Diagnostic methods in intestsinal parasites

1. Diagnostic methods in
parasitology
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Dr. Roma A Chougale
Dept. of Microbiology
D.Y Patil Medical College, Kolhapur

2. Lab diagnosis in parasitic infections
• Detection of parasite or its stage (trophozoite,
cyst, egg, larva, adult etc.) in different
specimens
• Culture and egg counting
• Serological techniques and immunodiagnosis
• Molecular methods – DNA probes, PCR
• Imaging techniques
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3. Intestinal parasites found in feces
• Entamoeba histolytica
• Giardia lamblia
• Cryptosporidium parvum
• Isospora belli
• Cyclospora cayetanensis
• Balantidium coli
• Diphyllobothrium latum
• Taenia species
• Hymenolepis nana
• Schistosoma mansoni
• Schistosoma japonicum
• Fasciola hepatica
• Fasciolopsis buski
• Clonorchis sinensis
• Ascaris lumbricoides
• Ancylostoma duodenale
• Necator americanus
• Enterobius vermicularis
• Strongyloides stercoralis
• Trichuris trichiura
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4. Stool examination
• Collection – wide mouthed, clean, leak-proof
container without contamination of urine, water
or disinfectants
• Time of collection –
- Close to the onset of symptoms
- Before anti-parasitic therapy
- First morning sample
- Free from oil, barium, bismuth
- i/c/o barium enema – after 1 week
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5. • Three specimens for eggs/larvae, six for
amoebiasis or giardiasis
• Avoid purgatives with oil base
• Liquid stools – examine within 30 minutes
for trophozoites
• Semi-formed stools – in 60 min
• Formed stools – only cysts, examine up to
24 hrs
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6. Macroscopy:
1. Mucoid bloody stool – Amoebic dysentery,
Intestinal schistosomiasis and invasive
balantidiasis
2. Colour: Dark red= Upper GIT bleeding and
bright red= Lower GIT bleeding
3. Frothy pale offensive stool(Steatorrhoea or fat in
stools)- Giardiasis
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7. Preservation of stool
• To maintain morphology of protozoa, eggs
• Analysis in reference lab
• Prevent further development i/c/o
helminthic eggs, larva
• Trophozoite motility – lost
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8. Methods of preservation
1. Formalin
- 10% formalin saline solution
- 3 parts formalin + 1 part stool
- Long shelf life
- Maintains eggs, larvae, cysts
- Not adequate for trophozoites
2. Polyvinyl alcohol (PVA)
- Preserves trophozoites well
- 3 parts PVA + 1 part stool
- Not adequate for helminth eggs, larvae, coccidia, microsporidia
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9. 3. Merthiolate-iodine-formalin (MIF) solution
- Stains and fixes parasitic cysts, eggs, larvae
- Not suitable for trophozoites
- Preserves specimen for > 1 year
4. Schaudinn’s solution
- Mercuric chloride, ethyl alcohol, glacial acetic acid,
glycerol, D/W
- Trophozoites, cysts – well preserved
- Used for fixing stool smears before permanent staining
(e.g. Trichrome stain)
- Not adequate for helminth eggs, larvae, coccidia,
microsporidia
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10. Examination of stool
• Macroscopic examination
- Consistency, colour, odour
- Presence of blood, mucus
- Adult helminths – Ascaris, Enterobius, segments of tapeworms
- Trophozoites (liquid stool), cysts (semiformed or formed)
- Coccidian oocysts (Cryptosporidium parvum) – any type of stool
- Helminthic eggs (any stool)
- Blood & mucus – amoebic dysentery, schistosomiasis, invasive
Balantidium coli infection, heavy infection with Trichuris trichiura
- Fat coloured, frothy stool – giardiasis
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11. • Microscopic exam –
1. Saline wet mounts
2. Iodine wet mounts
3. Smear after concentration
4. Permanent stained smears
i. Iron hematoxylin stain
ii. Trichrome stain
iii. Modified acid fast stain
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12. Saline wet mount
• Avoid air bubbles
• Thick enough so that
newspaper print can be read
through it
• 1st low power, then high power
• Detects trophozoites, cysts,
eggs, larvae
• Motile trophozoites –
Entamoeba, Giardia, B. coli
Larva of Strongyloides
sterocralis
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13. Iodine wet mount
• Five times diluted soln of Lugol’s iodine is used.
• Lugol’s iodine contains –
1. Potassium iodide 10 gm
2. Iodine crystals 5 gm
3. Distilled water 100 ml
• Potassium iodide renders elemental iodine soluble in water by
forming triiodide.
• Useful to see nuclear character of cysts, trophozoites
• Glycogen mass – stained brown, chromatin bars – not stained
• Trophozoite motility – not seen
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15. Cyst of E. histolytica – iodine mount
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16. Permanent stained smears
• For cytological details
• E. histolytica, Giardia etc.
• Prepared from fresh stools or PVA preserved stool
1. Iron hematoxylin
2. Trichrome stain
3. Modified acid fast stain – 5 -10% H2SO4 – For
oocysts of Cryptosporidium parvum, Isospora,
Cyclospora
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17. Iron hematoxylin stain
Iron hematoxylin stain
Trichrome stain (E. histolytica)
Modified Z-N
(Cryptosoporidium and
Isospora belli)
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18. Stool concentration methods
• To separate intestinal protozoa & helminthic eggs
from fecal debris
Flotation
techniques
Sedimentation
techniques
1. Saturated salt flotation,
Sp. Gravity – 1.2
2. Zinc sulfate centrifugal
flotation, Sp. Gravity –
1.8
3. Sheather’s sugar
centrifugal flotation
1. Simple sedimentation
2. Formalin ether
sedimentation
3. Acid ether sedimentation
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21. Eggs that float in saturated salt solution
• Fertilized egg of Ascaris
• Ankylostoma duodenale
• Trichuris trichiura
• Enterobius vermicularis
• Hymenolepis nana
F A T E H
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A B
C
D
E
F
D
G
H
C
G
A– Fertilized egg of Ascaris lumbricoides
B– Giardia lamblia
C- Taenia saginata/solium
D- Unfertilized egg of Ascaris lumbricoides
E—Egg of H. nana
F--- Larva of Strongyloides sterocrolis
G—Egg of Trichuris trichuria
H – Egg of Ancylostoma
Egg of
Enterobius
vermicularis

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Microscopic Character Amoebic Dysentery Bacillary Dysentery
RBCs In clumps Discrete or in Rouleaux
Pus cells Few Numerous
Macrophages Few Numerous, many have
RBCs and may mimic EH
Eosinophils Present Sacrce
Charcot-Leyden crystals Present Absent
Pyknotic bodies Present Absent
Ghost cells Absent Present
Parasites Trophozoites of EH Absent
Bacteria Many motile bacteria Few or absent
Difference b/w amoebic and bacillary dysentery

25. Quantification of worm burden
• Direct smear egg counting (2 mg) – wet
mount preparation
• Stoll’s method
– 4 gm feces + 56 ml of N/10 NaOH
– Mix to make uniform suspension
– 0.075 ml is removed with pipette to make wet
mount
– Count the eggs and multiply by 200
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26. How to detect eggs of
Enterobius vermicularis ?
1. Scotch cellulose
adhesive tape method
2. NIH swab
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27. • Examination of urine
– Trichomonas vaginalis – in centrifuged urine sediment
– Eggs of S. hematobium, Dioctophyma renale
– Microfilaria of W. bancrofti in c/o chyluria
• Examination of sputum
– Eggs of P. westermani, Capillaria aerophila
– Trophozoites of E. histolytica in c/o pleuro-pulmonary
amoebiasis
– Fragments of laminated membrane and free scolices of E.
granulosus in c/o pulmonary hydatid cyst
– A. lumbricoides, A. duodenale, N. americanus, S.
stercoralis – during migratory phase
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28. • Examination of aspirates
– Liver aspirates – amoebic liver abscess, hydatid cyst
– Proctoscopic aspirates and scrapings – amoebic ulcers
– Duodenal aspirates – G. lamblia, larvae of S. stercoralis
– Aspirates from L.N., spleen, liver, bone marrow, CSF –
African trypanosomiasis (T. brucei), leishmaniasis, Chagas
disease (T. cruzi), toxoplasmosis
• Examination of CSF
– N. fowleri, Acanthmoeba spp., Balamuthia spp.
– T. brucei gambiense and T. brucei rhodeiense –
sleeping sickness
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29. Biopsy examination
• Skin biopsy – Leishmania, Onchocerca volvulus,
Mansonella, encysted larva of Spirometra
• L.N. biopsy – Trypanosoma brucei, T. cruzi,
Toxoplasma, filarial nematodes
• Muscle biopsy – T. solium, larvae of Trichinella
spiralis, Ancylostoma, Toxocara, sarcocysts of
Sarcocystis lindemanni
• Gastric biopsy – Anisakis simplex
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30. • Colon and rectum biopsy – eggs of S. japonicum, S.
mansoni, trophozoites of E. histolytica and B. coli
• Brain biopsy – trophozoites of E. histolytica, N. fowleri,
Acanthamoeba spp., Balamuthia spp., cysticercus
cellulose of T. solium, hydatid cyst of E. granulosus
• Corneal scrapings – Acanthamoeba keratitis
(trophozoites)
• Liver biopsy – E. histolytica, L. donovani, E. granulosus
• Lung biopsy – E. histolytica, E. granulosus, P.
westermani, Capillaria aerophila
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31. Culture of stool for parasites
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33. Immunodiagnostic
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Molecular diagnosis
Disease/Parasite Test format
Amoebiasis Nested PCR , Real time PCR,
Biofire film assay
Giardiasis PCR, Biofire film assay
Cryptosporiodiosis PCR, Biofire film assay
Intestinal Taeniasis PCR
Hydatid cyst PCR, PCR-RFLP
Hook worm PCR and Real time PCR
assays
Strongyloides stercoralis Real time PCR
Ascaris lumbricoides Multiplex PCR

35. Intradermal skin test
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Skin test showing immediate hypersensitivity
Hydatid cyst (Casoni’s test)
Ascariasis
Strongyloidiasis
Trichinellosis (Bachman test)
Imaging techniques
Disease Imaging method
Amoebic liver abcess USG
Hydatid cyst X-ray, USG, CT scan and MRI
Neurocysticercosis CT scan and MRI

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