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Laboratory diagnosis of parasitology-1.pptx
- 1. Laboratory diagnosis of Parasitology Dr.BALAJI.S 2nd yearPG Department of Microbiology SVMC, Tirupati.
- 2. Direct methods Immunologicalmethods Molecular methods Stool ICT PCR Urine IHAT DNA probes Blood LAT Sputum IFAT CSF ELISA Tissue biopsy and CFT Aspirates Diagnostic methods
- 3. Parasites and theirrelated disease Entamoeba histolytica: Amoebic dysentery Giardia lamblia: Diarrhoea Leishmania donovani: Kala-azar Plasmodium: Malaria Trypanosoma cruzi: Chagas disease Toxoplasma gondii: Encephalomyelitis Taenia solium: Cysticercosis Echinococcus granulosus: Hydatid disease Ascaris lumbricoides: Diarrhoea Ancyclostoma: Diarrhoea Strongyloides: Diarrhoea Trichuris trichiura: Diarrhoea Enterobius vermicularis: Diarrhoea Wuchereria bancrofti: Filaria Trichomonas vaginalis: Vaginal infection
- 4. Examination of Stool Collectionof Fresh Stool Specimen All stool specimens should be collected in a suitable, clean, wide mouthed container like a plastic container At least three stool specimens collected on alternate days The specimen should not be contaminated with water, urine, or disinfectants.
- 5. Liquid stools shouldbe examined or preserved within 30 minutes of passage(trophozoites) Soft stools should be examined or preserved within 1 hour of passage (trophozoites and cysts) Formed stool should be examined or preserved within 24 hours of passage(cysts)
- 6. Preservations of specimens 5or 10% formalin – preserves protozoal cysts, helminthic eggs and larvae Merthiolate Iodine Formalin-used both as stain and preservative Sodium acetate- Acetic acid formalin – fixative which gives better result when stained with iron haematoxylin Polyvinyl alcohol – preservative for protozoal cysts and trophozoites in stool.
- 7. Macroscopic examination ofstool consistency Liquid stool – trophozoite (motile) Semisolid stool – cysts and trophozoites Formed stool – cyst forms Helminthic eggs and larvae are found in any type of stool. Presence of parasite Adult worm – ascaris lumbricoides, Ancylostoma duodenale, Trichuris trichiura, Enterobius vermicularis, intestinal flukes. Tapeworm segments. Blood and mucus Suggestive of amoebic dysentery
- 8. Microscopic examination The salinewet mount is the standard preparation and is made by emulsifying a small quantity of stool in a drop of (0.85%) saline placed on a slide and applying a coverslip (22 mm × 22 mm) on top, avoiding air bubbles. The entire field under coverslip should be systematically examined with low power objective (10X) under low light intensity. Any suspicious object may then be examined with the high power objective.
- 9. Wet mount Use Advantage Disadvantage Salinewet mount Preliminary identification of cysts Demonstrate helminthic eggs, motile trophozoites, larvae Can demonstrate viable, motile trophozoite Chromatoid body seen in cysts Saline stained cysts does not show nuclei, cytoplasm and glycogen material Lugol’s Iodine Further identification of protozoal cysts Iodine stained cysts show brown dot nuclei, yellowish cytoplasm and brown glycogen material Trophozoite killed with iodine so motile trophozoite not seen
- 10. Parasites and theirdevelopmental stages found in stool Cysts/Trophozoites Eggs Larvae Adult worm Entamoeba histolytica Giardia lamblia Balantidium coli Sarcocystis spp. Cyclospora cayetanensis Cryptosporidium parvum Cestodes Taenia spp. Hymenolepsis nana Hymenolepsis diminuta Diphyllobothrium latum Nematodes Trichuris trichiura Enterobius vermicularis Ascaris lumbricoides Ancylostoma duodenale Necator americanus Strongyloides stercoralis Taenia solium Taenia saginata Diphyllobothrium latum Ascaris lumbricoides Enterobius vermicularis Trichinella spiralis Trematodes Schistosoma spp. Fasciolopsis buski Fasciola hepatica Clonorchis sinensis Opisthorchis spp.
- 12. Bile stained eggs Ascarislumbricoides(fertilized egg) Trichuris trichiura Taenia species Non bile stained eggs Enterobius vermicularis Hymenolepsis nana Hymenolepsis diminuta Diphyllobothrium latum
- 13. Permanent Stained Smears Thetwo methods - Wheatley’s trichrome stain and the iron-hematoxylin stain. Permanent stained smears are examined normally under oil immersion (100X) objective. Confirmation of the intestinal protozoan, both trophozoites and cysts, is the primary purpose of this technique. Helminthic eggs and larvae take up too much stain and usually cannot be identified. Permanent smear can be prepared with both fresh and polyvinyl alcohol preserved stool specimen.
- 14. Trichrome stain (Wheatley’smethod) The trichrome technique of Wheatley for stool specimens is a modification of Gomori’s original staining procedure for tissue. It is a quicker and simpler method, which produces uniformly well-stained smears of the intestinal protozoa, human cells, yeast cells, and artefact material in about 45 minutes or less. Helminth eggs and larvae can not be stained. Microsporidia spores can not be seen using the Wheatley Trichrome Stained smear.
- 15. Trichrome stain IronHematoxylin stain Principle Uses chromotrope dyes to give contrast between cytoplasm, nuclei and chromatoid bodies Uses as a mordent with hematoxylin to give sharp nuclear detail Staining time Shorter, easier,quicker Longer, more labor - intensive Nucleus Moderate contrast Excellent contrast Cytoplasm Good differentiation of cytoplasm and chromatoid bodies Cytoplasm appears darker less contrast than nuclei Appearance Cytoplasm : blue/green Nuclei : red/purple chromatoid bodies: red Cytoplasm : Grey to black Nuclei : Dark black /blue Use Common for routine diagnosis of intestinal protozoa Used when precise nuclear morphology is required(research)
- 16. Hot method (ModifiedZN stain) Cold method (Kinyon’s stain) Organisms detected Cryptosporidium, Isospora, Cyclospora Cryptosporidium, Isospora, Cyclospora Heating Recquired (carbol fuchsin applied with heat) Not recquired Decolorizer 5% sulphuric acid(mild) 1% sulphuric acid Appearance of oocysts Pink – red against blue background Pink – red against blue background Advantage Better penetration and contrast Simple, safe and avoids heating
- 17. Concentration Methods When theparasites are scanty in stools, routine microscopic examination may fail to detect them. It is then necessary to selectively concentrate the protozoan cysts and helminth eggs and larvae. Concentration may be done using fresh or preserved feces.
- 18. Technique Principle TypesAdvantage Disadvantage Floatation method The suspending fluids used are denser(has high specific gravity) than the parasitic forms, this helps in separation of helminthic eggs and protozoal cysts from faecal debris, parasite rise to surface. Saturated salt solution method Zinc sulphate floatation method Saturated magnesium sulphate solution method Sugar floatation method Both surface film and sediments can be examined for parasite Some helminthic eggs do not float in salt solution High specific gravity of the fluid may cause distortion in the morphology of eggs and cysts. Helminthic eggs which does not float in salt solution: operculated eggs of trematodes, eggs of Taenia species and unfertilised eggs of Ascaris lumbricoides, larvae of Strongyloides stercoralis
- 19. Technique Principle TypesAdvantage Disadvantage Sedimentation method Suspending fluids used are lighter than the parasitic forms. So the parasite sink to the bottom so it can be recovered from the sediment. Sedimentation by gravity or by centrifugation. Formalin ether sedimentation method Simple gravity sedimentation method Formalin –Acetone sedimentation method Easy to perform More sensitive Morphology of eggs and cysts are preserved Formalin fixes the eggs, larvae and cysts so that they become non- infectious Sediment preparation contains more faecal debris Ether is extremely flammable and highly volatile
- 20. Quantification of wormburden There are two methods Direct smear egg count Stoll’s method Direct smear method Two mg of faeces is mixed in a small drop of saline on a slide and cover the cover slip. Examined under low power of the microscope and count the eggs and calculate the number of eggs/gm of faeces.
- 21. Stoll’s technique forcounting helminth egg 4 gm of stool and 56 mL of N/10 sodium hydroxide 0.075 mL on slid Multiply result in 200 Number in 1 gm
- 22. NIH swab Eggs aredeposited in peri anal and perineal skin. The cellophane part is used for swabbing by rolling over the perianal area. Spread over glass slide and examined microscopically. This procedure should be repeated on three successive days.
- 23. Scotch cellulose adhesivetape method The adhesive surface of the tape is then pressed against the perineal skin at several places. Then placed adhesive – side – down on the slide for examination. A drop of toluene or xylol may be placed between the tape and the slide to clear the preparation.
- 24. Urine Examination Large volumeof urine samples should be allowed to settle for 1–2 hours. About 50 mL of the bottom sediment of the sample is taken for centrifugation. The highly concentrated sediment after centrifugation is examined for direct wet mount microscopy. May show eggs of Schistosoma and Trichomonas vaginalis. Microfilaria may be detected from chylous urine in lymphatic filariasis.
- 25. Examination of Blood Bloodexamination is the routine diagnostic method in malaria, filariasis, African trypanosomiasis, and babesiosis. Two methods Wet preparation Stained blood smear Parasites found in peripheral blood film Protozoa Nematodes Plasmodium spp Wuchereria bancrofti Babesia spp Brugia malayi Trypanosoma spp Loa loa Leishmania spp Mansonella spp
- 26. Wet preparation A dropof anticoagulated blood is placed on a clean glass and cover slip is put over it. This preparation is examined microscopically for parasite such as Trypanosomes and Microfilariae.
- 27. Stained blood smears Threetypes of blood smear Thin blood smear Thick blood smear Thin and thick blood smear on same slide
- 28. Features Thin smearThick smear Preparation Small drop of blood spread into a thin film, fixed with methanol Large drop of blood spread thick, not fixed (RBC lysed during staining) Purpose Species identification Screening of presence of malaria parasite (more sensitive) Parasite density detection Detects parasite when parasitemia is moderate to high Detects parasites even at low density RBC morphology Preserved – useful for differentiating plasmodium species Destroyed (RBCs hemolysed) so species identification difficult. Advantage Provides detailed morphology of parasite and RBC Increased sensitivity by examining larger blood volume Disadvantage Less sensitive at low parasitemia Cannot accurately identify species alone
- 29. Staining of bloodsmears Thick or thin smears are stained with Romanowsky’s stain. These stain include Giemsa stain Leishman’s stain Field’s stain Jaswant singh and Bhattacharjee stain Theses stains are combination of methylene blue and eosin.
- 30. Quantitative buffy coat(QBC): This involves collection of the blood in a capillary tube coated internally with acridine orange stain, centrifugation and then examination of the buffy coat region under fluorescence microscopy. This is extremely useful for the detection of the malaria parasites and microfilariae
- 31. Quantitative buffy coat(QBC):
- 32. Concentration of blood: Thisis useful for the detection of microfilariae from the blood specimen. Various concentration methods are: Sedimentation technique Gradient centrifugation Membrane filtration Knott concentration
- 33. Types of buffycoat concentration methods Method procedure Gradient centrifugation Venous blood is collected in heparinised tube 4ml of Ficoll – hypaque solution is taken in a 15ml centrifuge tube and mixed with equal volume of blood Centrifuged Middle Ficoll – hypaque layer is examined for the Microfilariae. Membrane centrifugation 5ml heparinised blood is collected in syringe. Blood is filtered through the membranes. Microfilaria sticking to the surface is examined. Knott centrifugation 10ml of 2% formalin is collected in a 15ml centrifuge tube. 1ml of venous blood is collected aseptically and is add to the tube and mixed thoroughly Blood and formalin suspension is centrifuged at 200g for 2 mins Sediment is collected and then examined for microfilariae
- 34. Diethylcarbamazine Provocation Test Oraladministration of diethylcarbamazine (DEC; 100 mg or 2 mg/kg of body weight) brings about mobilization of microfilariae into peripheral blood. After 30 mins, the capillary blood is collected by finger prick for demonstration of microfilariae by direct wet mount or staining. This is a great advantage for surveys. But the drug may cause febrile reactions, particularly in brugiasis.
- 35. Sputum Examination Sputum isexamined for the detection of egg of paragonimus westermani. Parasites found in sputum P.Westermani eggs Rarely migrating larvae of ascaris E.Histolytica (trophozoites in case of pulmonary abscess) Rarely migrating larvae of strongyloides Pneumocystis jirovecii Rarely migrating larvae of Ancylostoma duodenale Rarely migrating larvae of necator americanus
- 36. CSF examination Direct microscopicexamination of unstained and stained smears of CSF is useful for detection of Naegleria fowleri Acanthamoeba species Balamuthia mandrillaris Trypanosoma brucei
- 37. Examination of aspirates Theaspirate from liver is useful in diagnosis of amoebic liver abscess and hydatid cyst. Duodenal aspirates may reveal trophozoites of Giardia lamblia.
- 38. Duodenal Capsule Technique(Entero test) Entero test is a simple method of sampling duodenal contents. The device is composed of a length of nylon yarn coiled inside a gelatine capsule. The end of the yarn is affixed to the patient’s face. The capsule is then swallowed and the gelatin dissolves in the stomach. The weighted string is carried into the duodenum by peristalsis. Bile-stained mucus is then retrieved after 3–4 hours and duodenal contents adherent to the yarn is scrapped off and examined under microscope. Usually 4–5 drops of material is obtained. Entero test is used for detecting trophozoites of Giardia, larvae of Strongyloides, eggs of liver flukes, and oocysts of Isospora
- 39. Examination of biopsy Musclebiopsy Cysticerci of Taenia solium Larvae of Trichinella spiralis Sarcocysts of sarcocystis lindemani Liver biopsy Entamoeba histolytica Leishmania donovani Echinococcus granulosus Brain biopsy Entamoeba histolytica Naegleria fowleri
- 40. Culture Methods Many parasitescan now be grown in culture, but this has not become a routine diagnostic method in parasitic infections. It is sometimes employed for accurate identification of the parasite species. parasites Culture medium Entamoeba histolytica Boeck and Drbhohlav diphasic medium Balantidium coli balamuth’s medium Giardia lamblia Axenic culture Trichomonas vaginalis Trypticase serum media Acanthamoeba spp. Naegleria fowleri Agar plates Leishmania spp. Trypanosoma spp. NNN(Novy – MacNeal – Nicolle) medium Plasmodium spp. RPMI medium(Roswell Park Memorial Institute Medium)
- 41. Animal inoculation Animal inoculationis not a routine diagnostic procedure in parasitic infection. It is useful in some parasites such as Toxoplasma gondii(intraperitoneal inoculation of mice) Leishmania donovani(intraperitoneal inoculation of hamsters) Trypanosoma species(intraperitoneal inoculation or tail vein inoculation in mice)
- 42. Xenodiagnosis This method involvesthe diagnostic infection of a vector, in which the parasite multiplies and can be demonstrated. In Trypanosoma cruzi diagnosis may be established by letting the vector reduviid bug feed on suspected patients. In 4–5 weeks, live flagellate forms can be seen in the feces of the bugs.
- 43. Immunological Diagnosis Amoebiasis Invasive amoebiasis,particularly in liver abscess, serology is very useful. IHA is most widely employed. Titers of 1:256 or more are significant in cases of amoebic liver abscess and have prognostic value. E. histolytica test was able to detect galactose lectin (galnac) antigen in almost all patients of amoebic liver abscess Antigen detection in parasitic disease Galactose lectin antigen Entamoeba histolytica Giardia- specific antigen 65 Giardia lamblia WKK and rk39 antigen Leishmania donovani HRP- 2 antigen Plasmodium falciparum Vivax specific LDH Plasmodium vivax 200 kDa Ag and OG43A antigen Wuchereria bancrofti
- 44. Giardiasis ELISA and indirectimmunofluorescence test (IIF) have been developed for detection of Giardia. Commercially available ELISA (ProSpec T/Giardia) Kit detects Giardia- specific antigen 65 (GAS 65). The sensitivity of the test is 95% and specificity is 100%, when compared to conventional microscopy.
- 45. Trypanosomiasis Serological tests usedto detect trypanosomiasis are IHA, indirect fluorescent antibody (IFA), and ELISA. Specific antibodies can be demonstrated by IFA and ELISA in CSF sample. Specific antibodies can be identified in the serum by these tests about 2–3 weeks after infection.
- 46. Leishmaniasis IHA, CounterImmuno Electrophoresis (CIEP), and DOT-ELISA are usually positive in Kala-azar. Complement test using Witebsky Klingenstien, and Kuhn (WKK) antigen from the acid-fast Kedrowsky bacillus are relatively less sensitive. IFA test is positive very early in the disease, even before the appearance of symptoms and becomes negative within 6 months of cure. rK 39 micro ELISA test is a qualitative immuno chromatographic assay for detection of antibodies to Leishmania.
- 47. Malaria IIF, ELISA, andIHA are sensitive and specific, but are not useful for diagnosis of acute malaria because antibodies persist for some years after cure. A negative test may, however help to exclude malaria. Molecular assays such as antigen capture for detection of HRP-2 and pLDH have been applied for developing rapid dip-stick tests.
- 48. Toxoplasmosis Serological tests offerthe most useful diagnostic method in toxoplasmosis. The original Sabin-Feldman dye test, though very specific and sensitive, is no longer in use. IIF, IHA, and CFT were other useful tests. The dye test remains positive for life, while CFT becomes negative soon after active infection. At present, ELISA is routinely used in Toxoplasma serology. It is very informative, as it provides titers of IgM and IgG antibodies separately for better interpretation of the results.
- 49. Cryptosporidiosis • IFA andELISA using purified oocysts as antigens have been used to detect circulating antibodies specific to Cryptosporidium parvum . • but extensive cross-reactions limit their use in diagnosis.
- 50. Trichinosis Bentonite flocculation slidetests and CFT become positive 3–4 weeks after infection. IIF becomes positive even earlier. ELISA is also available. Demonstration of seroconversion is diagnostic.
- 51. Filariasis IHA and bentoniteflocculation tests with antigen from Dirofilaria immitis gives positive reaction in patients, and high titers in tropical pulmonary eosinophilia. But cross reactions are frequent. Immunochromatographic filariasis card test (ICT) is a new and rapid filarial antigen test that detects soluble Wuchereria bancrofti antigens in the serum of infected humans.
- 52. Echinococcosis Several serologicaltests have been developed using hydatid fluid or scolex antigens from hydatid cysts in sheep. IHA, IIF, CIEP, and ELISA are very sensitive. Cross-reactions occur with cysticercosis.
- 53. Skin test Skin testDisease Casoni’s test Hydatid disease Montenegro test or leishmanin test Kala azar Frenkel’s test Toxoplasmosis Fairley’s test Schistosomiasis Bachman intradermal test Trichinellosis
- 54. Molecular Methods DNA probeis a highly sensitive method for the diagnosis of malaria. It can detect even less than 10 parasite/µL of blood. Drug resistances in malaria are detected now by PCR techniques. B1 gene of Toxoplasma gondii can be detected by PCR of the amniotic fluid in case of congenital toxoplasmosis. PCR have been developed for detection of filarial DNA from patients blood. Specific 17 kDa and 27 KDa sporozoite antigens are employed for epidemiological studies in cryptosporidiosis using western blot technique.
- 55. Imaging technique X –ray – May show calcified cysts (hydatid cyst). USG – detects cystic lesions (hydatid cyst in liver). CT scan / MRI –May show Granulomatous Amoebic Encephalitis).
- 56. Recent updates Kerala hasreported 80 cases and 21 deaths in the Primary Amoebic Meningoencephalitis (PAM) A rare but highly fatal brain infection caused by Naegleria fowleri.
- 57.