Diagnosis of Parasites

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 General laboratory techniques
1. Parasitic (morphological) diagnosis
Macroscopic
Microscopic
2. Immunological diagnosis
Antibody detection
Antigen detection
3. Molecular diagnosis
4. Culture
5. Xenodiagnosis

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 1-Parasitic (morphological) diagnosis
Laboratory procedures detect organisms within clinical specimens using
morphological criteria,
A. Macroscopic-using our naked eye
Eg: Stool specimen can be examined with the naked eye for
presence of some adult worms
E.g. Ascaris, Taenia species, E.vermicularis and gravid Taenia
species
B. Microscopic
Majority of intestinal, blood, urinary and skin parasites are usually detected
microscopically
Use different body specimen such as blood, stool, urine, CSF etc
Stained or unstained preparation
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 2-Immunodiagnosis
Is based on the detection of
A. Antibody detection
Ab is produced in response to a particular parasitic infection.
The Ab. may persist for a long period of time in the serum after an
infection has ended
antibody tests are may not able to distinguish between past or present
infection
B. Antigen detection
Ag. is excreted by parasites and can be found in the serum, urine, CSF,
feces or other specimens.
Antigen tests provide evidence of present infection

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 Immunodiagnostic techniques are required
Parasites live in the tissue of internal organ can not easily obtained for
examination.

Parasites can be found in specimens only in certain stages of infection,

e.g., in the acute stage not in the chronic stage.

Parasites are present intermittently or in too few numbers to be easily

detected in the specimens.

The techniques used to detect parasites are complex or time consuming.

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3-Culture
Growth medium is provided for a specific parasite.
A specimen is tested for the presence of an infectious agent able to
grow within that medium
Culture allows identification of infectious organisms by examining
their
microscopic features,
by detecting the presence of substances produced by pathogens,
and
by directly identifying an organism by its genotype
Relatively few of protozoa and helminths parasites, can be cultured.
eg. E.histolytica, T.vaginalis, T.cruzi and Leishmania species
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4. Xenodiagnosis (XD): XD is a diagnostic procedure which uses the vector
which acts as a biological culture medium for the detection of T. cruzi in the
blood of infected man and other mammals.
5. Molecular Diagnosis
Microscopic examination is still considered the gold standard for the
diagnosis of parasitic diseases.
the stool specimen can be analyzed using molecular techniques such as
polymerase chain reaction (PCR).
PCR amplified fragments can be analyzed by:
Using restriction fragment length polymorphisms (RFLP) or
DNA sequencing if further characterization is needed.

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 Examination of stool
Purpose of examining stool:
To identify intestinal parasitic infection associated
Severe anemia especially in pregnant & child
Series ill-health
Persistent diarrhea
Weight loss, malabsorption
Impairment of development etc
To identify chronic infections with serious complication
To identify parasitic causes of blood and mucus
To assist in surveillance & control of parasitic infection
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 Examination of stool
Macroscopic examination of stool
color
consistency
Abnormal elements
Odor
Volume
Microscopy examination of stool
for blood, mucus, fat, or parasites
Immunological
Ag-detection
Ab detection
Molecular technique
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 Examination of stool
Detection of parasites depends on proper collection and fixation of samples
Stool collection container should be
clean, dry, water-proof, wide-mouth container
Sufficient amount should be collected and
Series of 3 specimens should be collected and examined properly
Appropriate precautions!
NO contamination with H2O - could bring free-living protozoans
NO contamination with urine - destroys trophozoites
Collect before antibiotics and anti parasite treatment - may obscure/decrease
number of organisms

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 Direct wet mount
Fresh Stool Examination Materials:
Primarily motile protozoans in liquid/soft stools Microscope
Wet prep - small amount of stool in Microscope slides
saline: detect worm eggs/larvae & refractile Cover slips
protozoan cysts Sodium chloride
Iodine: shows nuclear detail Solution bottle with pipette
Wooden stick
Fresh stool
Gloves

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1. Use cleaned microscope Slides
2. Place a drop of saline
3. Take a small amount of stool
4. Mix stool with saline
5. Note: Mistake:Too much stool
6. Place coverslip Avoid air bubble
7. Examine by microscopy: use
10x objective
ova/larva/cyst/trophozoite

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 Ova, cysts and trophozoites can be identified as per their characteristic
features
Reporting of result
Species of parasite, stage of parasite
and parasitic load should be indicated
No O/P seen (If the result is
negative)
Ova of A.lumbricoids 20/slide or
Trophozote stage of P.falciparum 3+
or (200parasite/ml blood)
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 Preservation
Required if the sample will not be delivered to lab. immediately
Preserve protozoan and helminthic egg/larvae morphology
Prevents development of worm eggs/larvae
Common preservatives of stool
5 - 10% formalin - wet mount/concentrate; cysts, eggs, larvae can be
preserved for long time.
Sodium-acetate-acetic acid formalin (SAF): for concentration &
permanent stain
Merthiolate-iodine-formalin (MIF): preserves protozoan/worms in
wet mount & concentration (NOT for permanent stain)
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 Concentration techniques
Fecal concentration techniques are used to concentrate and quantify fecal
parasites when a direct wet mount fails to reveal the presence of parasites.
Concentrate the parasites using gravity or centrifugation
Can detect parasites in small amount of sample
Removes most of debris
Protozoan trophozoites do not survive
Detects protozoan cysts, worm larvae/eggs
Fecal concentration techniques include:
The sedimentation method
The floatation method

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 Concentration techniques
Sedimentation method
Uses the principle of gravity or centrifugation to allow for the recovery of all
protozoa, eggs and larvae present
The sediment preparation contains more fecal debris
Example: the formalin-ether method
Floatation method
Separates protozoan cysts and certain helminth eggs through the use of a liquid
with a high specific gravity
Provides a clearer preparation without much debris compared to the sedimentation
method
Examples:
zinc sulfate floatation technique
saturated sodium chloride method
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 Staining of Parasites
Staining is a means of providing color to different components of a
Parasite/cell
Parasites can be easily detected after staining
Acid fast staining- Intestinal parasites eg. Tenia Spp.
Giemsa stain- for blood parasites eg. Plasmodium spp.

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Staining with Giemsa Stain

Immediately before use dilute the Giemsa as required

3% for 30 min
10% for 10 min
Prepare Thin and Thick films then allow to dry
Thin films must be fixed in absolute methanol for at least 30
seconds(1-2min)
Stain
Wash
Drain & dry
Examine under microscopy using oil immersion objective(100X)
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