LABORATORY DIAGNOSIS OF PARASITIC INFECTIONS Lecturer. Mohamed El-Sakhawy

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

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

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

URINE EXAMINATION Membrane filtration technique air 10 ml urine Nucleopore filter + Saline Eggs of Schistosoma

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

URINE EXAMINATION

STOOL EXAMINATION

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

Staining the saline preparation with methylene blue

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.

STOOL EXAMINATION Scanty infection Concentration 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

STOOL EXAMINATION Saline sedimentation Mesh wire gauze Saline Emulsify Conical flask 10 g stool Sediment

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

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

STOOL EXAMINATION Permanent Stained smears Iron haematoxylin stain Trichrome stain Modified Ziehl Neelsen stain (Crptosporidum.)

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

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

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

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

NaOH Sputum Centfifuge

floor Edge

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

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

BLOOD EXAMINATION QBC technique RBC +parasite Acridine orange centrifuge RBC Microhaematocrit tube Malaria, Filaria, Trypanosomes

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

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

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

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

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

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

INDIRECT IMMUNOLOGICAL METHODS Double Electro Immuno Diffusion Line of ppt Electric current +ve Ab Ag -ve Buffered gel

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

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

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

10 X Objective

Relative sizes of helminth eggs as seen in microscope field using the 10 objective (with 10 eye-pieces). Eggs are as seen in a saline preparation. 1. E.vermicularis, 2. A.lumbricoides, 3. S.stercoralis larva (motile), 4. Hookworm, 5. T.trichiura, 6. D.latum, 7. O.sinensis, 8. Fasciola sp, 9. S.mansoni, 10. Paragonimus sp, 11. S.japonicum, 12. S.intercalatum, 13.Taenia sp, 14.V.nana, 15. H.dimunuta.

40 X Objective

Relative sizes of trophozoites and cysts of intestinal protozoa, common nematode eggs and larva of Strongyloides as seen in microscope field using the 40 objective (with 10 eyepieces). 1. I.belli oocyst, 2. A lumbricoides egg, 3. Leucocytes, 4. E.histolytica/E.dispar cyst, 5. E.histolytica trophozoite (motile), 6. Red cells, 7. S.stercoralis larva (motile), 8. E.coli cyst (mature), 9. G.lamblia cyst, 10. C.mesnili cyst, 11. Hookworm egg, 12. G.lamblia trophozoite (motile). Iodine preparation: 13. E.coli cyst, 14. I.buetschlii cyst, 15. E.histolytica/E.dispar cyst, 16. V.nana cyst, 17. T.trichiura egg, 18. Blastocystis hominis, 19. G.lamblia cyst.

Non-parasitic structures found in faeces: Care must be taken not to report as parasites those structures that can be normally found in faeces such as: muscle fibres, vegetable fibres, starch cells (stain blue-black with iodine), pollen grains, fatty acid crystals, soaps, spores, yeasts, and hairs . Large numbers of fat globules may be seen in faeces when there is malabsorption. Charcot Leyden crystals (breakdown products of eosinophils) can sometimes be seen in faeces (also in sputum) in parasitic infections. They appear as slender crystals with pointed ends, about 30–40m in length

Structures found in faeces that required differentiation from parasites.

Image illustrating Red Blood Cells in slide preparation. Image illustrating Fat Globules in slide preparation Image illustrating Yeast Cells in slide preparation Note similarity to parasitic oocysts. Image illustrating Vegetable cell in slide preparation.

Image illustrating Vegetable Spiral in slide preparation. Image illustrating a Vegetable Spiral in slide preparation. Such spirals may appear similar to proglottids. Image illustrating Vegetable cell in slide preparation.

Image illustrating geranium pollen cells in slide preparation. Image illustrating pollen in slide preparation that could be mistaken for a Taenia egg. The shell is thinner, of non-uniform thickness, and no hooks are visible. Image illustrating pollen resembling a Hymenolepis nana egg. Hooks and polar filaments are not visible. Image illustrating geranium pollen cells in slide preparation. Image illustrating pollen in slide preparation using a color filter

Image illustrating vegetable hairs in slide preparation. Image illustrating peach hair in slide preparation. Note the similarity to Strongyloides stercoralis. Image illustrating vegetable hairs in slide preparation.