Reference Population at risk for contracting parasites

Clinical Parasitology Lecture Individuals in underdeveloped areas and

countries
2nd edition, Refugees
Immigrants
Elizabeth A. Zeibig, PhD, MLS (ASCP) Visitors who are immonucompromised
Individuals living in close quarters
Children who attend day care centers

Chapter 1 Mode of transmission

Parasites - Parasite gains entry into an

unsuspecting host vary by specific
- Organism that live on and obtain
parasite species
their nutrients from another
Mode of Parasite Transmission
organisms
- A field known as parasitology. Ingestion of contamination food
- Responsible for the invasion in the or drink
body (Infection) invasion on the Hand-to-mouth transfer
Insect bite
body (Infestation) and disease. Entry via drilling through the skin
Unprotected sexual relations
Parasitic Mouth-to-mouth contact
Droplet contamination
- The escalation of disease caused by Eye contact with infected
the presence of parasites because of swimming water
global travel tends to result in higher
parasite recovery rates. Types of
Parasite
Disease
Obligatory Cannot survive outside of a host
- Characteristics symptoms, emerged, Parasite
determining an effective means of Facultative Capable of existing independently
healing infected persons become a Parasite of a host
priority. Endoparasite Established inside of a host

Vector Ectoparasite Established in or on the exterior

surface of a host
- Responsible for transmission,
parasite control and elimination also
became important.
Types of
Host Parasitism Two different species
Accidental Other than the of organism that is
or incidental normal one that is beneficial to one at the
host harboring a parasite others expense
Commensal The association
between two different
Definitive Adult sexual phase of organism in which one
host parasite development benefit and has a
occurs neutral effect on the
other
Intermediate Larval asexual phase Pathogenic Parasite that has
host of parasite demonstrated the
development occurs ability to cause
disease
Reservoir Harboring parasites
host that are parasitic for
humans and from Symbiosis
which humans may
become infected - When parasite infect a host
- Primary function of the host is to
Transport Responsible for carry on the parasites life cycle.
host transferring a
parasite from one
location to another
*Parasites have an amazing capability to
Carrier Parasite-harboring adapt to their host surrounding
host that is not
exhibiting any *Parasite alter their antigenic makeup so that
clinical symptoms the host will not recognize the modified
but can infect others parasite as foreign and thus the initiation of
an immune response does not occur.
Parasite-Host
Relationship *some parasite require only definitive host
Terms while others require one or more
intermediate host.
Symbiosis Living together of two
living organism *Parasitic Life Cycle have three common
Commensali Association of two component; mode of transmission,
sm different species of
Infective stage, diagnostic stage.
organism that is
beneficial to one and
neutral to the other
Mutualism Two different
organisms that is
beneficial to both
Parasite life cycle consists of two common Stool
phase;
- most commonly submitted sample
(a) Route a parasite follows when in or on for such studies
the human body - Typical stool analysis consist of;
macroscopic and microscopic
(b) The route a parasite follows
technique
independently of the human body provides
crucial information pertinent to *A process to remove fecal debris, which is
epidemiology, prevention and control. often resembles parasitic forms, is
performed on a portion of sample after a
preservative is added to it.
Infective stage

- Morphologic form that invades

O&P
humans
- Parasite recovery method
Diagnostic stage - O stands for Ova and P stands for
- Can be detected via laboratory parasite
retrieval methods.

Symptoms associated with parasitic disease *Specimen including blood, tissue biopsies,
processes
CSF, sputum, urine, and genital material
Diarrhea may also be examined for the presence of
Fever parasites
Chills
Abdominal pain
Abdominal cramping
Elephantiasis Giemsa stain
Anemia
Vitamin deficiency - The procedure of choice for blood
Bowel obstruction sample submitted for parasite study
Edema
Enlargement of major organs
Cellophane tape preparation
Skin lesions
blindness - Methodology for recovery of
pinworm eggs, and the enterotest
Parasite treatment options parasite are among the traditional
antiparasitic medication tests.
change in diet
vitamin supplement Artifacts/confusers
fuid replacement
blood transfusion - Commonly encountered
bed rest
 - Often referred to as Pseudoparasites
may be present in the sample
submitted for parasite study

Protozoa

- Single-celled eukaryotic animal

- Measure 12 to 18 microns, a measure
defined as one millionth of a meter

Metazoa

- Multicellular worms

Anthropods

- Animalia

Ascaris Lumbricoides egg

- Member of the subkingdom metazoa

which includes multicellular
organism such as parasitic worm

*Scientific names for parasites are written in

italics and consist of two components; genus
and species.

Giardiasis

- Refers to the disease or condition

associated with giardia internalis

*Group of parasites in each classification

table are organized by; kingdom and
subkingdom, phylum and subphylum.
Bolongon, Marjorie B. MLS 3-3 *Morphologic forms of protozoa and
helminths may be detected from a properly
Parasitology Lecture Aug. 29, 2017 collected and prepared stool specimen

*When present, the protozoa forms known

Chapter 2 as trophozoites and cyst may be recovered
from these samples.

*Parasite are often shed enter and

O&P subsequently passed in the stool, they may
not appear in a stool specimen on a daily
- Most common procedure performed
basis; therefore, multiple specimens are
in the area of parasitology is the
recommended for adequate detections.
examination of a stool specimen for
Ova and parasite *Typical stool collection protocol consist of
three specimens, one specimen collected
Ova
every other day or a total of three collected
- Refers to the egg stage of select in 10 days.
parasites
*One exception is in the diagnosis of
Parasite amebiasis, in which up to six specimens in
14 days is acceptable.
- Encompasses the other morphologic
forms that may be present. *stool sample from patients whose therapy
includes barium, bismuth or mineral oil
should be collected in a clean watertight
container with a tight-fitting
*Two general components associated with
this routine parasitology procedure;
Macroscopic and microscopic
examination 2 to 5g

- The acceptable amount of stool

required for parasite study, often
Microscopic examination referred to as the size of a walnut
- Consist of three possible *Urine should not be allowed to contaminate
components, each which is detailed the stool specimen because it has been
in the selections that follow a known to destroy some parasite.
discussion of collection, transport
and fixative for preservation. *Stool should not be retrieved from the
toilet bowl water because free-living
protozoa and nematodes may be confused
with human parasites
*To demonstrate the motility of Stool preservatives and applicable
protozoan trophozoites, a fresh specimen laboratory procedures
is required Preservative Concentration Permanent Antigen
stain Tests
10% formalin + - +
SAF + + (Iron +
Trophozoite stage hematoxylin)
SVA + + (trichrome or
- iron -
- Sensitive to environmental
hematoxylin)
changes and on release from the Modified PVA + + (trichrome or +
body, disintegrates rapidly (zinc) - iron -
hematoxylin)
- Usually found in liquid stool, it Single-vial + (trichrome or
+ +
its recommended that liquid system iron -
specime be examined within 30 hematoxylin)
minutes of passage.

Formed stool *some laboratory prefer to use a two-vial

fixative system; others use a single vial
- Specimens are not likely to contain system.
trophozoites; therefore, they can be
held for 24hrs following collection *If other tests are ordered, such as fecal
immunoassay, the laboratory must ensure
Freshly collected stool sample that the fixative is compatible for use with
these techniques.
- Which is immediately submitted to
the laboratory, is the ideal specimen *Some fixatives contain mercury and
for parasitic examination. When this disposal regulations for these compound
is not possible, the sample must be could affect the laboratorys decision of
preserved to maintain its integrity. which fixatives to use in ther testing
protocols.
Fixative

- Substances that preserve the

morphology of protozoa and prevent Formalin
further development of certain
helminth eggs and larvae. - Has been used for many years as an
all-purpose fixative for the recovery
*The ratio of fixative to stool is important of protozoa and helminthes
for the successful recovery of parasite and, - Two concentrations are commonly
whatever fixative is used, the recommended used; 5% concentration preserves
ratio is three parts fixative to one part stool protozoan cysts and a 10%
concentration preserves helminthes
*The specimen must be fixed in the
eggs and larvae.
preservative for at least 30 minutes before
processing begin.
 - May be routinely used for direct Schaudinn solution contains mercury
examination and concentration chloride
procedures, but not for permanent
smear *Many laboratories choose to use a two-vial
- There are three primary advantages system a formalin vial for the
for use of formalin; (1) it is easy to concentration technique and a PVA Vial for
the stained slide.
prepare; (2) It preserves specimens
for up to several years; and (3) it
has a long shelf life
Sodium Acetate Formalin (SAF)
*Disadvantage of formalin is that it does not
preserve parasite morphology adequately for - A viable alternative to the use of
permanent smears. PVA and schaudinn fixative
- Can be used for performing
concentration techniques and
permanent stained smears.
Polyvinyl alcohol (PVA)
- some laboratories have adopted this
- Compromised of a plastic powder fixative because it only requires a
that acts as an adhesive for the stool single vial, and it is mercury-free
specimen when preparing slides for - Easy to prepare, has a long shelf life,
staining and can be used for preparing smears
- Most often combined with schaudinn for staining with the modified acid-
solution, which usually contains zinc fast stain to detect coccidian oocysts
sulfate, copper sulfate, or mercuric - Another limiting factor of SAF is in
chloride as a base. the choice of permanent stains made
Advantages from this fixative.
Trophozoites and cysts of the
*Disadvantages
protozoa, as well as most helminthes
eggs, may be detected using this Because of the adhesive properties of
fixatives SAF are not good, the addition of
It can be used for preparation of a albumin to the microscope slide may
permanent stained smear PVA- be necessary to ensure adhesion of
preserved specimens have a long shelf the specimen to the slide
life when stored at room temperature Protozoa morphology from SAF-
Disadvantages preserved specimen is not as clear in
Although concentration techniques can permanent stain as when mercury-
also be performed from a PVA- containing preservatives are used.
preserved specimen, the recovery of
certain parasites is not as effective as
when formalin is used.
Modified Polyvinyl Alcohol procedure or if the patient is on antibiotic
therapy.
- Other alternatives to mercury-base
PVA are the use of substitute *The range of colors varies including black
compounds containing copper to green to clay and color in between
sulfate or zinc sulfate
*The color of normal stool is brown and
unusual color, such as purple, red or blue
Advantage
typically suggest that a patient is on
They can be used for concentration
particular medication
methods and permanent stained
smear. *The surface should be examine first for
parasite such as pinworms, tapeworms
*Zinc sulfate fixatives
proglottids and adult worms
- Provide better results than copper
*Sample containing adult worms may be
sulfate reagents.
carefully washed through wire screen. This
process allows for the retrieval and
examination of the parasites for
Alternative Single-Vial systems identification purposes.
- Free of formalin and mercury and *Blood and/or mucus in loose or liquid stool
can be used for concentration may suggest the presence of amebic
techniques and permanent stained ulcerations in the large intestine. Bride red
smears blood on the surface of formed stool usually
- Can also be used for performing associated with irritation and bleeding.
fecal immunoassay
- Do not provide the same quality of Macroscopic
preservation as mercury-based
- Should be examined for the presence
fixatives and organism identification
of gross abnormalities
will be more difficult from
- To perform must receive a fresh,
permanent stained slides.
unpreserved stool specimen.
- The consistency or degree of
moisture in stool specimen may
Processing serve as an indication of the types of
*Samples are examined from two potential parasite present.
perspectives, macroscopic and microscopic *Soft or liquid stool may suggest the
* Color of stool is important because it may presence of protozoan trophozoites.
indicate the condition of the patient, such as *protozoan cysts are most likely to be
whether a patient has recently had a special found in fully formed stools
*helminthes eggs and larvae may be found Microscope
in liquid or formed stools
- Most important piece of equipment
Macroscopic examination of stool specimens; in the parsitology laboratory
Possible Descriptive terms
Consistency Possible Gross Ocular micrometer
term color Appearance
Terms - Special designed ocular piece
Hard Dark Conspicuously equipped with a measuring scale
Brown fibrous - The diagnostic stages of parasites
Soft Black Fiber scanty to
detected microscopically are
moderate
Mushy Brown Colloidal measured in units known as microns
(homogenous) defined as a unit measuring 0.001
Loose Pale brown Scanty mucus [10] millimeter.
Diarrheic Clay Much mucus - Use to measure object observed
Watery, Yellow Mucus with microscopically accurately
liquid scanty blood - Is a disk that is inserted into the
Formed Red- Others eyepiece of the microscope. The disk
brown (blood,
equipped with a line evenly divided
barium)
Semiformed Green, into 50 to 100 units
other
Calibration

- Performed with the use of a stage

Microscopic Examination micrometer containing a calibrated
scale divided into 0.01 mm units
- To detect presence of parasite in a
- This procedure involves aligning the
stool specimen
eyepiece and stage scale on the
- Microscopic examination of stool for
microscope
ova and parasites involves three
distinct procedures, direct wet
preparations, concentrated
technique resulting in Direct wet preparation
concentrated wet preperations, and
- Defined as a slide made by mixing a
a permanently stained smear small portion of unfixed stool (stool
*If the specimen received in fixative, the with no added preservatives) with
direct wet preparation can be eliminated saline or iodine and subsequent
from the O&P procedure; the examination of the resultant mixture
concentrate and permanent stain under the microscope
techniques are performed. - Other parasite stage that might be
observed in a direct wet preparation
 include protozoan cysts, oocysts, *It is recommended to use direct saline and
helminth eggs, and larvae direct iodine wet preparations on each
- Perform only on fresh specimen sample that requires this component of
testing.

Trophozoite mortality

- Can only be demonstrated in fresh Concentration methods

specimens, especially those of a
liquid or soft consistency. If the - Examination of concentration of the
specimen is received in the fecal specimen
laboratory in a fixative, this Concentration technique
procedure can be eliminated from the
Q&P assay - Provide the ability to see any
parasites present clearly.
- Can be performed on fresh or
preserved stool
Direct Saline wet preparation
- Two types of concentration methods
- Made by placing a drop of 0.85% available; sedimentation and
saline on a glass slide and mixing floatation. These techniques use
with small portion of unfixed stool differences in specific gravity and
using a wooden applicator stick or centrifugation to separate the parasite
another mixing tool. from the fecal debris and increase
- Oil immersion is not usually their recovery.
recommended on wet specimen
unless the cover slip is sealed to the Sedimentation techniques
slide. - Parasites are concentrated in the
- A temporary seal can be prepared sediment of the tube following
using a hot paraffin-petroleum jelly centrifugation and the sediment is
mixture around the edges of the examined microscopically.
cover slip.
Floatation techniques
Direct iodine wet preparation
- Parasite are less dense than the
- Enhance the detail protozoan cysts solutions used and during
- Is made using a drop of iodine in centrifugation, they float to the
place of saline. surface
Iodine Formalin-ethyl acetate sedimentation
- Kills any trophozoites present procedure

- Most widely used sedimentation

technique
 - Base on specific gravit - Design to confirm the presence of
- Ethyl acetate is added to a saline- protozoa cysts and/or trophozoites
washed formalin-fixed sample and
the tube is then centrifuged.
Advantages *Permanent stain smear is not the
Provides good recovery of most method use of choice for the
parasites and is easy to perform identification of helminths eggs or larvae
Disadvantages because these parasites often stain too
Is that the preparation contains more dark or appear distorted.
fecal debris than a floatation
technique and is more challenging to *Helminth eggs or larvae are best
the microscopist detected and identified using
concentration technique

*two common stain used for routine

Zinc sulfate flotation technique O&P testing include trichrome and iron
hematoxylin
- Also based on differences in specific
gravity between the sample debris,
which in this case is heavy and sink
to the bottom of the test tube and Wheatley trichrome
potential parasite, which are lighter
- Most widely used permanent stain
and float towards the top of the tube.
- Uses reagent with a relatively long
Advantages shelf life and the procedure is easy to
More fecal debris is removed and it perform
yields a cleaner preparation
Disadvantage Iron hematoxylin
Some helminth eggs are very dense
- May be used instead of the trichrome
and will not float.
technique
*concentrated wet preparations are referred - Excellent morphology of the
to as concentrated saline preparations and intestinal protozoa
concentrated iodine wet preparations - Detection of acid-fast parasites in
addition to the other protozoa
normally recovered using the iron
Permanent stains hematoxylin stain

- Final procedure in the O&P Specialized stains

examination is the preparation and - Important permanent stain procedure
examination of permanent stain for the detection of the oocysts of
smear cryptosporidium, as well as those of
Isospora and Cyclospora
 Disadvantage Sigmoidoscopy material
They do not detect oocysts of the
- Helpful for detecting E. Histolytica.
coccidian parasite or spores of
Material from ulcer obtained by
microsporidia.
aspiration or scraping should be
examined by direct wet preparations
and permanent stains
*Alternative test have been developed that
are often referred to as rapid methods, or Cellophane tape preparation
stool-screening method. These methods
- The specimen of choice for the
can be obtained as kits that contain
detection of Enterobius vermicularis
monoclonal anti body
(pin worm) eggs

Blood
Duodenal material
- Parasite that may be recovered in
*Parasite that reside in the small intestine blood including; leishmania
may be more difficult to recover in a stool donovani and trypanosome spp.,
specimen plasmodium and babesia Spp. And
microfilariae.
*the specimen may be collected by
nasogastric intubation or by the enteric Collection and handling
capsule test
- Blood specimens for parasite study
*Parasite that can be observed in this type of must be collected by aseptic
specimen include; giardia intestinalis technique
trphozoites, cryptosporidium spp., - Most laboratories use venipuncture
Isopora belli, strongyloids stercoralis and specimens collected with an
eggs of Fasciola hepatica or clonorchis anticoagulant.
sinesis - Blood specimens should be collected
in tubes containing Ethylene diamine
tetraaetic acid (EDTA).
Enterotest *If Malaria is suspected, it is best to
- Simpler method for collecting prepare smear within 1 hour of
duodenal material without requiring collection, because storage of blood for a
intubation. The patient swallow a longer period leads to distortion and
gelatin capsule that contain a coiled possible loss of malaria parasite
length of yarn.
 *Once the blood sample has been Wrights stain
collected, two types of smears may be
made; thick and thin. - which contains the fixative and stain
in one solution
Thick smears - typically yields only satisfactory
results
- Frequently satisfactory for screening
purposes, particularly when malaria Giemsa stain
is suspected
- Primarily used when parasite are few - In which the two are separated
in number or when thin smears are - Thus considered the preferred stain
negative because it allows for the detection of
parasite detail necessary for species
Advantages
identification
Increase the ability to detect the
malaria parasite Knot technique
Disadvantages
Is that red blood cells have been - Designed to concentrate blood
lysed and it is not possible to assess specimen suspected of containing
the morphology of parasite that are low numbers of microfilariae
detected.
Buffy coat slide
Thin Smear
Buffy coat - layer of white blood
- Provide the best view of the malaria cells between the plasma and red
parasite in the RBC blood cells that results from
extracted from blood specimens,
Dehemoglobinized thick smear stained , stained with giemsa stain
and microscopically examined for
- Typically have much higher
leishamania and trpanosoma
concentration of parasite than thin
smear Cultures
Permanent stain Leishmania Spp and trypanosome
- Two permanent stains commonly cruzi uses Novy-MacNeal-Nicolle
used for the detection of blood (NNN) medium.
parasites; Wrights stain and NNN slant
Giemsa stain
- Inoculated by the addition of a single
drop of collected blood or ground
tissue
Cerebrospinal Fluid Eye specimen

- Specimen may be collected for the - Acanthamoeba keratitis is best

diagnosis of amebic condition diagnosed by the collection and
associated with select ameba examination of corneal scrapings
- A wet preparation can be prepared to - Acanthameoba T, gondii,
search for the presence of the micrcosporidia and Loa Loa are
characteristics morphologic forms of also potential eye pathogens
Naegleria fowleri and
Acanthamoeba spp. And the
trypomastigote stage of Mouth scraping
trpanosoma spp.
- Sample choice for detection of E.
ginggivalis and trichomonas tenax
are helpful for the recovery of
CFS sendiment
parasites such as N. Fowleri
- Is inoculated to the medium, sealed
and incubated at 35 C Skin Snips

- Useful for detection of onchocerca

Hepatic abscess
volvulus may be made using of two
- Material is the specimen of choice collection techniques
for patients suspected of liver
abscesses caused by E. Histolytica Culture methods

- Not a common means of detecting

Sputum
parasites
- Typically collected and tested from
patients suspected of being infected Xenodiagnosis
by the fluke - Techinique used for the diagnosis of
changas disease
Urine

- Specimen of choice for Immunologic testing

demonstrating the motile - Usually considered as an adjunct or
trophozoites. supplement to standard laboratory
protocols
Alternative techniques for the
diagnosis of T. Vaginalis include
antigen detection methods using
latex agglutination and EIA
procedures