Lecture Notes: MEDICAL PARASITOLOGY KRetoriano2017

Definition of Terms:
Parasites Organisms that live on and obtain their nutrients from another organism and obtain their nutrients
from another organism
Parasitology Area of biology concerned with the phenomenon of dependence of one living organism on another.
Medical Primarily concerned with the animal parasites of humans and their medical significance as well as their
Parasitology importance in human communities (Belizario, 2004)
Tropical Branch of medicine which deals with tropical disease and other special medical problems of tropical
Medicine regions.
Tropical Disease Majority are parasitic disease or illnesses which is indigenous to or endemic in a tropical area but may
also occur in sporadic or endemic proportions that are not tropical.

PARASITIC RELATIONSHIPS
Symbiosis Living 2 organisms of different species in which both members are so dependent upon each other that
life apart is impossible. Involve also protection or other advantages to one or both partners.
Mutualism Living 2 organisms of different species in which both members benefit from each other but life without
the other is still possible.
Ex. Termites and the flagellates (Trichonympha campanula) that synthesize cellulose for their digestive
system. This enzyme helps the termites digest the wood that they eat.
Commensalism Two living organisms of different species in which one member benefits but the other member is
neither benefited nor injured.
Parasitism One of the members lives at the expense of the other and causes harm (pathogenic).

Types of Parasitism
According to Habitat:
Endoparasites Organism lives in the body of the Erratic Parasite Organism that lives in an organ different
host (infection) from the one it usually parasitize or not its
Ectoparasites Organism that lives outside the body usual habitat.
of the host or exterior surface of the
host. (infestation)
According to effect of the Parasite to the Host:
Pathogenic Cause injury to the host by its Nonpathogenic Does not cause injury to the host.
mechanical, traumatic and toxic
activities
Others:
Obligate Organism that is completely Spurious Organism that pass the alimentary tract w/o
Parasite dependent on is host or cannot infecting the host
survive outside the host
Facultative Able to live as an independent Pseudoparasite- Artifact mistaken as parasite.
Parasite organism or as a parasite at its own.
Exist in a free-living state or may
become parasitic when needed.
Intermittent Parasite that visits and leaves the Permanent - Parasite which lives its entire life from the
Parasite host at intervals. A.K.A. temporary Parasite time of hatching to death in a single host or
parasite. remains in the body of the host for its entire
life.
Periodic Parasite in which its larval stage Transitory Parasite which passes its larval period of
Parasite develops in a host different from that Parasite development within the body of the host
of the adult. while the adult is free-living
Accidental / Parasite which occasionally occurs in Hematozoic Parasite living inside the RBC.
Incidental an usual host Parasite
Parasite
Cytozoic Parasite living inside the cells of issue Coezolic Parasite living in the body cavities
Parasite Ex. Isosphora hominis Parasite Ex. Mansonella ozzardi
Enterozoic Parasite living inside the lumen of the Temporary Lives on the host only for a short period of
Parasite intestines. Parasite time.

Types of Host
Definitive / Final Harbors the adult or sexual stage of the parasite where the parasite attains its sexual maturity.
Host Humans are always the definitive host expect in Malaria.
Intermediate (1st and 2nd intermediate host) harbors the larva or asexual stage of the parasite. Host that harbors
Host the larval asexual stage or parasite development.
Incidental Host Host not necessary in the arrested stage of development or host other the normal one that is
or Accidental harboring the parasite.
Host
Paratenic Host Harbors the parasite in an arrested stage of development. The parasite do not develop into further
or later stages. However the parasite is still alive enabling it to infect a susceptible host. Important in
knowledge of the parasite life cycle.
Dead-end Host Host in w/c parasite cannot be transmitted further.
Reservoir Host Animal host that harbors the same parasite as man or parasitic to man. They harbour the definitive,
intermediate and paratenic host.
Carrier Host Host that carries the parasite inside but shows no signs and symptoms of infections.
Transport Host Host responsible for transferring a parasite from one location to another.

Types of Vectors
Vector – responsible for transmitting the parasite from one host to another
Biologic Vector Transmits the parasite only after the latter has completed its development within the host.
Mechanical or The sole purpose is only to transmit the parasite regardless of its stage.
Phoretic Vector

Exposure and Infection

Carrier Harbors a particular pathogen without manifesting any signs and symptoms.
Exposure Process or inoculating an infective agent.
Infection Establishment of the infective agent in the host that leads to the manifestation of signs and
symptoms or invasion of the body.
Infestation Invasion of the body
Pre-patent Period Period between infection or acquisition of the parasite and evidence or demonstration of infection.
or Biologic
Incubation Period
Patent Period or Period between infection and manifestation of signs and symptoms.
Clinical Incubation Ex. Diarrhea, fever, chills, abdominal pain and abdominal cramping.
Period
Autoinfection Result when an individual infected becomes his own direct source of infection.
Superinfection or Happens when an infected individual is further infected with the same species leading to massive.
Hyperinfection Infection with the parasite or increase in the worm burden.
Ex. Strongyloides – increase deposition Rhabditiform larvae resulting to its transformation to
Filariform larvae in the gut.

Infective Stage Stage that is infective to host (human).

Diagnostic Stage Stage that can be identified, detected or measured by different laboratory procedures.
Distribution of Disease:
Endemic When a disease in human population maintains a relatively steady, moderate level
Epidemic There is a sharp rise in the incidence or an outbreak of disease.
Hyperendemic If the prevalence of a disease in a community is high.
Sporadic If the disease appears only occasionally in one or few members of the society.
Pandemic When the disease covers extensive area of infection

Method of Parasite Transmission

Thru skin by direct penetration
Anthropod Vectors: Mosquitoes
Flies
Fleas
Ticks
Bugs
Thru ingestion of contaminated food, a. Ingestion of embryonated egg: _______________________.
water and milk (hand to mouth b. Ingestion of embryo in infected flesh: _______________________.
transfer) c. Ingestion of encysted larvae in infected flesh: _______________________.
Thru Sexual Activity

Thru the respiratory tract from

contaminated air or water
Thru placenta or congenital
transmission
In transmamamary infection with ___________________ the parasite may be
transmitted through mother’s milk.
Thru contact with animals Cats
Dogs
Rodents
Contact / Direct Transmission / mouth Enterobius vermicularis
to mouth Triichominas vaginalis
Autoinfection The individual becomes his own source of infection.

Intranasal

Inhalation (Airborne)
Eye contact with infected swimming
water

Epidemiologic Measures:
Epidemiology Study of patterns, distribution and occurrence of disease
Incidence Number of new cases of infection appearing in a population in a given period of time.
Prevalence Number in a population estimated to be infected with a particular parasite species at a given time.
Expressed as percentage.
Cumulative Percentage of individuals in a population infected with at least one parasite.
Prevalence
Intensity of Measured directly by counting expelled worms or indirectly by counting the eggs in the faces.
Infection / Expressed as eggs per gram.
Worm Burden
Morbidity Disease
Mortality Death
 Treatment
Selective Involves individual-level deworming with selection for treatment on a diagnostic of infection or an
Treatment assessment of the intensity of infection or base on presumptive grounds.
Targeted Group-level deworming where the (risk) group to be treated (without prior diagnostic) may be defined by
Treatment age, sex or other social characteristics irrespective of infection status.
Universal Population-level deworming in which the community is treated irrespective of age, sex, infection status or
Treatment other social characteristics.

Other Terminology:
a. Deworming – use if anti-helminthic drugs in an individual or public health programs.
b. Cure Rate – refers to the number (expressed as percentage) of previously positive subjects found to
be egg-negative on examination of a stool or urine sample using a standard procedure at a set time
after deworming.
c. Egg-reduction Rate – percentage fall or decrease in egg counts after deworming based on
examination of a stool or urine sample using a standard procedure at a set time after treatment.
d. Coverage – proportion of the target population reached by an intervention.
e. Drug Resistance – genetically transmitted loss of susceptibility to a drug in a worm population that
was previously sensitive to the appropriate therapeutic dose.
f. Efficacy – effect of drug against an infective agent in ideal experiment conditions and isolated from
any context.
g. Effectiveness – measure of the effect of a drug against an infective agent in a particular host, living
in a particular environment with specific ecological, immunological and epidemiological determinants.
h. Elimination – a reduction in a zero of the incidence of a specified disease in a defined geographic area
as a result of deliberate efforts. Continued intervention or surveillance measures are still required.
i. Eradication – defined as a permanent reduction to zero of the worldwide incidence of infection cause
by a specific agent as a result of deliberate efforts. Surveillance and measures are no longer needed.

New Parasite Laboratory Diagnostic Techniques

1. Direct Fluorescent Antibody (DFA)
2. Enzyme Immunoassay (EIA)
3. Indirect Fluorescent Antibody (IFA)
4. Late Agglutination (LA)
5. PCR
6. Rapid Immunochromatographic Technique

SPECIMEN COLLECTION

3 Phases of Quality Assurance

1. Pre-analytic Collection, handling (guidelines) labelling and specimen transport.
2. Analytic Testing.
3. Post-Analytic Reporting and interpretation of results
Example: No ova or Parasite seen

Recommended Number of Collection: ____________________.

GUIDELINES for collection:
1. If patient is under Barium, Bismuth or Mineral Oil therapy, collection should be before or after 5-7 days upon
completion of therapy.
2. If patient is under antibiotic or anti-malaria therapy, sample should be collected after 2 weeks upon completion
of therapy.
3. Specimen should be collected in a clean, water tight-fitting lid – put in a zip lock plastic bag.
4. Acceptable stool sample is 2 to 5 grams or size of a walnut.
a. Water destroys schistosomes eggs and amebic trophozoites.
5. Specimen containers should have:
• Patient’s Name • Date and time of collection
• Identification Number • Other information: diagnosis, travel history, clinical findings
• Physician’s Name
 6. Water stools should be processed within 30 minutes – usually have trophozoites
7. Formed stools can be processed for 24 hours (cyst) if not add a preservative.

REMINDER: wear gloves at all times! Follow Universal Precaution or OSHA guidelines.

Fixatives
Recommended Ratio: _____________________________

Preservative Comments Advantages & Centrations Permanent Antigen Test

Disadvantages Stain
Formalin • 5% - preserves cyst Advantage: Used Not applicable Used
• 10% - preserves 1. Easy to prepare
helmith egg 2. Preserves specimen for
years
3. Long shell-life

Disadvantage:
1. Morphologic structure not
preserved well for stained
smears.
2. Toxic
Polyvinyl • Composed of plastic Advantage: Used but Applicable: iron Not used
Alchohol poweder 1. Used for both protozoan recovery of hematoxylin or
• Usually combined and helminth eggs parasite is trichome
with Schaudinn 2. Ideal for permanent smears not that
solution (with zinc 3. Longer shelf life at room effective
sulfate, copper sulfate, temperature
or mercuric chloride)
Disadvantage:
1. Not effective as formalin
(lower recovery of parasite)
2. Mercury is toxic
Sodium Acetate Used for concentration Advantage: Used Applicable: iron Used
Formalin (SAF) techniques and 1. Easy to prepare hematoxylin
permanent stained 2. Long shelf life
smears 3. Used for modified acid-fast
stain (coccidians)

Disadvantage:
1. May require albumin to
ensure adhesion of specimen
to slide.
2. Morphology of protozoans
is not clear in permanent
stained smears when mercury
containing fixatives is used.
Modified PVA Contain copper sulfate Advantage: Used Applicable: iron Used (+/-)
(zinc) and zinc sulfate 1. Used for both concentration hematoxylin or
and permanent stained smears. trichome
Zinc sulfate provide
better Disadvantage:
1. Quality of preservation of
protozoan morphology is not
the same with mercury-based
fixatives
Alternative Single Free of formalin and Disadvantage: Used Used (+/-)
Vial System mercury used for 1. Quality not the same with
concentration technique mercury based fixatives
and permanent stain.
Guidelines for Specimen Processing
1. Sample are examined macroscopically: color and consistency
a. Samples should be fresh and unpreserved
b. Soft or Liquid: Protozoans
c. Formed: Helminth eggs, larvae and Protozoan cyst
2. Samples are examined microscropically:
3. Adult worms can be washed with a wire screen

Macroscopic Examination of Stool Specimens

Color Consistency Gross Appearance
Dark Brown Hard Conspicuously fibrous
Black Soft Fiber scanty to moderate
Brown Mushy Colloidal (homogenous)
Pale Brown Loose Scanty mucus
Clay Diarrheic Much mucus
Yellow Watery, liquid Mucus with scanty blood
Red Brown Formed Other (eg. Blood barium)
Green Semi-formed

Color Comment
Brown Normal
Purple or Red Medication
Bloody Amebic ulcerations
Bright red Irritation or bleeding

Microscopic Examination
Fresh Specimen: direct wet smear, concentration technique and permanent stained smear
Fixed Specimen: concentration technique and permanent stained smear
Occular Meter: microns ( u or um – defined as a unit measuring 0.001 (103) millimeter or 10−6 meter.
• Disk that is inserted into the eyepiece of the microscope
• Disk is equipped with a line evenly divided into 50 or 100 units.
• Calibration is repeated ANNUALLY.
• Calibration is by use of a stage micrometer containing a calibrated scale divided into 0.01 mm units. Calibration
involves alingning the eyepiece and stage scales on the microscope followed by determining the values of lines
superimposed to the right of the zero point with a simple calculation.
• REPORTING: Scientific Name with stage of parasite ; WBC – reported semiquantitatively rare, few moderate & many

SEE CHAPTER 2 pages 35-40 for the detailed procedure

Methods Comments Procedure

Direct Wet - Place a drop of 0.85% saline on a glass slide (3 x 2)
Preparation inches) + small portion of unfixed stool using a
wooden applicator stick.
- Put a 22-mm square cover slip on the slide
- Scan using low power (10x)
- OIO not used, applicable only if a temporary seal is
used (hot paraffin-petroleum jelly or Vaseline)
Direct Iodine Wet
Preparation
Concentration
technique

Formalin-Ethyl Acetate Sedimentation

Procedure:
1. Ethyl acetate + saline-washed formalin fixed sample
→centrifuge
Ova-sediment; Fecal Debris – Supernatant
Advantage: good recovery and easy to perform
Disadvantage: more fecal debris
Acid Ether Concentration Technique

Procedure:
1. Stool in acid solution
2. Centrifuge tube by passing thru 2 layers of gauze (to
separate the solid portion)
3. Add ether and place rubber stopper then shake and
centrifuge
4. Examine the sediments, ring out the debris in order
to get the sediment.
Zinc Sulfate Flotation Technique Procedure:
1. Zinc Sulfate + stool → centrifuged
Fecal Debris – sediment ; Cyst – supernatant

Advantage: cleaner preparation & easier microscopy

Disadvantage: not recommended for helminth eggs
Permanent Stain ____________ - most widely used permaninth stain
Advantage: long shelf life and easy to perform

____________- excellent for morphology of intensial

protozoa

____________ - use of Carbol-Fuchisn to detec acid-

fast parasite aside from protozoans

Appearance of Select Apperance of Select Appearance of Select Appearance of Microsporidia

Protozoan Protozoan Structures and Protozoan Structures, Yeast on Modified Trichome Stain
Structures and Background Background Material on and Background Material on
Materials on Iron Hematoxylin Stain Modified Acid-Fast Stain
Trichome Stain
Structure of Appearance Structure Appearance Oocysts of Pink to Structure or Appearance
Materials or Material Cryptoporidium Red Material
and Isospora
Cyptoplasm Light pink Protozoa Blue or Oocysts of Variable; Spores of Pink to Red
of E. or blue Cytoplasm Purple Cyclospora clear to Microsposirida with Clear
histolytica green pink to interior
trophozoite red
and cyst
Cytoplasm of Purple tint Protozoa Blue or Oocysts of Variable; Polar Tubule Red
E. coli cyst nuclear Purple Cyclospora clear to horizontal or
material pink to diagonal bar
red
Nuclear Bright red Debris and Light blue, Background Blue or Bacteria, Pink to red
Chromosomes to red- background sometimes Material Light red Yeast, Debris
purple material with pink
tint
Degenerated Light green Background Green
 Parasites
Background green

Stool Screening Methods

Antigen and Antibody Detection Methods:
EIA IHA
DFA IFA
Membrane Flow Cartridge Technique (Immunochromatographic) Latex Agglutination
Bentonite Flocculation Test – Trichinellosis PCR
Card Agglutination Rapid
Complement Fixation
Immunoblot

1. EIA
2. DFA
3. Membrane Flow Cartridge Techniques
4. Bentonite Flocculation Test
Immunological Testing:
Other Intestinal Specimens
- Performed when DFS is negative
Duodenal Material Procedure:
- Patient swallow a gelatin capsule that contains a coiled length of yarn
- Capsule dissolves in the stomach and the string is carried in the duodenum
- The free end of the string is attached to the patient’s neck of cheek with tape
- After 4-hr incubation period the yarn is pulled out of the patient, then the bile stained mucous material is
brought on the string
Sigmoidoscopy
Material
Cellophane Tape
Preparation

Other Specimens
Blood Collection site: ___________
Anticoagulant: ___________

Types of processing procedures:

1. Thick and Thin smears – Think: Parasite Identification, Thick Malarial count – gold standard
2. Permanent stain – Wright stain (fixative + stain = 1 solution); Giemsa (Fixative and stain are separate)

Appearance of Select Parasitic Structure and Background Material on Giemsa Stain

Leishmania, Red Filariae
Trypanosome, Malaria
& Babesia structure
nuclear structure
Cytoplasm Blue Nuclei Blue to Purple
Schuffner’s dots Red Sheath Clear, may not
stain
Background Material
RBC Pale Red
WBC Purple
Neutrophilic Pink-Purple
Granules
Eosinophilic Purple-Red
Granules

3. Knott technique
-1 ml of blood + 10 ml of 2% formalin → centrifuge (1 min @ 500xg) → stain by Giemsa
4. Buffy Coat Slides – Buffy coat is a layer of blood by Giemsa for Leishmania and Trypanosoma extrarcted by
capillary pipet
Specimen: citrated blood or oxalated blood
 Tube: Wintrobe tube centrifuge for 30 minutes @ 100 x g
5. Culture
NNN (Novy-Macneal-Nicole) medium – 1 drop of blood is inoculated or ground tissue
- examined at 400X magnification; negative cultures should be held for 1 month.

CSF and other

sterile fluid

Ex. fluid in cyst,

aspirates,
peritoneal fluid,
pleural fluid &
bronchial
washings

Tissue and
Biopsy
Specimens /
Tissue Apirates

Sputum

Urine and
Genital
Secretions

Eye Specimens

Mouth Scrapings
and Nasal
Discharge

Urine

Skin Snips

Animal
Inoculation and
Xenodiagnostic
Summary of Conventional Methods of Examination:
Specimen Method
Examination of Stool Macroscopic or Physical
Examination
Microscopic Direct Fecal Smear (DFS)
Examination
Kato – Thick Smear
Concentration
Sedimentation procedures:
1. Acid Ether Concentration Technique (AECT)
2. Formalin Ether Concentration Technique (FECT)
Flotation Procedure:
1. Zinc Sulfate (ZnSO4) flotation
2. Brine Flotation (uses saturated NaCl to float the ova)
3. Sheather’s sugar flotation (for coccidian oocyst like Cryptosporidium parvum)

Stool Culture Methods

1. Corpo culture
2. Harada – Mori or the test tube culture method
Egg counting procedures
1. Kato-katz or the Cellophane covered thick smear
2. Stoll Egg Count
Perianal Swab
1. Cellulose Tape or Scotch tape method
Staining of Stool specimen
1. Iron – Hematoxylin
2. Trichome
3. Chlorazol Black E (Khon’s)
4. Phosphotungstic Acid-Hematoxylin

Examination of Blood Finger-Prick Blood 1. Wet / Fresh Preparation

Sample 2. Stained Smears (Thick and Thin smear
Number of parasites / Number of WBCs (200) x 800 = No. of parasites / uL
3. Capillary Tube Method (Buffy Coat Films and Quantitative buffy coat (QBC)
Veneous Blood 1. Knott’s Concentration
2. Membrane Filtration
Examination of Gross or Macroscopic 1. Color
Sputum 2. Consistency
Microscopic 1. Wet mount
Examination 2. Sputum Concentration
Examination of Urine
Examination of Tissue Liver Aspirates
Aspirates
Duodenal Aspirate Entero – String Test
Cutaneous or Skin
Aspirates
Examination of Tissue Muscle and Rectal
Biopsy Materials Biopsy

PROTOZOANS KBretorian207

History
Fedor Losch (1875) First identified taxonomy of Amoeba coli (E. histolytica) in St. Peterburg, Russia
Clifford Dobell (1919) Description of 3 species as being human activities.
E. histoloytica – 4 nuclei
E. coli – 8 nuclei
E. gingivalis – in oral cavity
Von Prowazek (1912) Identified E. hartmanii, morphologically similar in size with other amoeba.
Emile Brumpt (1925) Identified E. dispar, no ability to cause disease in man and animals
Sargeaunt & Williams Ameba isolates from:
a. asymptomatic – non-pathogenic zymodemes
b. amebic dysentery – pathogenic zymodemes
 General Characteristic of Protozoans:

Endoplasm
▪ Inner portion of the cell that surrounds the nucleus and consist of moderately dense granular protoplasm in
which undigested food is contained in the food vacuoles. Food synthesis takes place in endoplasm and food may
be stored in the form of glycogen or protein (chromatoidal bodies). Mitochrondria, golgi apparatus, microsomes
and endoplasmic reticulum are also present in endoplasm.
Karyosome (nucleolus, Single mass or aggregate of granules, may be near the center of the call or at times
endosome) surrounded by chromatin particles that appears to be arranged on a achromatic
network or chromatin granules lining the nuclear membrane.
Contractile Vacoules Maintain normal osmotic pressure by collecting excess water within their cytoplasm
and expelling it outside.
Nuclear Membrane Connects the nucleoplasm to the endoplasm
○ nucleus – lies within the cytoplasm of the cell.

Nuclear Membrane Connects the nucleoplasm to the endoplasm

Nucleus – lies within the cytoplasm of the cell.

Endoplasm – inner portion of the cell that surrounds the nucleus and consists of
moderately dense granular protoplasm in which undigested food is contained in the
food vacuoles. Food synthesis takes place in endoplasm and food may be stored in
the form of glycogen or protein (chromatoidal bodies). Mitochrondia, golgi
apparatus, microsomes and endoplasmic reticulum are also present in endoplasm.

Ectoplasm
▪ envelopes the endoplasm or the outer portion
▪ function as locomotor apparatus for the procurement and ingestion of food, respiration discharge of metabolic
wastes and protection of organism.
Pseudopobia / pseudopods Locomotion of amebae; “false feet”
Flagella Locomotion of flagellates; hair-like projections of the cytoplasm arising in the
kinetoplast functions as locomotory organelles
Cilia Locomotion of ciliates; arise from the basal granules within the ectoplasm and
distributed over the surface of the body, functions for locomotions.
Cytostome Specialized “cell mouth”, located laterally near the anterior end of the body
Cytopyge Responsible for food wastes are excretion discharge.
Cell Anus Present in ciliates
Excretory Vacoules Collects fluid wastes that are ejected from the cell.
Plasma Membrane In trophozoite stage it functions as the control portion of the intake and output of
food secretions, excretions and maintains normal concentration of the plasma
substance by being permeable to some substances and impermeable to others.

Process asexual multiplication where the nucleus of the parent cell divides mitotically resulting to two identical daughter
cell is _________________________________.
 Encystation Excystation
Factors responsible: Factors responsible:

1. Deficiency or overabundance of food supply 1. Osmotic changes in the medium

2. Excess of catabolic products of the organism of 2. Enzymatic action of the enclosed organism on the
associated bacteria inner surface of the cyst wall.
3. Marked changed in pH 3. Change in favourable pH, food supply, oxygen and
4. Dessication of the medium enzymatic action to host tissues.
5. Depletion or excess supply of oxygen
6. Overpopulation

General Life Cycle

▪ Trophozoite stage > precystic stage > cycstic stage > metacystic stage

TWO MAJOR STAGE OF DEVELOPMENT

TROPHOZOITE CYST

How to Differentiate different species?

1. Size
2. Number of nuclei
3. Endosome
4. Glycogen vacuole
5. Peripheral Chromatin
6. Karyosome

Specimen Consideration: ______________________________________________________________________________.

Laboratory Diagnosis: _________________________________________________________________________________.