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

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Presentation on theme: "LAB ORATORY DIAGNOSIS OF PARASITIC DISEASES DEPARTEMENT OF PARASITOLOGY FACULTY OF MEDICINE UNIVERSITAS PADJADJARAN DEPARTEMENT OF PARASITOLOGY FACULTY."— Presentation transcript:

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

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

4 PHYSICAL EXAMINATION ANAMNESIS ASPESIFIC CLINICAL FEATURES (Symptom and Sign) CLINICAL FEATURES (Symptom and Sign) SPESIFIC LABORATORY EXAMINATION INTRODUCTION -Fever -Gastrointestinal symptoms -Fever with respiratory system -Neurological symptoms -Fever and meningitis / Encephalitis -Cutaneous symptoms -Fever -Gastrointestinal symptoms -Fever with respiratory system -Neurological symptoms -Fever and meningitis / Encephalitis -Cutaneous symptoms

5 INTRODUCTION DEPEND ON :  Habitat Parasite  Parasite distribution DEPEND ON :  Habitat Parasite  Parasite distribution 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 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 TYPE OF SPECIMEN : 1.Stool 2.Blood 3 Urine 4 Sputum 5. Vaginal discharge, urethral discharge 6. Skin scrapings 7. Liquor Cerebro Spinal 8.Tissue biopsy 9.Nasal discharge 10.Corneal scrapings TYPE OF SPECIMEN : 1.Stool 2.Blood 3 Urine 4 Sputum 5. Vaginal discharge, urethral discharge 6. Skin scrapings 7. Liquor Cerebro Spinal 8.Tissue biopsy 9.Nasal discharge 10.Corneal scrapings LABORATORY EXAMINATION 3 IMUNODIAGNOSIS 4 IMPORTANT

6 INTRODUCTION FECAL SPECIMENS  Immediatelly have to examine :  Liquid specimens should be examined within 30 minute of passage  Soft (semiformed) specimens 1 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 endure without preservation than intestinal protozoa  Immediatelly have to examine :  Liquid specimens should be examined within 30 minute of passage  Soft (semiformed) specimens 1 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 endure without preservation than intestinal protozoa

7 INTRODUCTION FECAL SPECIMENS  The specimens should not be contaminated with  Water – because water may contain free- living organisms that can be mistaken for 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 orally or as a suppository) is usually inadequate for obtaining a purged specimen.  The specimens should not be contaminated with  Water – because water may contain free- living organisms that can be mistaken for 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 orally or as a suppository) is usually inadequate for obtaining a purged specimen.

8 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  Repeating fecal examination after therapy :  Ascariasis, 2-3 weeks after therapy  Protozoa infection, 3-4 weeks after therapy  Taeniasis, 5-6 weeks after therapy

9 EXAMINATION OF HELMINTH PARASITE SPECIMENS ( most important ) SPECIMENS ( most important )  FECAL SPECIMENS  BLOOD AND TISSUE SPECIMENS & &

10 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 and Ancylostoma duodenale  Strongyloides stercoralis  For examination of helminth egg  Most important for examination intestinal nematode including “Soil Transmitted Helminths” :  Ascaris lumbricoides  Trichuris trichiura  Hook worm : - Necator americanus and Ancylostoma duodenale  Strongyloides stercoralis

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

12 LABORATORY TECHNIQUE FOR EXAMINATION OF HELMINTH PARASITE

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

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

15 MICROSCOPIC QUALITATIVE pSTOLL DILLUTION METHOD pKATO – KATZ CELLOPHANE THICK SMEAR METHOD QUANTITATIVE EXAMINATION TECHNIQUE OF INTESTINAL HELMINTH Click “Esc” button When finished pDIRECT WET SMEAR METHOD pFLOTATION METHOD pMODIFICATION MERTHIOLATE IODINE FORMALDEHYDE (MIF) pCELLOTAPE TAPE METHOD pFORMALDEHYDE ETHER SEDIMENTATION (RITCHIE’S METHOD) pMETODA KATO

16 pFast pSevere infection pReagens  NaCl Physiologis (0,9%), or  Eosin 2% DIRECT DIRECT WET SMEAR METHOD MICROSCOPIC QUALITATIVE Click “Esc” button When finished

17 DIRECT WET SMEAR METHOD MICROSCOPIC QUALITATIVE 1-2 drops NaCl 0,9% or eosin 2% 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 Click “Esc” button When finished Place 2 mg faeces on microscope slide

18 MICROSCOPIC QUALITATIVE SALINE WET SMEAR METHOD Place 2 mg faeces on microscope slide Emulsify 2 mg faeces in NaCl 0,9% drop Place coverslip over the suspension 1-2 drops NaCl 0,9% Emulsify 2mg faeces in the saline drop Place coverslip over suspension Examine using the lower power objective 10x or 40x

19 MICROSCOPIC QUALITATIVE FLOATATION METHOD pBerdasarkan BJ telur < BJ larutan pBerguna untuk infeksi ringan pLarutan yang dipergunakan :  NaCl jenuh, atau  Gula jenuh FLOTATION METHOD WITH CENTRIFUGATION WITHOUT CENTRIFUGATION Click “Esc” button When finished

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

21 MICROSCOPIC QUALITATIVE NaCl saturated faeces Stirring glass filtered Centrifuge 100 rpm for 5 minutes Transfer 10 gr faeces and emulsify in 200 ml NaCl saturated until homogenous solution 10 gr. faeces cc NaCl saturated FLOATATION METHOD WITH CENTRIFUGATION

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

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

24 NaCl saturated faeces Stirring glass filtered centrifuge 100 rpm For 5 minutes Centrifuge 100 rpm for 5 minutes 10 gr.faeces cc NaCl saturated MICROSCOPIC QUALITATIVE FLOATATION METHOD WITH CENTRIFUGATION

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

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

27 pFor identification eggs from intestinal helminth, Ameba and Giardia lamblia pSolution 1 :  250 ml aquadest  200 ml thimerosal (1:1.000)  25 ml formalin  5 ml glycerin pSolution 2 :  Fresh lugol solution5% MERTIOLAT IODIN FORMALIN MODIFICATION (MIF MODIFICATION) MICROSCOPIC QUALITATIVE

28 MERTHIOLAT MERTHIOLAT IODIN FORMALIN MODIFICATION (MIF MODIFICATION MODIFICATION ) pIt’s good for staining and conservation of cyst of intestine protozoa and worm egg MICROSCOPIC QUALITATIVE

29 (+) 7 ml. ether (4 0 C) MERTHIOLAT IODIN FORMALIN MODIFICATION (MIF MODIFICATION ) Gelas pengaduk 5 ml base solution 1 0,5 ml base solution 2 0,5 gr faeces filtered Shake until homogenous centrifuge x/mnt 1 minutes shake until homogenous MICROSCOPIC QUALITATIVE Add 7 ml ether (4 0 C) Open the tube, let it 2 minutes, centrifuge for 1 minutes ( rpm)

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

31 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 MICROSCOPIC QUALITATIVE

32 CELLOTAPE METHOD pTo examine the egg of Enterobius vermicularis pChildren 1-10 years old pDoing in the morning before take a bath or wash the anus with water after defecating pTransparent plastic plaster (2 x 1,5 cm)patched to skin around the anus pPress the plaster, then lift slowly pPatched to the object glass, examine under the microscope MICROSCOPIC QUALITATIVE

33 CELLOTAPE METHOD MICROSCOPIC QUALITATIVE

34 Concentration Method ppractical, 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 rpm for 1 mnt  Throw away the supernatan, take the sediment with pipette  Place the sediment on microscope slide, place coverslip on microscope slide MICROSCOPIC QUALITATIVE

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

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

37 CELLOPHANE THICK SMEAR METHOD (KATO METHOD) pPractical, simple, and cheap pCan be used in mass examination pExamination needs more stool, so the eggs can be found much more pMorphology of egg is clear MICROSCOPIC QUALITATIVE

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

39 CELLOPHANE THICK SMEAR METHOD (KATO METHOD) pTechnique :  Take 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 minutes  Examine under the microscope MICROSCOPIC QUALITATIVE

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

41 pSolution used :  NaOH 0,1 N (faeces solvent) or  KOH 10% pGood for severe and moderate infection pLess good for mild infection STOLL METHOD MICROSCOPIC QUANTITATIVE

42 MICROSCOPICQUANTITATIVE Shake Let it 1 night or 3-4 hours but shake it longer faeces NaOH solution 0,1N STOLL METHOD 56 ml60 ml

43 MICROSCOPICQUANTITATIVE Shake and take 0,15 ml STOLL METHOD

44 MICROSCOPICQUANTITATIVE pFormula : amount of egg in 1 gram feces = amount of seen egg (miroscopic) x 100 STOLL METHOD

45 MICROSCOPICQUANTITATIVE ENUMARATION pNaOH = 56 ml, feces 4 ml ~ 4gr p4 gr feces in 60 ml por 1 gr feces in 15 ml pIn the 0.15 ml of stool solution, can be found y eggs pSo, in the 15 ml, is found y x 100 eggs(~ 1 gr stool) STOLL METHOD

46 MICROSCOPICQUANTITATIVE Infection level based on amount of egg in each gram feces and amount of helminth pMILD pMODERATE pSEVERE Ù< Ù Ù> A. lumbricoides Ù5 or less Ù6-25 Ù> 25 pMILD pMODERATE pSEVERE A. duodenale Ù< Ù Ù> Ù20 or less Ù Ù> 100 pMILD pMODERATE pSEVERE N. americanus Ù< Ù Ù> Ù50or less Ù Ù> 200 AMOUNT OF HELMINTH AMOUNT OF EGG PER-GRAM FAECES INFECTION LEVEL Infectid by SOURCE : PARASITIC DISEASES PROGRAME. WHO. GENEVA, 1981 STOLL METHOD

47 KATO-KATZ METHOD MICROSCOPICQUANTITATIVE pTolls :  Object glass  cellotape, thick 40 , 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

48 KATO-KATZ METHOD MICROSCOPICQUANTITATIVE  Malachite-Green solution :  100 ml aquadest  100 ml glycerin (in order to make the feces clear)  1 ml malachite green solution pSoak the cellotape in solution for > 24 hours

49 KATO-KATZ METHOD MICROSCOPICQUANTITATIVE FILTERING FAECES  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 PRINTING FAECESMAKING PREPARAT

50 KATO-KATZ METHOD MICROSCOPICQUANTITATIVE Filtering faeces Oily papper Wire netting press faeces (5 gr) TEKAN Wire netting faeces (5 gr) Object glass Holed carton Filtered faeces printed Printed faeces Object glass Soaked cellotape Printing faecesMaking THE SPECIMEN EXAMINE UNDER THE MICROSCOPE

51 KATO-KATZ METHOD KATO-KATZ METHOD MICROSCOPICQUANTITATIVE WHO (1981) pEGG PRODUCTION PER-DAY :  A. lumbricoides:  A. doudenale:  N. americanus: pWEIGHT OF FAECES PER-DAY  Children: 70 gram  Adult : 2 x children

52 FORMOL ETHER SEDIMENTATION METHOD (RITCHIE) MICROSCOPICQUALITATIVE p(0,5 ml faeces) + (1-2 ml aquadest), shake, + (10-12 ml aquadest), shake pFilter pCentrifuge rpm, 1 mnt p+ (1 ml formalin 10%) + (formalin 10% sampai volume 8 ml), let it 10 mnt p+ (3ml ether), shake second pCentrifuge rpm, 1-2 mnt pThrow away the supernatan, place the sediment on microscope slide, examine under the microscope

53 MICROSCOPICQUALITATIVE shake 0,5 ml. faeces 1-2 ml. aquadest shake ml. aquadest filter centrifuge rpm, 1 minute pSaring FORMOL ETHER SEDIMENTATION METHOD (RITCHIE) pcentrifuge rpm, 1 minute

54 MICROSCOPICQUALITATIVE + (1 ml formalin 10%) + (formalin 10% until volume 8 ml), let it 10 minutes LET IT (10 minutes) 3 ml. ether 1 ml. Formalin 10% + Formalin 10% until volume 8 ml SHAKE (15-20 seconds) CENTRIFUGE rpm, 1-2 minutes FORMOL ETHER SEDIMENTATION METHOD (RITCHIE) Throw away the supernatan, place the sediment on microscope slide, examine under the microscope

55 USING FORMALIN SOLUTION 3,5 % OR 4% CONSERVATION AND STORAGE METHOD EGG AND ADULT WORM IN FAECES pMaking formalin solution 3,5 % Or 4% :  1 part of formalin solution 35% or 40% mixed with 9 part of running water, then put into closed bottle pPut the faeces into the bottle and closed tightly pFor organ that attacked by parasite, wash cleanly before put into the bottle

56 Laboratory tecknique for examination of Filaria sp. BLOOD AND TISSUE NEMATODE (Blood specimens ) ¶ DIRECT METHOD F Blood taken of from finger tip F Thin blood smear  Qualitative  Determine microfilariae in peripheral blood vessels  Not for species identification F Thick blood smear  Quantitative  Measured blood releasefrom finger tip with mikropipet (160 m), make thick blood smear. · CONCENTRATION METHOD F Take blood vein F More sensitive than direct method Click “esc” button When finished

57 SUBJECT MATERIAL SUBJECT MATERIAL HOW TO EXAMINE AND INTERPRET INTESTINAL PROTOZOA - Direct wet mount - Modified Merthiolate-Iodine -Formalin (MIF) HOW TO EXAMINE AND INTERPRET BLOOD PROTOZOA - Thin Blood smear with Giemsa staining - Thick Blood smear with Giemsa staining EXAMINATION FOR Trichomonas vaginalis HOW TO PREPARE PERMANENT (FIXED) MOUNT - Blood parasites - Trichomonas vaginalis - Ameba, using Heidenhein method and staining METHODS FOR PREPARING COLOR STAININGS HOW TO EXAMINE AND INTERPRET INTESTINAL PROTOZOA - Direct wet mount - Modified Merthiolate-Iodine -Formalin (MIF) HOW TO EXAMINE AND INTERPRET BLOOD PROTOZOA - Thin Blood smear with Giemsa staining - Thick Blood smear with Giemsa staining EXAMINATION FOR Trichomonas vaginalis HOW TO PREPARE PERMANENT (FIXED) MOUNT - Blood parasites - Trichomonas vaginalis - Ameba, using Heidenhein method and staining METHODS FOR PREPARING COLOR STAININGS

58 (1). Direct wet mount exam Purpose : For quick and simple examination Purpose : For quick and simple examination LAB METHODS FOR INTESTINAL PROTOZOA LAB METHODS FOR INTESTINAL PROTOZOA - For trophozoite form of ameba, use 2% eosin solution - For cyst and nucleus of amoeba use lugol solution (2% of Iodine + 3% potasssium Iodine) - For cyst and nucleus of amoeba use lugol solution (2% of Iodine + 3% potasssium Iodine)

59  Using a small stick, add small amount of feces on top of an object glass  Add few drops of physiological saline solution NaCl/lugol/eosin 2% on top Preparation :  Distribute/spread evenly and cover with coverglass  Examine under low power microscope LAB METHODS FOR INTESTINAL PROTOZOA (1). Direct wet mount examination

60 (2). Modified Method Merthiolate-Iodine-Formaline (MIF) Purpose : Good for diagnosis of Ameba and Giardia in feces LAB METHOD FOR INTESTINAL PROTOZOA Click “Esc”button When finished

61 Ingredients used : Basic solution (1) : –250 ml aquadest –200 ml tincture of merthiolate –25 ml formaldehyde Basic solution (2) : –lugol 5 % (not to be kept > 3 weeks) Both basic solutions should be kept in brown colored bottle Modified Method Merthiolate-Iodine-Formaline (MIF) LAB METHOD FOR INTESTINAL PROTOZOA Click “Esc”button When finished

62  Acidic in character  Foul smelling  Produce less mucus compared to bacillary dysentery, less sticky  With or without blood (blood may be found in solid feces)  In some cases mucosal wall may come out  Acidic in character  Foul smelling  Produce less mucus compared to bacillary dysentery, less sticky  With or without blood (blood may be found in solid feces)  In some cases mucosal wall may come out CHARACTERISTIC OF FECES WITH AMEBA MACROSCOPIC

63 CHARACTERISTIC OF FECES WITH AMEBA MACROSCOPIC Source :A Colour Atlas of Clinical Parasitology. Tomio Yamaguchi. Alih Bahasa : Lesmana Padmasutra, dkk. Feces of amebic dysenteric patient, plenty of trophozoite stage

64 CHARACTERISTICS OF FECES WITH AMEBA MACROSCOPIC Source : Color Atlas of Medicine and Parasitology W. Peters & H.M. Gillers Feces of amebic dysenteric patient, loose, slimy and bloody feces

65 CHARACTERISTICS OF FECES WITH AMEBA MICROSCOPIC  Plenty of bacteria  Entamoeba histolytica (+) containing erythrocytes  Erythrocytes in reuleaux (chain) formation  Charcot-Leyden crystals (unspecific for ameba dysentery, can be found also in other parasitic infection e.g. Strongyloides stercoralis) – from eosinophil desintegration  Pus less abundant compared to bacillary dysentery, if no secondary infection  Macrophages in bacillary dysentery mimics Entamoeba histolytica but nucleus and pseudopods differs  Cyst found in carrier patients and cases with light infection  Plenty of bacteria  Entamoeba histolytica (+) containing erythrocytes  Erythrocytes in reuleaux (chain) formation  Charcot-Leyden crystals (unspecific for ameba dysentery, can be found also in other parasitic infection e.g. Strongyloides stercoralis) – from eosinophil desintegration  Pus less abundant compared to bacillary dysentery, if no secondary infection  Macrophages in bacillary dysentery mimics Entamoeba histolytica but nucleus and pseudopods differs  Cyst found in carrier patients and cases with light infection

66 CHARACTERISTIC OF FECES WITH AMEBA microscopic Charcot-Leyden crystals (From eosinophil desintegration) Source;Color Atlas of Medical Parasitology. Prayong Radomyos, dkk.

67 Blood slides with Giemsa staining Purpose Purpose : For the examination of blood protozoa, e.g. : Plasmodium, Trypanosoma, Babesia etc. Prepared in two stages : (1). Prepare the blood smear (2). Do the color staining EXAMINATION METHODS FOR BLOOD PROTOZOA

68  Place blood slide in upright position, allow to dry, keep away from dust or small insects. EXAMINATION METHOD FOR BLOOD PROTOZOA Blood slides with Giemsa staining Preparation of thin blood film

69 EXAMINATION METHOD FOR BLOODPROTOZOA EXAMINATION METHOD FOR BLOOD PROTOZOA Blood slides with Giemsa staining Staining procedure Fix with methyl alcohol (3-5) minutes Stain with standard Giemsa for 45 minutes Wash slowly with tap water ALLOW TO DRY

70 EXAMINATION METHOD FOR BLOODPROTOZOA EXAMINATION METHOD FOR BLOOD PROTOZOA Thick blood smear with Giemsa stain Purpose : Rapid examination of blood protozoa (for mass survey and screening ) Conducted in two stages : (1). Prepare thick blood film (2). Staining the blood film

71 EXAMINATION METHOD FOR BLOODPROTOZOA EXAMINATION METHOD FOR BLOOD PROTOZOA (1). Preparation of thick blood smear Thick blood smear with Giemsa stain  Blood is prepared similar to thin blood smear  Place 1-2 drops of blood on a glass slide  Spread evenly, forming a circle of 1 - 1,5 cm in diameter  Allow to dry, keep away from dust or insect

72 EXAMINATION METHOD FOR BLOODPROTOZOA EXAMINATION METHOD FOR BLOOD PROTOZOA (2). Staining procedure ALLOW TO DRY Stain with standard Giemsa for 45 minutes Rinse slowly with tap water Thick blood smear with Giemsa stain NO FIXATION !!!

73 THIN BLOOD SMEAR – Morphology and stages of Plasmodium clearly defined – Erythrocytes are intact (due to fixation) – Slow to prepare – Used for examination of moderate and heavy infection THICK BLOOD SMEAR – Morphology and stages of Plasmodium not clearly defined – Erythrocytes are lysed, only stroma from erythrocytes are seen – Preparation and staining are faster (can be used for mass survey examination) – Commonly used for light infection COMPARISON OF THIN AND THICK BLOOD SMEAR EXAMINATION METHOD FOR BLOODPROTOZOA EXAMINATION METHOD FOR BLOOD PROTOZOA

74 EXAMINATION OF Trichomonas vaginalis EXAMINATION OF Trichomonas vaginalis Methods of examination : (1). Direct examination (2). Culture Methods of examination : (1). Direct examination (2). Culture Direct method : (1). Direct wet mount (2). Staining

75 EXAMINATION OF Trichomonas vaginalis EXAMINATION OF Trichomonas vaginalis Sample for examination : (1). Women : vaginal, or urethral discharge (using vaginal spiculum) (2). Men : discharge from urethra or prostate, and centrifugated urine sample

76 DIRECT EXAMINATION Materials used : -Test tube containing 5% glucose (in physiological saline solution) - Cotton bulb Cotton bulb Glucose 5% Examination for Trichomonas vaginalis

77 Terima kasih …………………. April 2005


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