Diagnosis

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Diagnosis

  1. 1. LABORATORY DIAGNOSIS OF PARASITIC INFECTIONS By: Dr.Maha jaafar
  2. 2. Case diagnosis <ul><li>History (Age, occupation, residency, previous infection) </li></ul><ul><li>Complaint </li></ul><ul><li>Clinical examination </li></ul><ul><li>Invesigations </li></ul><ul><li>- Laboratory investigations </li></ul><ul><li>- Radiology </li></ul><ul><li>- Surgical intervention (Exploratory) </li></ul>Provisional diagnosis Confirm the diagnosis
  3. 3. DIAGNOSIS DIRECT INDIRECT MOLECULAR Urine Stool Sputum Biopsy Blood Aspirates PCR DNA probes IHAT LAT IFAT ELISA CFT DEIDT
  4. 4. Acetic acid RBC haemolysis Clear ova Ether Dissolve fat M.f URINE EXAMINATION MACROSCOPIC MICROSCOPIC colour white smoky Sedimentation concentration Membrane filtration Chyluria Blood Filaria S. haematobium
  5. 5. URINE EXAMINATION <ul><li>SEDIMENTATION </li></ul><ul><li>CONCENTRATION </li></ul>15-20 min Centrifuge (2 min) Clean conical glass receptacle
  6. 6. URINE EXAMINATION Membrane filtration technique air 10 ml urine Nucleopore filter Eggs of Schistosoma + Saline
  7. 7. URINE EXAMINATION HELMINTHES PROTOZOA ARTHROPODES <ul><li>S. haem.egg </li></ul><ul><li>E. vermic. egg </li></ul><ul><li>S. mansoni egg </li></ul><ul><li>Mf (Ov, Wb) </li></ul><ul><li>H sand </li></ul><ul><li>T. Vag troph </li></ul><ul><li>Pthirus pubis </li></ul><ul><li>L. higher deptera </li></ul>
  8. 8. URINE EXAMINATION Egg viability Live eggs Dead eggs <ul><li>Well defined miracidium </li></ul><ul><li>Flickering F cells </li></ul><ul><li>Hatching moving miracidium </li></ul><ul><li>Dark colour </li></ul><ul><li>Granulated </li></ul>
  9. 9. STOOL EXAMINATION MACROSCOPIC MICROSCOPIC OTHERS <ul><li>Consistency </li></ul><ul><li>Colour </li></ul><ul><li>Composition </li></ul><ul><li>Culture </li></ul><ul><li>Cellophane tape </li></ul><ul><li>Baeremann tech. </li></ul><ul><li>Ova quantitaion (Stoll & Kato) </li></ul>Temprory Permanent Diect saline smear Iodine smear Concentration techniques Sedimentation Floatation Saline Formol ether Sat saline Zinc sulphate Sheather’s sugar
  10. 10. STOOL EXAMINATION MACROSCOPIC EXAMINATION COLOUR CONSISTENCY COMPOSITION Adult PARASITES Pale=Steatorrhea (G.l) <ul><li>Liquid (Troph) </li></ul><ul><li>Formed (Cyst) </li></ul><ul><li>Semi formed (Cyst) </li></ul>?? Blood ?? Mucus (dysentry) *Ascaris worm *E. vermicularis *T. saginata
  11. 11. STOOL EXAMINATION MACROSCOPIC MICROSCOPIC OTHERS <ul><li>Consistency </li></ul><ul><li>Colour </li></ul><ul><li>Composition </li></ul><ul><li>Culture </li></ul><ul><li>Cellophane tape </li></ul><ul><li>Baeremann tech. </li></ul><ul><li>Ova quantitaion </li></ul><ul><li>(Stoll & Kato) </li></ul>Temprory Permanent Diect saline smear Iodine smear Concentration techniques Sedimentation Floatation Saline Formol ether Sat saline Zinc sulphate Sheather’s sugar
  12. 12. STOOL EXAMINATION Temporary <ul><li>Saline smear </li></ul><ul><li>Iodine smear </li></ul>saline Iodine 1% <ul><li>Huge number of: </li></ul><ul><li>Eggs </li></ul><ul><li>Protozoal troph. Motility </li></ul><ul><li>(Amoeb, flagellates) </li></ul><ul><li>Huge number of: </li></ul><ul><li>Cyst morphological details </li></ul>
  13. 13. STOOL EXAMINATION Scanty infection Concentration techniques Sedimentation Floatation <ul><li>Heavy eggs (Ascaris egg) </li></ul><ul><li>Operculated eggs (Trematodes) </li></ul><ul><li>Larvae (Strong sterc.) </li></ul><ul><li>Cysts </li></ul><ul><li>Non Operculated eggs </li></ul><ul><li>Trematodes ( S. m.) </li></ul><ul><li>Cestode </li></ul><ul><li>Nematode( Hookworms,Trichostong) </li></ul><ul><li>Cysts </li></ul>
  14. 14. STOOL EXAMINATION MACROSCOPIC MICROSCOPIC OTHERS <ul><li>Consistency </li></ul><ul><li>Colour </li></ul><ul><li>Composition </li></ul><ul><li>Culture </li></ul><ul><li>Cellophane tape </li></ul><ul><li>Baeremann tech. </li></ul><ul><li>Ova quantitaion </li></ul><ul><li>(Stoll & Kato) </li></ul>Temprory Permanent Diect saline smear Iodine smear Concentration techniques Sedimentation Floatation Saline Formol ether Sat saline Zinc sulphate Sheather’s sugar
  15. 15. STOOL EXAMINATION Saline sedimentation 10 g stool Saline Mesh wire gauze Conical flask Sediment Emulsify
  16. 16. STOOL EXAMINATION Formol Ether Sed. Conc. 10% Formalin 1 g stool Sediment formalin debris Ether Thorough mixing Ether <ul><li>Ether adsorbs fecal debris & floats. </li></ul><ul><li>Formalin fixes & preserves the specimen. </li></ul>Conical flask centrif. tube
  17. 17. STOOL EXAMINATION MACROSCOPIC MICROSCOPIC OTHERS <ul><li>Consistency </li></ul><ul><li>Colour </li></ul><ul><li>Composition </li></ul><ul><li>Culture </li></ul><ul><li>Cellophane tape </li></ul><ul><li>Baeremann tech. </li></ul><ul><li>Ova quantitaion </li></ul><ul><li>(Stoll & Kato) </li></ul>Temprory Permanent Diect saline smear Iodine smear Concentration techniques Sedimentation Floatation Saline Formol ether Sat saline Zinc sulphate Sheather’s sugar
  18. 18. STOOL EXAMINATION Tin container 20 min Centrif. 2 min Seive Clean light eggs & cysts Floatation concentration Sat saline Zn sulphate Sheather’s sugar <ul><li>Cestode eggs (non op) </li></ul><ul><li>Nematode eggs????? </li></ul><ul><li>Hookworms??????? </li></ul><ul><li>Trichostong ؟؟؟؟؟؟؟؟؟؟؟ </li></ul><ul><li>Egg of S.m. </li></ul><ul><li>Eggs of small tapeworms </li></ul><ul><li>Cysts </li></ul><ul><li>Crypto, Iso. oocysts </li></ul>
  19. 19. STOOL EXAMINATION MACROSCOPIC MICROSCOPIC OTHERS <ul><li>Consistency </li></ul><ul><li>Colour </li></ul><ul><li>Composition </li></ul><ul><li>Culture </li></ul><ul><li>Cellophane tape </li></ul><ul><li>Baeremann tech. </li></ul><ul><li>Ova quantitaion </li></ul><ul><li>(Stoll & Kato) </li></ul>Temprory Permanent Diect saline smear Iodine smear Concentration techniques Sedimentation Floatation Saline Formol ether Sat saline Zinc sulphate Sheather’s sugar
  20. 20. STOOL EXAMINATION Permanent Stained smears <ul><li>Iron haematoxylin stain </li></ul><ul><li>Trichrome stain </li></ul><ul><li>Modified Ziehl Neelsen satin (Crpto.) </li></ul>
  21. 21. STOOL EXAMINATION MACROSCOPIC MICROSCOPIC OTHERS <ul><li>Consistency </li></ul><ul><li>Colour </li></ul><ul><li>Composition </li></ul><ul><li>Cellophane tape </li></ul><ul><li>Culture </li></ul><ul><li>Baeremann tech. </li></ul><ul><li>Ova quantitaion </li></ul><ul><li>(Stoll & Kato) </li></ul>Temprory Permanent Diect saline smear Iodine smear Concentration techniques Sedimentation Floatation Saline Formol ether Sat saline Zinc sulphate Sheather’s sugar
  22. 22. 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
  23. 23. 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
  24. 24. STOOL EXAMINATION Baermann’s technique Warm water Stool/soil seive Glass funnel clamp 30 min 25-50 CC <ul><li>centrifuge </li></ul>Detec. Of Nematode L. /stool, soil
  25. 25. STOOL EXAMINATION Cultures for Nematode larvae <ul><li>Filter paper culture </li></ul><ul><li>Scanty infection </li></ul><ul><li>Larvae of: </li></ul><ul><li>St. stercoralis (A,L) </li></ul><ul><li>Hookworms </li></ul><ul><li>Trichostrong </li></ul>Water Sealed petri dish Filter paper Slide
  26. 26. DIAGNOSIS DIRECT INDIRECT MOLECULAR Urine Stool Sputum Biopsy Aspirates Blood PCR DNA probes IHAT LAT IFAT ELISA CFT DEIDT
  27. 27. NaOH Sputum Centfifuge SPUTUM EXAMINATION MACROSCOPIC MICROSCOPIC Appearance Concentration Bloody (Parag) Rusty brown (Parag)
  28. 28. Parasites/sputum P living in lung P migrating in lung P resulting from rupture of <ul><li>P. westermani eggs </li></ul><ul><li>St. stercoralis </li></ul><ul><li>Ascaris </li></ul><ul><li>Hookworm (filariform L) </li></ul><ul><li>Hydatid cyst (sand) </li></ul><ul><li>Amoebic abcess (troph) </li></ul>
  29. 29. BIOPSY SPECIMEN SKIN SNIP MUSCLE BIOPSY RECTAL BIOPSY O. Volvulus mf T. Spiralis larvae Schistosoma egg <ul><li>Raise skin by needle </li></ul><ul><li>Slice by scissors </li></ul><ul><li>Put snip in normal saline </li></ul><ul><li>Examine </li></ul>Muscle digestion with HCl + pepsin
  30. 30. floor Edge ASPIRATES EXAMINATION CSF Duodenal aspirates BM aspirates Cutanoeus ucler <ul><li>Afr. Tryp. (trypom) </li></ul><ul><li>FLA (troph) </li></ul><ul><li>M.f. of loa loa </li></ul><ul><li>L. of T spiralis </li></ul><ul><li>G. lamb troph </li></ul><ul><li>Crypto oocyst </li></ul><ul><li>St sterc. Rh L. </li></ul><ul><li>Fasciola eggs </li></ul><ul><li>L. donovani a,ast. </li></ul><ul><li>T. cruzi amast. </li></ul><ul><li>P. falciparum. </li></ul><ul><li>Leishmaniasis </li></ul>D. intubation D capsule (Enterotest) Direct stain (amast) NNN (promast) <ul><li>Lumbar puncutre </li></ul><ul><li>Centrifuge </li></ul><ul><li>Examine sed. </li></ul>
  31. 31. DIAGNOSIS DIRECT INDIRECT MOLECULAR Urine Stool Sputum Biopsy Aspirates Blood PCR DNA probes IHAT LAT IFAT ELISA CFT DEIDT
  32. 32. BLOOD EXAMINATION Blood films Buffy coat films QBC technique Knott’s conc. tech. Thin Thick
  33. 33. BLOOD EXAMINATION BLOOD FILMS <ul><li>Thin </li></ul><ul><li>Thick </li></ul>Bld drop spread Air dry methyl alcohol Geimsa Air dry Geimsa Circular motion Malaria, Babesia, Filaria, Tryp.
  34. 34. BLOOD EXAMINATION Buffy coat film centrifuge RBC WBC (BC) plasma Citrated bld 30 min Air dry Fix spread Geimsa Tryp., L. donovani
  35. 35. BLOOD EXAMINATION QBC technique centrifuge RBC RBC +parasite Microhaematocrit tube Acridine orange Malaria, Filaria, Trypanosomes
  36. 36. BLOOD EXAMINATION KNOTT’S CONC. TECHNIQUE 10 ml 1 ml Air dry fix Geimsa <ul><li>Citrated bld </li></ul>Formalin 2 % sediment 2 min centrifuge Filaria
  37. 37. INDIRECT IMMUNOLOGICAL METHODS <ul><li>Scanty infection. </li></ul><ul><li>Tissue parasite no portal of exit (Hydatid dis.) </li></ul><ul><li>Migratory stage (Fasciola) </li></ul><ul><li>Chronic infection fibrosis (Bilharziasis) </li></ul>
  38. 38. INDIRECT IMMUNOLOGICAL METHODS Antigen detection Antibody detection <ul><li>More specific </li></ul><ul><li>More accurate. </li></ul><ul><li>Active infection </li></ul><ul><li>Early </li></ul><ul><li>Quantitative </li></ul>Ab remain in serum for months even after cure
  39. 39. INDIRECT IMMUNOLOGICAL METHODS <ul><li>IHAT </li></ul><ul><li>LAT </li></ul>+ Sensitized Sheep’s RBC (O–ve) Ag Patient’s serum (?? AB) Agglutination + Agglutination Ag Latex particle Patient’s serum (?? AB)
  40. 40. INDIRECT IMMUNOLOGICAL METHODS INDIRECT FLUORESCENT ANTIBODY TEST parasite Patient’s serum (?? AB) Anti human AB fluorescein
  41. 41. INDIRECT IMMUNOLOGICAL METHODS ELISA OPD OPD Flat bottom plastic micrititre plate Ag Patient’s serum (?? AB) Anti human AB Peroxidase E AB
  42. 42. 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
  43. 43. INDIRECT IMMUNOLOGICAL METHODS Double Electro Immuno Diffusion +ve -ve Ag Ab Buffered gel Electric current Line of ppt
  44. 44. 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.
  45. 45. DIAGNOSIS DIRECT INDIRECT MOLECULAR Urine Stool Sputum Biopsy Blood Aspirates PCR DNA probes IHAT LAT IFAT ELISA CFT DEIDT
  46. 46. MOLECULAR BIOLOGICAL TECHNIQUES DNA Probes DNA Probe Commercially prepared DNA sequence Radio active material Nitrocellulose paper Sample (Serum/ stool) ?? parasite +ve parasite Hybridization Radioactivity
  47. 47. MOLECULAR BIOLOGICAL TECHNIQUES Polymerase Chain Reaction (PCR) Single stranded DNA Replication Detection T cruzi, T gondii

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