enteric fever, 332
Facultative parasites, 152
Enteric hepatitis, 320
Fascioliasis, 231
Enteric viruses, 323
Fasciolopsiasis, 235
Enteritis necroticans, 337
Fasciolopsis buski, 233–235
Enteritis, 313
Fifth disease
Enterobiasis, 245–246
See Erythema infectiosum
Enterobius vermicularis, 244–246
Filariasis, 259–261
Enterococcus faecalis, 365
Filtration, 82
Enterococcus, 64
Fimbriae
Enterocolitis, 332
See Pili
Enterotoxin, 328
Fitz Hugh Curtis Syndrome, 350
Enzyme linked immunosorbent
assay (ELISA), 357
Flaccid paralysis, 387, 391
Flagella, classiffication of, 33
Eosinophils, 104
Flavivirus, 393
Epidemic typhus, 421
Fleming, Alexander, 5
Epidermophyton, 278
Flora, 51–56
Epiglottitis, 302
Flukes, 223
Epitope, 101
Fluorescent Treponemal Antibody
Epstein Barr virus (EBV), 416 Absorption (FTA ABS), 348
Erratic parasites, 152 Focal infection, 144
Erysipelas, 272 Folliculitis, 270
Erythema infectiosum, 402 Fomites, 68
Erythrogenic toxin, 291 Food borne botulism, 387
Eschar, 269, 421 Forschemer spots, 400
Escherichia coli, 330–331
Fractional sterilization, 79
enteroaggregative/adherent E. coli, 331 Freezing, 82
enterohemorrhagic E. coli, 331 FTA ABS
enteroinvasive E. coli, 331 See Fluorescent Treponemal Antibody
enteropathogenic E. coli, 330 Absorption
enterotoxigenic E. coli, 330 Fulminant infection, 142
Eukaryotes, 20 Fungal meningitis, 384
Eumycotic mycetoma, 279 Fungal skin infections, 276–279
Exanthems, 397 Fungicidal, 77
Excystation, 169 Furuncle, 270
Exfoliative toxin, 271 Fusion protein, 39
 Index 52

G Hapten, 101
HDCV
Gamma hemolysis, 14
See Human diploid cell vaccine
Gas gangrene, 275
HDV
Gastritis, 313
See Hepatitis D Virus
Gastroenteritis, 290, 313
GB virus C Healthcare associated infection, 62
See Hepatitis G Virus Heat, 78–82
General purpose media, 14 types of, 78–82
Genetic exchange, 95 Heavy metals, 87
Genital herpes, 347, 353 Helicobacter pylori, 54, 317
Geophilic, 278 Hemagglutinin, 398
German measles, 399 Hemolytic reactions, 14
Giardia lamblia, 174–175 Hemolytic uremic syndrome, 331
Giardiasis, 175–176 Hemorrhagic colitis, 331
Gingivitis, 315 Hemorrhagic cystitis, 290
Glycocalyx, 30 Hepatitis, 313, 318–321
Gonorrhea, 349–350 Hepatitis A Virus (HAV), 318–319
Granulomatosis infantiseptica, 383 Hepatitis B Virus (HBV), 319
Granulomatous amebic encephalitis, 181 Hepatitis C Virus (HCV), 319–320
Granulomatous meningitis, 383 Hepatitis D Virus (HDV/Delta Virus), 320
Granulomatous reactions, 347 Hepatitis E Virus (HEV), 320
Hepatitis G Virus (HGV/GB virus C), 320
H Herman’s sign, 412
Herpes gladiatorum, 282
H antigen Herpes labialis (fever blister or cold sore), 282
See Hemagglutinin
Herpes simplex infections, 281
H & E stain, 277 Herpes simplex virus (HSV), 283, 353, 374
HAART Herpes zoster, 405
See Highly active anti retroviral treatment
Herpetic whitlow, 282
HAV Heterotrophs, 41
See Hepatitis A Virus
HEV
HBV See Hepatitis E Virus
See Hepatitis B Virus
HGB
HCV See Hepatitis G Virus
See Hepatitis C Virus Higly active anti retroviral
Haemophilus ducreyi, 352 treatment (HAART), 357
Haemophilus influenza biogroup aegyptius, 372 Histoplasma capsulatum, 137, 384
Haemophilus influenzae, 301 HIV
Halitosis, 316 See Human immunodeficiency virus
Halogens, 87 Hooke, Robert, 4
Halophiles, 44 Hookworm infection, 250–251
Handwashing, 63–64 Hordeolum, 270
Hansen’s bacillus, 388 Hortaea werneckii, 277
524 Index

Host, 139 Incubation period, 144

Hot air oven, 82 Indigenous flora, 3
HPV Infant botulism, 387
See Human papilloma virus Infection, 133, 136
HSV classification of, 144
See Herpes simplex virus control of, 61
Human diploid cell vaccine (HDCV), 393 factors of, 136–137
Human herpes virus, 6, 401 modes of transmission, 138–140
Human Herpesviridae, 143, 281, 353, 401, 416–417 portal of exit, 137
Human immunodeficiency virus (HIV), 355
Infectious diseases, 4
Human papilloma virus (HPV), 280, 354
classification of, 142–144
Humoral immunity, 109
stages of, 144–145
Hydatid cyst, 218
Infectious hepatitis, 318
Hydatidosis, 218
Hydrophobia, 392 Infectious mononucleosis, 416–417
Hymenolepiasis, 216 Infective stage, 169
Hymenolepis nana, 214–215 Infestation, 152
Hypersensitivity reactions, 115–123 Influenza, 293–295
antigens of, 293
immunologic types of, 293
I
Influenza, immunologic types of, 293
Iatrogenic infection, 62 Influenza A, 293
Immune complex mediated hypersensitivity, Influenza B, 293
120–121 Influenza C, 293
Immune response, 108–109 Infrared rays, 82
Immune system, 102 Inspissation, 79
Immunity, 106–109, 113–114
Intermediate hosts, 152
adaptive, 108
Intermittent parasites, 152
cell mediated, 113
Interstitial pneumonia, 298
innate, 106
Intestinal capillariasis, 257
Immunization, 124
Isolation, 67
types of, 124
Ixodes, 424
Immunoassay, 297
Immunofluoresence
See Immunoassay J
Immunogen, 101 Japanese B Encephalitis, 393
Immunoglobulins, 110–112 Jenner, Edward, 5, 124, 405
classes of, 111–112
Immunologic, 142 K
Immunology, 3, 101
Immunopathology, 156 Kanagawa hemolysin, 327
Impetigo, 271–272 Keratitis, 181
Incidental parasites, 152 Kinyoun stain, 12
Incineration, 81 Klebsiella pneumoniae, 303
Inclusion conjunctivitis, 372 Klebsiella, 29
 Index 52

Koch, Robert, 5, 134 Malassezia furfur, 276

Koch’s postulates, 5, 134–135 MALT
Koplik’s spots, 398 See Mucosa associated lymphoid tissue
Mannitol Salt agar, 15
L Mastoiditis, 293
Measles virus, 375
Lag phase, 45 Medical asepsis, 63
Large intestines, 321 Membrane attack complex, 107
Late congenital syphilis, 348 Meningitis, 302, 379
Latent disease, 143 Meningococcemia, 381
Latent syphilis, 348 Meningococcus, 381
Leeuwenhoek, Anton von, 4 Meningoencephalitis, 379
Legionella pneumophila, 303–304 Mesophiles, 43
Legionnaire’s disease, 304 Mesosomes, 34
Leishmania braziliensis complex, 186–187 Metazoa, 157–158, 207
Leishmania donovani complex, 184–185 Microaerophiles, 43
Leishmania tropica complex, 187–188 Microbial ecology, 51
Leprosy, 388–390 Microscopy, 273
two forms of, 389 Microsporum, 278
Leprosy, two forms of, 388 Mononucleosis like syndrome, 418
lepromatous, 388 Mucosa associated lymphoid tissue (MALT), 317
tuberculoid, 388 Mumps, 316–317
Leptospira interrogans, 422 Mutualism, 133
Leptospirosis, 422–423 Mycetoma, 279
Lipopolysaccharide, 31, 303 Mycobacterium africanum, 306
Lister, Joseph, 5 Mycobacterium avium intracellulare
Listeria monocytogenes, 382–383 complex, 306, 357
Living cells, 19 Mycobacterium bovis, 306
Lobar pneumonia, 298 Mycobacterium leprae, 388
Localized infection, 144 Mycobacterium tuberculosis, 306, 338–339
Loeffler’s syndrome, 243 Mycolic acid, 32, 306
Logarithmic phase, 45 Mycology, 3
Löwenstein Jensen medium, 15 Mycoplasma pneumoniae, 302–303
Lyme disease, 423 Myelitis, 379
Lymphocytes, 103
Lymphogranuloma venereum, 351–352 N
Lyophilization, 82
Naegleria, 181–183
Nasopharyngitis, 290–291
M Natural killer cells, 104
MacConkey’s agar, 15 Necator americanus, 249–251
Macrophages, 103 Necrotizing fasciitis, 272
Macule, 269 Negri bodies, 392
Malaria, 197–199 Neisseria gonorrheae, 347, 349, 373
526 Index

Neisseria meningitides, 381–382 Parainfluenza, 293

Nematodes, 239–264 Paralytic poliomyelitis, 390
Neonatal herpes, 353 Paralytic shellfish poisoning, 24
Nervous system infections, 379–394 Paramyxoviridae, 297
Neutrophiles, 44 Parasites, 151–152
Nodules, 269, 397 Parasitic infections, 160
Nomarski, Georges, 9 Parasitism, 133, 151–152
Non communicable disease, 143 Parasitology, 3, 151
Non paralytic poliomyelitis, 390 Paratenic hosts, 152
Normal flora, 3, 51, 53 Parenteral hepatitis, 319
Norwalk virus, 324 PAS stain
Nosocomial, 143 See Periodic acid–Schiff stain
Nosocomial infections, 273 Pasteur, Louis, 5
Nosocomial pneumonia, 299, 304 Pasteurization, 5, 78
Nucleoid, 34 Pathogen associated molecular patterns, 107
Pathogenesis, 155
O Pathogenic stage, 169
Pathogenicity, 133
Obligate parasites, 152
Pediculosis pubis, 358
Occupational exposure, 62
Pediculus humanus, 425
Onychomycosis, 278
Penicillin notatum, 5
Open flame, 81
Peptidoglycan, 30
Ophthalmia neonatorum, 350, 373
Periodic acid–Schiff (PAS) stain, 277
Opisthotonus, 386
Periodontal diseases, 315–316
Oral thrush, 316
Periodontitis, 315
Organotrophs, 42
Perioral erythema, 271
Orientia tsutsugamushi, 421
Peripheral lymphoid organs, 103
Ornithodorus, 425
Permanent parasites, 152
Oropharyngeal tuberculosis, 338
Personal protective equipment (PPE), 62, 64–67
Orthomyxoviridae, 293
Pertussis, 305–306
Oseltamivir, 295
stages of, 305
Osmophiles, 44
Pertussis, stages of, 305
Osmotic pressure, 84
catarrhal, 305
Osteochondritis, 274
convalescent, 305
Otalgia, 292
paroxysmal, 305
Otitis externa, 292
Pharyngitis, 290
Otitis media, 292
Pharyngoconjunctival fever, 290
Otorrhea, 292
Phenolic compounds, 86
Phthirus pubis, 358
P Phycology, 3
Papules, 269, 397 Pili, 33
Paragonimiasis, 233 Pityriasis versicolor, 276
Paragonimus westermani, 231–233 Pityrosporum orbiculare, 27
 Index 52

Plasmodium spp., 193–199 R

Platelets, 104
Rabies, 391–393
Platyhelminthes, 207
Radiation, 83
Pneumococci, 300
Rashes, 347
Pneumocystis jiroveci, 357
Red flame, 81
Pneumonia, 295, 298–304, 399
Regan Lowe charcoal, 305
classifications of, 298
Relapsing fever, 424–425
Pneumonia, classifications of, 298
Reservoir hosts, 152
atypical, 298
Retrovirus, 355
typical, 298
Reverse isolation, 68
Poliomyelitis, 390–391 Reverse transcriptase, 355
Poliovirus, 390
Reye’s syndrome, 295
Pollution, 134
Rhinovirus, 289
Polyarthritis, 405 Ribavirin, 298
Pontiac fever, 304
Ribosomes, 34
Post herpetic neuralgia, 405 Rickettsia akari, 421
Post poliomyelitis syndrome, 391 Rickettsia prowazekii, 421
PPE Rickettsia rickettsii, 420
See Personal protective equipment Rickettsia tsutsugamushi, 421
Prevotella intermedia, 316 Rickettsia typhi, 421
Primary amoebic meningoencephalitis, 182 Rickettsialpox, 421
Primary infection, 144 Ringworm, 278
Primary syphilis, 348 Risus sardonicus, 386
Prodromal period, 144 Ritter’s disease, 271
Proglottids, 207 Rocky Mountain Spotted Fever, 420
Prokaryotes, 3, 20 Rohrer, Heinrich, 10
Proteus mirabilis, 364 Roseola infantum, 401–402
Proteus vulgaris, 292 Rotavirus, 323
Protozoa, 23, 169–202 Rapid plasma reagin (RPR), 348
properties of, 170 Respiratory syncytial virus (RSV), 297–298
Pseudohyphae, 316 RPR
Pseudomembrane, 296 See Rapid plasma reagin
Pseudomonas aeruginosa, 273–274, 304 RSV
Psychrophiles, 43 See Respiratory syncytial virus
Pustules, 269, 397 Rubella, 399–401
Pyelonephritis, 366 Rubivirus, 400
Pyemia, 134 Ruska, Ernst, 10
Pyoderma, 272
Pyogenic, 270 S

Saboraud’s dextrose agar, 15

Q Salmonella, 141, 332
QFever, 421 categories of, 332
528 Index

Salmonellae, 332 Staphyloccocal scalded skin syndrome, 271

Saprophytes, 4 Staphylococcus aureus, 270–272, 304, 326
SARS Staphylococcus epidermidis, 272
See Severe Acute Respiratory Syndrome Staphylococcus saprophyticus, 365
Scarlet fever, 291 Stationary phase, 46
Schistosomiasis, 226–227 Sterilization, 62, 77
Scrofula, 306 methods of, 78–87
Scrub typhus, 421 Streptococcal gangrene, 272
Secondary syphilis, 348 Streptococcal pyrogenic exotoxin, 291
Selective media, 15 Streptococcus mutans, 315
Sepsis, 62 Streptococcus pneumoniae, 64, 96, 300, 372
Septicemia, 134, 333 Streptococcus pyogenes, 272
Serratia marcescens, 365 Streptococcus viridans, 52
Serratia spp., 364 Strobila, 207
Serum bath, 79 Strongyloides stercoralis, 251–253
Serum hepatitis, 319 Strongyloidiasis, 254–255
Severe Acute Respiratory Syndrome (SARS), 289 Sty, 270
Sexually transmitted infections, 345–358 Subacute sclerosing panencephalitis (SSPE), 399
Shigella, 141, 334 Subclinical or inapparent infection, 144
types of, 334 Subcutaneous mycoses, 279
Shigellae, 334 Sudden infant death syndrome (SIDS), 388
SIDS Superficial mycoses, 276–277
See Sudden infant death syndrome Surface active agents, 85
Significant bacteriuria, 367 anionic, 85
Sinusitis, 291 cationic, 85
Skin infections, 269–282 Surgical asepsis, 63
types of, 269–282 Symbiosis, 133, 151
Skin infections, types of, 269–282 Syncytia formation, 398
bacterial, 269–276 Syphilis, 347–349
fungal, 276–279
viral, 280–282
Skin lesions, 269 T
Small intestines, 321 T Cell mediated hypersensitivity, 121–123
Sound waves, 83 Tachycardia, 299
Spanish flu, 294 Tachypnea, 299
Spastic paralysis, 386 Taenia saginata, 208–210
Sporadic disease, 143 Taeniasis, 211
Sporothrix schenckii, 279 Taenia solium, 210–211
Sporotrichosis, 279 Target mimicry, 96–97
Spotted fever group, 420 Target sites, modification of, 95–96
SSPE Tertiary syphilis, 348
See Subacute sclerosing panencephalitis Tetanospasmin, 386
Staining, 10–12 Tetanus, 386–38
 Index 52

Tetanus neonatorum, 386 Tuberculosis, 306–308

Thayer Martin agar, 15 Tuberculous meningitis, 383
Thermal death time, 78 Typhoid fever, 332
Thermophiles, 43 Typhus group, 421
Tinea barbae, 278
Tinea capitis, 278
Tinea corporis, 278 U
Tinea cruris, 278 Universal precautions, 67
Tinea manus, 278 Urethral discharge, 347
Tinea nigra, 278 Urethritis, 365
Tinea pedis, 278 Urinalysis, 366
Tinea unguium, 278
Urinary tract infections, 363–367
Tinea versicolor, 276 Urogenital tract infections, 351
Tinnitus, 292
Tonsillopharyngitis, 291
Toxemia, 134 V
Toxic and allergic phenomena, 156
Toxins, 141 Vaccine bath, 79
Toxoplasma gondii, 199–201 Vaccines, 123–126
Toxoplasmosis, 201–202 types of, 124–125
Trachoma, 372 Vaginal discharge, 347
Transduction, 95 Varicella zoster virus (VZV), 403
Transformation, 95 Variola, 405–407
Transitory parasites, 152 Variola virus, 406
Transmission based precautions, 68 Variolation, 405
Transport media, 15 Vector transmission, 139
Trematodes, 223–235 Vehicle transmission, 138
blood dwelling, 224–227 Verotoxin, 331
properties of, 223–224 Vesicles, 269, 397
tissue dwelling, 227–235 Vibrio cholerae, 336
Trench fever, 419 Vibrio parahaemolyticus, 327–328
Treponema pallidum, 347 Vincent’s disease, 315–316
Trichinella spiralis, 261–262 Viral exanthems, 397–408
Trichinellosis, 263–264 Viral gastroenteritis, 323–324
Trichinosis, 263–264 Viral pneumonia, 299
Trichomonas vaginalis, 176–177, 347 Viremia, 134
Trichomoniasis, 178 Viricidal, 77, 83, 87
Trichophyton, 278 Virology, 3
Trichuriasis, 248–249 Virulence, 134
Trichuris trichiura, 247–249 Viruses, 21
Trismus, 386 Visceral leishmaniasis, 185
Trypanosoma cruzi, 188–191 VZV
Trypanosoma spp., 188 See Varicella zoster virus
530 Index

W Y

Warts, 280–281 Yersinia enterocolitica, 335

genital and anogenital, 280
skin, 280
treatment of, 281
Z
Warty lesions, 347
Waterhouse Friderichsen syndrome, 381 Zanamivir, 295
White blood cells, 104 Zernike, Frits, 8
Wirtz Conklin stain, 276 Ziehl Neelsen stain, 12
Woolsorter’s disease, 308 Zika virus, 415
Wuchereria bancrofti, 258–259 Zoonotic infections, 13
The Authors

Dr. Fe A. Bartolome, MD, FPASMAP

Dr. Bartolome finished her Bachelor of Science in Psychology at the University of the
Philippines Diliman in 1981. She obtained her Doctor of Medicine degree at Our Lady
of Fatima University (OLFU) in Valenzuela City where she graduated 7th in her batch and
received the recipient of the Most Outstanding Clinical Clerk Award. She is a full time
faculty of the Department of Microbiology and Parasitology in OLFU Valenzuela and
teaches part time in the Department of Human Structural Biology as well as the Department
of Pathology where she is the officer in charge. She pursued a degree in Master of Arts in
Education major in Educational Management at the OLFU Graduate School where she
graduated Meritissimus (Summa Cum Laude). She also has PhD units in Molecular Biology
and Biotechnology from the University of the Philippines. She was the chairman of the
Curriculum Committee in OLFU College of Medicine and served as the Quality Assurance
Officer of the same college. She served as the President of the Philippine Academic Society for
Microbiology and Parasitology, Inc. (PASMAP, Inc.) for two terms. At present, she is a Fellow
and Secretary of the PASMAP, Inc. and is an Associate Professor I at the OLFU College of
Medicine where she has been teaching for the past 28 years. In addition to these, Dr. Bartolome
is also a certified instructor for Basic Life Support and Advanced Cardiac Life Support of the
American Heart Association.

Dr. Elizabeth P. Quiles, MD, FPASMAP

Dr. Quiles finished her Bachelor of Science major in Chemistry at the Far Eastern
University. She finished her Doctor of Medicine degree at Our Lady of Fatima University
(pioneer class). She completed 11 units in Master of Science major in Microbiology which
she took at the College of Public Health of the University of the Philippines in Manila.
She went on to enroll in Our Lady of Fatima University Graduate School where she
graduated with a degree of Master of Arts in Education major in Educational Management,
Bene meritus (Magna Cum Laude). At present, she is an Associate Professor I at OLFU
where she is a full time faculty and Officer in Charge of the Department of Microbiology
and Parasitology. She is also a part time faculty of the Department of Human Structural
Biology where she teaches Anatomy and Physiology in OLFU College of Nursing. She is
also a certified instructor for Basic Life Support and Advanced Cardiac Life Support of the
American Heart Association
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