LABORATORY DIAGNOSIS OF PARASITIC INFECTIONS

1. LABORATORY DIAGNOSIS OF PARASITIC INFECTIONS Lecturer. Mohamed El-Sakhawy

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

4. Ether Dissolve fat M.f Acetic acid RBC haemolysis Clear ova

5. SEDIMENTATION CONCENTRATION URINE EXAMINATION Clean conical glass receptacle 15-20 min Centrifuge (2 min)

6. URINE EXAMINATIONMembrane filtration technique air 10 ml urine Nucleopore filter + Saline Eggs of Schistosoma

7. URINE EXAMINATION HELMINTHES PROTOZOA ARTHROPODES • S. haem.egg • E. vermic. egg • S. mansoni egg • Micrfilaria (Ov, Wb) • H sand Tricomonas. Vaginalis troph • Pthirus pubis • L. higher deptera

8. URINE EXAMINATION

10. STOOL EXAMINATION

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

13. Staining the saline preparation with methylene blue

14. Lugol iodine–acetic acid solution causes the trophozoite forms to become nonmotile. Using a fine Pasteur pipette, allow a drop of methylene blue solution to run under the coverslip over the saline preparation (Fig. 7). This will stain the nuclei of any cells present and distinguish the lobed nuclei of polymorphs from the large single nuclei of mucosal cells. If a drop of eosin solution is added, the whole field becomes stained except for the protozoa (particularly amoebae), which remain colourless and are thus easily recognized.

15. STOOL EXAMINATIONScanty infectionConcentration techniques Sedimentation Floatation • Non Operculated eggs • Trematodes ( S. m.) • Cestode • Nematode(Hookworms,Trichostong) • Cysts • Heavy eggs (Ascaris egg) • Operculated eggs (Trematodes) • Larvae (Strong sterc.) • Cysts

17. STOOL EXAMINATIONSaline sedimentation Mesh wire gauze Saline Emulsify Conical flask 10 g stool Sediment

18. STOOL EXAMINATION Formol Ether Sed. Conc. Ether Ether debris 10% Formalin formalin 1 g stool Sediment Thorough mixing Conical flask centrif. tube • Ether adsorbs fecal debris & floats. • Formalin fixes & preserves the specimen.

20. STOOL EXAMINATION Clean light eggs & cysts Tin container Seive 20 min Centrif. 2 min

22. STOOL EXAMINATIONPermanent Stained smears • Iron haematoxylin stain • Trichrome stain • Modified ZiehlNeelsen stain (Crptosporidum.)

24. STOOL EXAMINATION Kato technique Mesh screen Hole Template Remove the template Cellophane soaked by glycerin (clears faeces( Egg count/ g stool Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni

25. STOOL EXAMINATION Stoll’s technique Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni 24 hr stool 60 CC 4 g Stool 56 CC NaOH Shake well 0.15 CC Egg count/ slide Eggs/1g= Eggs/slideX100 Erlynmeyer flask Egg/day=Eggs/1g X stool wt/g in 24 hrs

26. STOOL EXAMINATION Baermann’s technique Stool/soil seive 25-50CC Warmwater Glass funnel 30 min • centrifuge clamp Detec. Of Nematode L. /stool, soil

27. Filter paper culture STOOL EXAMINATION Cultures for Nematode larvae Filter paper Slide Sealed petri dish Water • Scanty infection • Larvae of: • St. stercoralis (A,L) • Hookworms • Trichostrong

29. NaOH Sputum Centfifuge

32. floor Edge

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

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

37. BLOOD EXAMINATIONQBC technique RBC +parasite Acridine orange centrifuge RBC Microhaematocrit tube Malaria, Filaria, Trypanosomes

38. BLOOD EXAMINATIONKNOTT’S CONC. TECHNIQUE • Citrated bld 1 ml 10 ml centrifuge Geimsa Air dry fix 2 min sediment Formalin 2 % Filaria

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

41. 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

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

43. INDIRECT IMMUNOLOGICAL METHODSELISA OPD Peroxidase E OPD Anti human AB Patient’s serum (?? AB) AB Ag Flat bottom plastic micrititre plate

44. INDIRECT IMMUNOLOGICAL METHODSCFT Sheep’s RBC Anti sheep AB +ve Ab No Sheep RBChaemolysis AB complement Patient’s serum (?? AB) -ve Ab haemolysis Ag Tube / microplate

45. INDIRECT IMMUNOLOGICAL METHODSDouble Electro Immuno Diffusion Line of ppt Electric current +ve Ab Ag -ve Buffered gel

46. INDIRECT IMMUNOLOGICAL METHODSImmunodiagnostic Strip Test (Dip Stick Test) Ag +ve -ve Pt bld (?Ag) Coloured dye Monoclonal Ab Nitrocellulose strip Malaria, Filaria, African tryp.

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

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

50. 10 X Objective