LABORATORY DIAGNOSIS OF PARASITIC INFECTIONS

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

DIAGNOSIS DIRECT Urine Stool Sputum Biopsy Blood Aspirates INDIRECT IHAT LAT IFAT ELISA CFT DEIDT MOLECULAR PCR DNA probes

URINE EXAMINATION MACROSCOPIC colour white Chyluria Filaria smoky Blood S. haematobium MICROSCOPIC Sedimentation concentration Membrane filtration Acetic acid RBC haemolysis Clear ova Ether Dissolve fat M.f

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

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

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

URINE EXAMINATION Egg viability Live eggs Well defined miracidium Flickering F cells Hatching moving miracidium Dead eggs Dark colour Granulated

STOOL EXAMINATION MACROSCOPIC Consistency Colour Composition MICROSCOPIC Temprory Diect saline smearIodine smearConcentration techniques Sedimentation SalineFormol ether Floatation Sat salineZinc sulphateSheather ’ s sugar Permanent OTHERS Culture Cellophane tape Baeremann tech. Ova quantitaion (Stoll & Kato)

MACROSCOPIC EXAMINATION COLOUR Pale=Steatorrhea (G.l) CONSISTENCY Liquid (Troph) Formed (Cyst) Semi formed (Cyst) COMPOSITION ?? Blood ?? Mucus (dysentry) Adult PARASITES *Ascaris worm *E. vermicularis *T. saginata STOOL EXAMINATION

MACROSCOPIC Consistency Colour Composition MICROSCOPIC Temprory Diect saline smearIodine smearConcentration techniques Sedimentation SalineFormol ether Floatation Sat salineZinc sulphateSheather ’ s sugar Permanent OTHERS Culture Cellophane tape Baeremann tech. Ova quantitaion (Stoll & Kato)

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

STOOL EXAMINATION Scanty infection Concentration techniques SedimentationFloatation Heavy eggs (Ascaris egg) Heavy eggs (Ascaris egg) Operculated eggs (Trematodes) Operculated eggs (Trematodes) Larvae (Strong sterc.) Larvae (Strong sterc.) Cysts Cysts Non Operculated eggs Trematodes ( S. m.) Cestode Hookworms,Trichostong) Nematode( Hookworms,Trichostong) Cysts

STOOL EXAMINATION MACROSCOPIC Consistency Colour Composition MICROSCOPIC Temprory Diect saline smearIodine smearConcentration techniques Sedimentation SalineFormol ether Floatation Sat salineZinc sulphateSheather ’ s sugar Permanent OTHERS Culture Cellophane tape Baeremann tech. Ova quantitaion (Stoll & Kato)

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

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

STOOL EXAMINATION MACROSCOPIC Consistency Colour Composition MICROSCOPIC Temprory Diect saline smearIodine smearConcentration techniques Sedimentation SalineFormol ether Floatation Sat salineZinc sulphateSheather ’ s sugar Permanent OTHERS Culture Cellophane tape Baeremann tech. Ova quantitaion (Stoll & Kato)

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

STOOL EXAMINATION MACROSCOPIC Consistency Colour Composition MICROSCOPIC Temprory Diect saline smearIodine smearConcentration techniques Sedimentation SalineFormol ether Floatation Sat salineZinc sulphateSheather ’ s sugar Permanent OTHERS Culture Cellophane tape Baeremann tech. Ova quantitaion (Stoll & Kato)

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

STOOL EXAMINATION MACROSCOPIC Consistency Colour Composition MICROSCOPIC Temprory Diect saline smearIodine smearConcentration techniques Sedimentation SalineFormol ether Floatation Sat salineZinc sulphateSheather ’ s sugar Permanent OTHERS Cellophane tape Culture Baeremann tech. Ova quantitaion (Stoll & Kato)

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

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

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

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

DIAGNOSIS DIRECT Urine Stool Sputum Biopsy Aspirates Blood INDIRECT IHAT LAT IFAT ELISA CFT DEIDT MOLECULAR PCR DNA probes

SPUTUM EXAMINATION MACROSCOPIC Appearance Bloody (Parag) Rusty brown (Parag) MICROSCOPIC ConcentrationNaOH Sputum Centfifuge

Parasites/sputum P living in lung P. westermani eggs P. westermani eggs P migrating in lung St. stercoralis St. stercoralis Ascaris Ascaris Hookworm (filariform L) Hookworm (filariform L) P resulting from rupture of Hydatid cyst (sand) Hydatid cyst (sand) Amoebic abcess (troph) Amoebic abcess (troph)

BIOPSY SPECIMEN SKIN SNIP O. Volvulus mf Raise skin by needle Raise skin by needle Slice by scissors Slice by scissors Put snip in normal saline Put snip in normal saline Examine Examine MUSCLE BIOPSY T. Spiralis larvae Muscle digestion with HCl + pepsin RECTAL BIOPSY Schistosoma egg

Lumbar puncutreLumbar puncutre Centrifuge Centrifuge Examine sed.Examine sed. floor Edge

DIAGNOSIS DIRECT Urine Stool Sputum Biopsy Aspirates Blood INDIRECT IHAT LAT IFAT ELISA CFT DEIDT MOLECULAR PCR DNA probes

BLOOD EXAMINATION Blood films ThinThick Buffy coat films QBC technique Knott ’ s conc. tech.

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

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

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

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

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

INDIRECT IMMUNOLOGICAL METHODS Antigen detection More specific More specific More accurate. More accurate. Active infection Active infection Early Early Quantitative Quantitative Antibody detection Ab remain in serum for months even after cure

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

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

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

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

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

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

DIAGNOSIS DIRECT Urine Stool Sputum Biopsy Blood Aspirates INDIRECT IHAT LAT IFAT ELISA CFT DEIDT MOLECULAR PCR DNA probes

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

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