Laboratory Diagnosis of parasitic infections

1. Laboratory Diagnosis of parasitic infections Professor Sudheer Kher

2. Learning Objectives Describe the principles, techniques, standards and recording of results and interpretation of different methods used in diagnosis of parasitic diseases

3. Case diagnosis • History (Age, occupation, residency, previous infection) • Complaint • Clinical examination • Invesigations - Laboratory investigations - Radiology - Surgical intervention (Exploratory) Provisional diagnosis Confirm the diagnosis

5. Laboratory Diagnosis of Parasitic Infections Purpose – Confirmation of clinical suspicion Identification of unsuspected infection Methods same as used in Bacteriology & Virology but significance of different methods varies. Isolation least important, morphological identification very important. Serology relatively less important

6. Morphological identification Examination of faeces – Gross Microscopy Saline mount Iodine Mount Thick smears – not commonly used Permanent stained smears Iron hematoxylene Whearley’s trichrome stain Concentration methods Floatation techniques Sedimentation techniques

7. Morphological identification Examination of Blood Thin Smear Thick smear Wet mount for microfilaria Stains used – Leishman / Giemsa

8. Cultivation of parasites Culture methods – Amoeba Leishmania & Trypanosoma Malarial parasite Animal inoculation – Not practical Xenodiagnosis – Vectors infected experimentally Immunological diagnosis

9. Serology – All tests available IHA ELISA CIEP IF CFT More useful in Amoebiasis Leishmaniasis Malaria Toxoplasmosis Trichinosis Filariasis Echinococcosis Skin Tests – Specificity low, cross reactions common Casoni’s test Leishmanin test Immunological diagnosis

10. Thin Thick BLOOD EXAMINATIONBLOOD FILMS Bld drop Circular motion spread Air dry Air dry methyl alcohol Geimsa Geimsa Malaria, Babesia, Filaria, Tryp.

11. BLOOD EXAMINATIONBuffy coat film plasma WBC (BC) centrifuge Air dry Fix 30 min RBC spread Geimsa Citrated bld Trypanosoma., L. donovani

13. STOOL EXAMINATION

14. Saline smear Iodine smear STOOL EXAMINATIONTemporary saline Iodine 1% • Huge number of: • Eggs • Protozoal troph. Motility • (Amoeba, flagellates) • Huge number of: • Cyst morphological details

15. STOOL EXAMINATIONScanty infectionConcentration techniques Sedimentation Floatation • Non Operculated eggs • Trematodes ( S. mansoni) • Cestode Tape worms • Nematode(Hookworm, Round worms) • Cysts • Heavy eggs (Ascaris egg) • Operculated eggs (Trematodes) • Larvae (Strongyloides stercoralis) • Cysts

16. Ether Dissolve fat Microfilaria Acetic acid RBC haemolysis Clear ova

17. NaOH Sputum Centfifuge

20. Edge

21. INDIRECT IMMUNOLOGICAL METHODS • Scanty infection. • Tissue parasite no portal of exit (Hydatid dis.) • Migratory stage (Fasciola) • Chronic infection fibrosis (Bilharziasis)

23. IHAT LAT INDIRECT IMMUNOLOGICAL METHODS Ag Ag + + Patient’s serum (?? AB) Latex particle Patient’s serum (?? AB) Sensitized Sheep’s RBC (O–ve) Agglutination Agglutination

24. INDIRECT IMMUNOLOGICAL METHODSINDIRECT FLUORESCENT ANTIBODY TEST fluorescein Anti human AB Patient’s serum (?? AB) parasite

25. MOLECULAR BIOLOGICAL TECHNIQUESDNA Probes Radio active material Commercially prepared DNA sequence DNA Probe Hybridization +ve parasite Nitrocellulose paper Sample (Serum/ stool) ?? parasite Radioactivity

26. MOLECULAR BIOLOGICAL TECHNIQUES Polymerase Chain Reaction (PCR) Single stranded DNA Replication Detection T cruzi, T gondii