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LAB ORATORY DIAGNOSIS OF PARASITIC DISEASES

LAB ORATORY DIAGNOSIS OF PARASITIC DISEASES. DEPARTEMENT OF PARASITOLOG Y FA C ULT Y OF MEDICINE UNIVERSITAS PADJADJARAN. INTRODUCTION. AN. DIAGNOSIS FOR PARASITIC INFECTION Anamnesa – ( The history should include details of the presenting complaint ) P hysical examination

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LAB ORATORY DIAGNOSIS OF PARASITIC DISEASES

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  1. LAB ORATORY DIAGNOSIS OF PARASITIC DISEASES DEPARTEMENT OF PARASITOLOGY FACULTY OF MEDICINE UNIVERSITAS PADJADJARAN

  2. INTRODUCTION AN DIAGNOSIS FOR PARASITIC INFECTION • Anamnesa – ( The history should include details of the presenting complaint ) • Physical examination • Laboratory examination espectially Parasitological diagnosis • Imunodiagnosis

  3. INTRODUCTION ANAMNESIS • Fever • Gastrointestinal symptoms • Fever with respiratory system • Neurological symptoms • Fever and meningitis / Encephalitis • Cutaneous symptoms CLINICAL FEATURES (Symptom and Sign) 1 SPESIFIC ASPESIFIC 2 PHYSICAL EXAMINATION 3 LABORATORY EXAMINATION

  4. INTRODUCTION LABORATORY EXAMINATION 3 DEPEND ON : • Habitat Parasite • Parasite distribution TYPE OF SPECIMEN : • Stool • Blood 3 Urine 4 Sputum 5. Vaginal discharge, urethral discharge 6. Skin scrapings 7. Liquor Cerebro Spinal • Tissue biopsy • Nasal discharge • Corneal scrapings IMPORTANT • Very important for examination Helminth parasite and Protozoa parasite • Repeating of Laboratory examination occasionally be needed *Should be understood about examination technique,morphology of parasite and life cycle of parasite IMUNODIAGNOSIS 4

  5. INTRODUCTION FECAL SPECIMENS • Immediatelly have to examine : • Liquid specimens should be examined within 30 minute of passage • Soft (semiformed) specimens1 hour • Formed specimens 24 hour after passage • If this time is not possible, the specimen should be placed in one of the available fixatives : • Formalin 10%, MIF (Merthiolate Iodine Formalin), PVA (Polyvinyl Alcohol) • Generally Helminthic eggs more endurewithoutpreservation than intestinal protozoa

  6. INTRODUCTION FECAL SPECIMENS • The specimens should not be contaminated with • Water – because watermay contain free-living organismsthat can be mistakenfor human parasites • Urine – may destroy motile organisms • Prior to examination, fecal specimens should never be incubated or frozen. • A chatartic with an oil base should not be used,and a stool softener (taken either orallyor as a suppository) is usually inadequate for obtaining a purged specimen.

  7. INTRODUCTION FECAL SPECIMENS • Repeating fecal examination after therapy : • Ascariasis, 2-3 weeks after therapy • Protozoa infection, 3-4 weeks after therapy • Taeniasis, 5-6 weeks after therapy

  8. EXAMINATION OF HELMINTH PARASITE SPECIMENS ( most important ) • FECAL SPECIMENS & • BLOOD AND TISSUE SPECIMENS

  9. ? EXAMINATION OF HELMINTH PARASITE FECAL SPECIMENS • For examination of helminth egg • Most important for examination intestinal nematode including “Soil Transmitted Helminths” : • Ascaris lumbricoides • Trichuris trichiura • Hook worm : - Necator americanus andAncylostoma duodenale • Strongyloides stercoralis

  10. EXAMINATION OF HELMINTH PARASITE QUALITATIVE EXAMINATION FECAL SPECIMENS DIRECT WET SMEAR ANOTHER QUALITATIVE EXAMINATION AND QUANTITATIVE EXAMINATION TO BE STUDIED IN FECAL EXAMINATION

  11. LABORATORY TECHNIQUE FOR EXAMINATION OF HELMINTH PARASITE

  12. FECAL SPECIMENS INTESTINAL HELMINTH LABORATORY TECHNIQUE FOR EXAMINATION HELMINTH PARASITE BLOOD AND TISSUE HELMINTH BLOOD AND TISSUE HELMINTH LABORATORY TECHNIQUE FOR EXAMINATION OF HELMINTH PARASITE Click “Esc” button When finished

  13. *Consistency (hard,formed,soft,diarrhea) *Colour *Odor *Foreign bodies (blood, mucus,parasite) MICROSCOPIC EXAMINATION QUALITATIVE QUANTITATIVE LABORATORY TECHNIQUE FOR EXAMINATION OF INTESTINAL HELMINTH MACROSCOPIC EXAMINATION EXAMINATION TECHNIQUE FOR HELMINTH PARASITE IN FAECES Click “Esc” button When finished

  14. QUALITATIVE MICROSCOPIC • DIRECT WET SMEAR METHOD • FLOTATION METHOD • MODIFICATIONMERTHIOLATE IODINE FORMALDEHYDE (MIF) • CELLOTAPE TAPE METHOD • FORMALDEHYDE ETHER SEDIMENTATION (RITCHIE’S METHOD) • METODA KATO • STOLL DILLUTION METHOD • KATO – KATZ CELLOPHANE THICK SMEAR METHOD QUANTITATIVE EXAMINATIONTECHNIQUEOF INTESTINAL HELMINTH Click “Esc” button When finished

  15. MICROSCOPIC QUALITATIVE DIRECT WET SMEAR METHOD • Fast • Severe infection • Reagens • NaCl Physiologis (0,9%), or • Eosin 2% Click “Esc” button When finished

  16. MICROSCOPIC QUALITATIVE DIRECT WET SMEAR METHOD Place 2 mg faeces on microscope slide Emulsify 2 mg faeces in NaCl 0.9% drop Place 1-2 drops NaCl 0,9% or Eosin 2% on microscope slide 1-2 drops NaCl 0,9% or eosin 2% Place 2 mg faeces on microscope slide Click “Esc” button When finished

  17. MICROSCOPICQUALITATIVE SALINE WET SMEAR METHOD Place 2 mg faeces on microscope slide Emulsify 2 mg faeces in NaCl 0,9% drop Emulsify 2mg faeces in the saline drop Place coverslipover suspension Examine using the lower powerobjective 10x or 40x 1-2 drops NaCl 0,9% Place coverslip over the suspension

  18. WITHOUT CENTRIFUGATION FLOTATION METHOD WITH CENTRIFUGATION MICROSCOPIC QUALITATIVE FLOATATION METHOD • Berdasarkan BJ telur < BJ larutan • Berguna untuk infeksi ringan • Larutan yang dipergunakan : • NaCl jenuh, atau • Gula jenuh Click “Esc” button When finished

  19. Ose stirring glass NaCl saturated 20 minutes faeces MICROSCOPIC QUALITATIVE FLOATATION METHOD WITHOUT CENTRIFUGATION Place coverslip over the solution Take down 1-2 drops of Surface layer with Ose and Place on microscope slide 10 gr faecesmixed with 200 ml NaCl saturated until homogenoussolution Take down1-2 drops thesurface layer with Ose Place coverslip over microcope slide 10 gr. faeces + 200 cc NaCl saturated Examine under low power objective

  20. Stirring glass filtered NaCl saturated faeces Centrifuge 100 rpm for 5 minutes MICROSCOPIC QUALITATIVE FLOATATION METHOD WITH CENTRIFUGATION Transfer 10 gr faecesand emulsify in 200 ml NaCl saturated untilhomogenous solution 10 gr. faeces + 200 cc NaCl saturated

  21. stirring glass filtered NaCl saturated faeces centrifuge 100 x/mnt 5 minutes MICROSCOPIC QUALITATIVE FLOATATION METHOD WITH CENTRIFUGATION Filtered with tea filter 10 gr. faeces + 200 cc NaCl saturated

  22. stirring glass filtered NaCl saturated faeces centrifuge 100 rpm For 5 minutes MICROSCOPIC QUALITATIVE FLOATATION METHOD WITH CENTRIFUGATION Decant into centrifuge tube 10 gr. faeces + 200 cc NaCl saturated

  23. Stirring glass filtered NaCl saturated faeces centrifuge 100 rpm For 5 minutes MICROSCOPIC QUALITATIVE FLOATATION METHOD WITH CENTRIFUGATION Centrifuge 100 rpmfor 5 minutes 10 gr.faeces + 200 cc NaCl saturated

  24. stirring glass filtered NaCl saturated faeces Centrifuge 100 rpm For 5 minutes MICROSCOPIC QUALITATIVE FLOATATION METHOD WITH CENTRIFUGATION Take down solution from surface layer with Ose 10 gr. faeces + 200 cc NaCl saturated

  25. Place solution on microscope slide and Place coverslip over the microscope slide. Examine under low power objective 10 x or 40 x. MICROSCOPIC QUALITATIVE FLOATATION METHOD WITH CENTRIFUGATION Place the solution on microscope slide Place coverslip over microscope slide

  26. MICROSCOPIC QUALITATIVE MERTIOLAT IODIN FORMALIN MODIFICATION (MIF MODIFICATION) • For identificationeggs from intestinal helminth, AmebaandGiardia lamblia • Solution 1 : • 250 ml aquadest • 200 ml thimerosal (1:1.000) • 25 ml formalin • 5 ml glycerin • Solution 2 : • Fresh lugol solution5%

  27. MICROSCOPIC QUALITATIVE MERTHIOLAT IODIN FORMALIN MODIFICATION(MIF MODIFICATION ) • It’s good for staining and conservation of cyst of intestine protozoa and worm egg

  28. (+) 7 ml. ether (40 C) filtered Shake until homogenous Gelas pengaduk centrifuge1.000-3.000 x/mnt 1 minutes 5 ml base solution 1 0,5 ml base solution 2 0,5 gr faeces MICROSCOPIC QUALITATIVE MERTHIOLAT IODIN FORMALIN MODIFICATION(MIF MODIFICATION ) Add 7 ml ether (40 C) Open the tube, let it 2 minutes, centrifuge for 1 minutes (1.000-3.000 rpm) shake until homogenous

  29. MICROSCOPIC QUALITATIVE MERTHIOLAT IODIN FORMALIN MODIFICATION(MIF MODIFICATION ) Place the sediment on microscope slide Place coverslip over microscope slide Throw away the supernatan, take sediment with pipette Examine under low power objective

  30. MICROSCOPIC QUALITATIVE CELLOTAPE METHOD • The egg adhere on perianal area, so rarely found in faeces (5 %). To find this parasite we need Scotch Adhesive tape Swab from Graham or Cellotape Method

  31. MICROSCOPIC QUALITATIVE CELLOTAPE METHOD • To examine the egg of Enterobius vermicularis • Children 1-10 years old • Doing in the morning before take a bath or wash the anus with water after defecating • Transparent plastic plaster (2 x 1,5 cm)patched to skin around the anus • Press the plaster, then lift slowly • Patched to the object glass, examine under the microscope

  32. MICROSCOPIC QUALITATIVE CELLOTAPE METHOD

  33. MICROSCOPIC QUALITATIVE Concentration Method • practical, simple, for ova examination in stool : • 1 gr faeces, put into the reaction tube, add aquadest, mixed until homogenous, put imyo the centrifuge tube • Centrifuge with velocity 3.000 rpm for 1 mnt • Throw away the supernatan, take the sediment with pipette • Place the sediment on microscope slide, place coverslip on microscope slide

  34. Put in faeces solution to the centrifuge tube Stirring glass Centrifuge 3.000 x/mnt, 1 minutes Aquadest 1 gr faeces Shake until homogenous MICROSCOPIC QUALITATIVE Concentration Method Throw away supernatan, take sediment with pipette

  35. MICROSCOPIC QUALITATIVE Concentration Method place coverslip on microscope slide Place sediment on microscope slide Examine under the microscope

  36. MICROSCOPIC QUALITATIVE CELLOPHANE THICK SMEAR METHOD(KATO METHOD) • Practical, simple, and cheap • Can be used in mass examination • Examination needs more stool, so the eggs can be found much more • Morphology of egg is clear

  37. MICROSCOPIC QUALITATIVE CELLOPHANE THICK SMEAR METHOD (KATO METHOD) Substance : • Solution • 100 ml aquadest/fenol 6% • 100 ml glycerin (supaya kotoran tinja jadi jernih) • 1 ml malachit green solution (supaya mata tidak silau) • Cellophane tape, 2,5 x 3 cm, soak in solution for >24 hours

  38. MICROSCOPIC QUALITATIVE CELLOPHANE THICK SMEAR METHOD (KATO METHOD) • Technique: • Take 20-50 mg faeces ( as large as red bean ) • Put on object glass, spread out • Cover with cellophane, pressing the faeces until flat and spread out under the cellophane • Drain the excessive fluid with filter paper • Let it 20-30 minutes • Examine under the microscope

  39. Soaked > 24 hours 100 part. Aquadest/fenol 6% 100 part. Glycerin 1 part Malachit green solution Cut the cellophane 2,5 x 3 cm Soaked cellophane faeces 20-50 gr faeces MICROSCOPIC QUALITATIVE CELLOPHANE THICK SMEAR METHOD (KATO METHOD)

  40. MICROSCOPIC QUANTITATIVE STOLL METHOD • Solution used : • NaOH 0,1 N (faeces solvent) or • KOH 10% • Good for severe and moderate infection • Less good for mild infection

  41. faeces 56 ml 60 ml NaOH solution 0,1N Let it 1 night or 3-4 hours but shake it longer Shake MICROSCOPIC QUANTITATIVE STOLL METHOD

  42. Shake and take 0,15 ml MICROSCOPIC QUANTITATIVE STOLL METHOD

  43. MICROSCOPIC QUANTITATIVE STOLL METHOD • Formula : amount of egg in 1 gram feces = amount of seen egg (miroscopic) x 100

  44. MICROSCOPIC QUANTITATIVE STOLL METHOD ENUMARATION • NaOH = 56 ml, feces 4 ml ~ 4gr • 4 gr feces in 60 ml • or 1 gr feces in 15 ml • In the 0.15 ml of stool solution, can be found y eggs • So, in the 15 ml, is found y x 100 eggs(~ 1 gr stool)

  45. AMOUNT OF EGG PER-GRAM FAECES Infectid by INFECTION LEVEL AMOUNT OF HELMINTH • MILD • MODERATE • SEVERE • < 7.000 • 7.000-35.000 • > 35.000 • 5 or less • 6-25 • > 25 A. lumbricoides • MILD • MODERATE • SEVERE • < 3.000 • 3.000-10.000 • > 10.000 • 20 or less • 21-100 • > 100 A. duodenale • MILD • MODERATE • SEVERE • < 2.000 • 2.000-7.000 • > 7.000 • 50or less • 51-200 • > 200 N. americanus MICROSCOPIC QUANTITATIVE STOLL METHOD Infection level based on amount of egg in each gram feces and amount of helminth SOURCE : PARASITIC DISEASES PROGRAME. WHO. GENEVA, 1981

  46. MICROSCOPIC QUANTITATIVE KATO-KATZ METHOD • Tolls : • Object glass • cellotape, thick 40 m, size 3 x 3 cm • Thick carton, make hole with fixed volume to print faeces as weight as 30 mg • Palm leaf rib, oily papper, wire netting

  47. MICROSCOPIC QUANTITATIVE KATO-KATZ METHOD • Malachite-Green solution : • 100 ml aquadest • 100 ml glycerin (in order to make the feces clear) • 1 ml malachite green solution • Soak the cellotape in solution for > 24 hours

  48. PRINTING FAECES MAKING PREPARAT • Put 5 gr faeces on the oily papper, put wire netting on it then press • Put the holed carton on the object glass, print the filtered faeces on holed carton • Lift the carton • Cover the faeces with soaked cellotape • Press, spread out • Let it 30 mnt • Examine under the microscope MICROSCOPIC QUANTITATIVE KATO-KATZ METHOD FILTERING FAECES

  49. Printing faeces Making THE SPECIMEN Holed carton press Wire netting Soaked cellotape faeces (5 gr) Object glass Filtered faeces TEKAN printed Oily papper Wire netting EXAMINE UNDER THE MICROSCOPE faeces (5 gr) MICROSCOPIC QUANTITATIVE KATO-KATZ METHOD Filtering faeces Printed faeces Object glass

  50. MICROSCOPIC QUANTITATIVE KATO-KATZ METHOD WHO (1981) • EGG PRODUCTION PER-DAY : • A. lumbricoides : 200.000 • A. doudenale : 10.000-25.000 • N. americanus : 5.000-10.000 • WEIGHT OF FAECES PER-DAY • Children : 70 gram • Adult : 2 x children

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