LABORATORY DIAGNOSIS OF

PARASITIC DISEASES
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Examination of Feces
 As many parasites inhabit in the intestinal tract, stool examination is the
most common diagnostic technique used for the diagnosis of parasitic
infections.

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Specimen Collection
 Stool specimens should be collected
in a wide-mouthed, clean, leak-proof,
screw capped containers and should
be handled carefully to avoid
acquiring infection from organisms
present in stool.
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Specimen Collection (Cont..)
Timing:
 Collected before starting anti-parasitic drugs and closer to the onset of
symptoms.

Frequency:
 At least three stool specimens collected on alternate days (within 10 days)
(except for intestinal amoebiasis - six specimens may be recommended).
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Specimen Collection (Cont..)
When to examine:
 Liquid stool specimens - examined within 30 minutes,
 Semisolid stools - within 1hr.
 Formed stools - up to 24 hours after collection.

For monitoring response to therapy:

 Repeat stool examination - 3 to 4 weeks after the therapy for intestinal
protozoan infection, and 5–6 weeks for Taenia infection.

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Specimen Collection (Cont..)
If delay in transport:
 Fecal specimens should be kept at room temperature;
 Preservatives (e.g. 10% formalin) - used to maintain the morphology of the
parasitic cysts and eggs.

Specimens other than stool:

 Perianal Swabs (cellophane tape or NIH swab)
 Duodenal Contents
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Macroscopic Examination
 Mucoid bloody stool: acute amoebic dysentery, intestinal schistosomiasis,
and invasive balantidiasis.
 Color: Dark red stool - upper gastrointestinal tract (GIT) bleeding and a
bright red stool - bleeding from lower GIT.
 Frothy pale offensive stool (containing fat) - found in giardiasis.

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Stool Consistency

 In liquid stool - trophozoites are usually

found.
 Semi-formed stool - both trophozoites and
cysts are found.
 Formed stool - cysts are mainly found.

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Microscopic Examination
 Includes direct wet mount examination

 Permanent staining methods.

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Direct Wet Mount (Saline and Iodine Mount)

 Drops of saline and Lugol’s iodine

are placed on left and right halves of
the slide respectively.
 A small amount of feces (~2 mg) -
mixed with a stick to form a
uniform smooth suspension.
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Direct Wet Mount (Saline and Iodine Mount)
(Cont..)
Method of screening of slide
 Cover slip - placed on the mount and
examined under low power objective
(10X) for detection of helminths eggs
and larvae; followed by high power
objective (40X) for protozoan cysts
and trophozoites.
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Direct Wet Mount (Saline and Iodine Mount)
(Cont..)
Method of screening of slide
 Cover slip - placed on the mount and
examined under low power objective
(10X) for detection of helminths eggs
and larvae; followed by high power
objective (40X) for protozoan cysts
and trophozoites.
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Normal constituents and artifacts found in
stool in wet mount examination

(A and B) Yeast cell resembling: (A) Giardia cyst; (B) Cryptosporidium oocyst; (C to E)
Fungal spore resembling: (C) Cyst of Entamoeba; (D and E) Cystoisospora oocyst; (F to
K) Pollen grain resembling: (F) Blastocystis (Trichrome staining); (G) Blastocystis
(saline mount); (H) Protozoan trophozoite; (I) Ascaris egg; (J) Clonorchis egg; (K) 13

Taenia egg; (L and M) Plant cell resembling: (L) Helminth eggs; (M)Hookworm egg;
Normal constituents and artifacts found in
stool in wet mount examination (Cont..)

(N) Plant hair resembling Strongyloides larva; (O) Diatoms; (P) Mite egg resembling
hookworm egg; (Q to S) Crystals: (Q and R) Pineapple juice crystals and kiwi
crystals; (S) Charcot-Leyden crystals; (T to W) Human cells resembling trophozoites:
(T) White blood cells; (U) Macrophages; (V and W) Epithelial cell; (X) Air bubbles; 14

(Y) Fat globules

Saline Mount
 Useful in the detection of trophozoites and cysts of protozoa, and eggs and
larvae of helminths.
 Advantages than iodine mount:
 Motility of trophozoites and larvae in acute infection.
 Bile staining property can be appreciated - bile stained eggs appear
golden brown and non-bile stained eggs appear colorless.

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Iodine Mount

Advantages:
 Nuclear details of protozoan cysts, helminthic eggs and larvae are better
visualized, compared to saline mount.

Disadvantages:
 Iodine immobilizes and kills the parasites, hence motility of the
trophozoites and helminthic larvae cannot be appreciated
 Bile staining property cannot be appreciated.

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Non-bile stained eggs
 Eggs of most of the intestinal parasites when they pass through intestine are
stained by bile.
 Exceptions:
 Enterobius
 Hookworm
 Hymenolepis nana

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Reporting of Wet Mount Examination
 Report is preliminary, and the result is reported as ‘presumptive
identification’; which should be confirmed only after examination of
permanent stained smear or immunoassay.

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Permanent Stained Smear
 Required for accurate detection of protozoan cysts and trophozoites by
staining their internal structures.
 Commonly used methods are:
 Iron-hematoxylin stain
 Trichrome stain
 Modified acid-fast stain - useful for coccidian parasites such as
Cryptosporidium, Cyclospora and Cystoisospora

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Concentration Techniques
 If the parasite output is low in feces (egg, cysts, trophozoites and larvae) and
direct examination may not be able to detect the parasites, then the stool
specimens need to be concentrated.
 These methods are also useful in epidemiological analysis and for assessing
the treatment response.
 The eggs, cysts and larvae are recovered after concentration procedures; but,
the trophozoites get destroyed.
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Commonly used concentration techniques

 Sedimentation techniques

 Floatation techniques

 Zinc sulphate flotation concentration technique

 Sheather’s sugar flotation technique

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Egg Counting (Egg Quantification) Methods

 Direct smear counting method of Beaver

 Kato-Katz thick film method

 Stoll’s method or dilution egg counting method

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Examination of Blood
 Useful in diagnosis of infection caused by blood parasites like Plasmodium,
Trypanosoma, Leishmania, Babesia, Wuchereria bancrofti, Brugia malayi,
Loa loa and Mansonella.

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Examination of Blood (Cont..)
 Various methods of examination of blood include:

 Direct wet mount examination

 Examination of blood smears (thin smear and thick smear)

 Quantitative buffy coat (QBC)

 Concentration of blood

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Microscopic Examination of Other Specimens
Specimen Morphological form Parasite

Peripheral blood smear Ring form, schizont Plasmodium spp.

gametocyte
Amastigote Leishmania spp.

Trypomastigote Trypanosoma spp

Microfilaria Filarial nematodes

Bone marrow, liver, lymph Tachyzoite Toxoplasma gondii

node, splenic aspirate
Amastigote Leishmania donovani

Liver aspirate Trophozoite Entamoeba histolytica

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Microscopic Examination of Others
Specimens (Cont..)
Specimen Morphological form Parasite

Lymph node aspirate Trypomastigote Trypanosoma spp.

Lymph node biopsy Adult worm Wuchereria bancrofti, Brugia

malayi
CSF Trophozoite Naegleria fowleri
Acanthamoeba

Trypomastigote Trypanosoma spp.

Urine Trophozoite Trichomonas vaginalis

Microfilaria Wuchereria bancrofti

Egg Schistosoma haematobium

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Microscopic Examination of Others
Specimens (Cont..)
Specimen Morphological form Parasite

Sputum Adult worm Paragonimus spp.

Egg Paragonimus spp.

Larva (migrating) Ascaris Strongyloides

Hookworm
Trophozoite Entamoeba histolytica

Duodenal aspirate Trophozoite Giardia lamblia

Larva Strongyloides stercoralis

Corneal scrapings Trophozoite Acanthamoeba spp.

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Microscopic Examination of Others
Specimens (Cont..)
Specimen Morphological form Parasite

Skin Amastigote Leishmania spp.

Microfilaria Onchocerca volvulus

Larva in skin ulcer Dracunculus medinensis

fluid
Muscle tissue Encysted larva Trichinella spiralis

Cysticercus cellulosae Taenia solium

Perianal area Egg Enterobius

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Immunodiagnostic Methods
 Detection of parasite specific antibodies in serum, and detection of
circulating parasitic antigen in the serum.
 These methods are useful when:
 Parasites are detected only during the early stages of the disease
 Parasites occur in very small numbers
 Parasites reside in internal organs and morphological identification is
not possible
 When other techniques like culture are time consuming
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Antibody Detection Tests
 Antibodies are detected in various parasitic infections mainly from serum,
sometime from other specimens such as CSF (neurocysticercosis) or
pleural fluid (paragonimiasis).

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Antibody Detection Tests (Cont..)
 Amoebic liver abscess: ELISA, detecting antibodies against 170 kDa of lectin
antigen.
 Visceral leishmaniasis: Detecting antibodies to rK-39 antigen by
immunochromatographic test (ICT)
 Toxoplasmosis: (i) Sabin-Feldman dye test- a complement mediated
neutralization test, which detects antibodies, (ii) Detection of specific IgM or
IgA or IgG antibodies by ELISA
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Antibody Detection Tests (Cont..)
 Cysticercosis: (i) ELISA, detecting antibodies against purified
glycoprotein antigens, (ii) Western blot, detecting antibodies against highly
specific 50–13 kDa lentil lectin-purified seven glycoprotein (LLGP)
antigenic fractions.
 Hydatid disease: (i) ELISA, detecting antibodies against B2t or 2B2t
antigen, (ii) DIGFA (Dot immunogold filtration assay)
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Antibody Detection Tests (Cont..)
 Lymphatic filariasis: (i) Flow-through assay, detecting antibodies against
recombinant filarial antigen (WbSXP-1), (ii) Brugia Rapid, detecting
antibodies against Recombinant B. malayi antigen (Bm-14).

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Antigen Detection Tests
 Amoebiasis: ELISA, detecting 170 kDa of lectin antigen in blood and
stool.
 Triage parasite panel: It is an ICT, that detects three antigens in stool:

 Giardia (alpha-1 giardin antigen)

 E. histolytica/ E. dispar (29 kDa Ag)

 Cryptosporidium (isomerase Ag) 34

Antigen Detection Tests (Cont..)
 Malaria: ICT format available detecting:
 Histidine rich protein-2 (Pf. HRP 2)— P. falciparum specific
 Parasite lactate dehydrogenase (pLDH) and aldolase - common to all
species
 Lymphatic filariasis: ELISA and ICT formats are available detecting
filarial antigens by using monoclonal Ab against Og4C3 and AD12
antigens.
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Molecular Methods
 Polymerase chain reaction (PCR) and real time PCR.

 LAMP assay: developed for visceral leishmaniasis and malaria.

 BioFire FilmArray: It is an automated multiplex nested PCR. The

gastrointestinal panel can simultaneously detect 22 enteric pathogens,
including 4 parasites such as—E. histolytica, G. lamblia, Cryptosporidium,
Cyclospora.
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Other Diagnostic Modalities
 Culture techniques

 Imaging techniques

 Intra-dermal skin tests

 Xenodiagnostic technique

 Animal inoculation methods

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