DIAGNOSTIC PROCEDURE IN PARASITOLOGY: DIAGNOSTIC PROCEDURE IN PARASITOLOGY SOMNATH 14.06.12 What is Parasite: What is Parasite A living organism which receives nourishment and shelter from another organism where it lives. ECTOPARASITES-Infestation Pediculus humanus ENDOPARASITES-Infection All protozoa and helminthes fungi bacteria virus Case diagnosis: Case diagnosis History (Age, occupation, residency, previous infection) Complaint Clinical examination Investigations Laboratory investigations Radiology & imaging Special tests Provisional diagnosis Confirm the diagnosis SPECIMEN FOR DIRECT DETECTION OF PARASITES: SPECIMEN FOR DIRECT DETECTION OF PARASITES 1)Examination of stool 2)Examination of other specimen from intestinal tract 3)Urine 4)Sputum, Aspirate, Biopsy materials 5)Blood History: History Leeuwenhoek in 1681 observed earliest protozoa in his own stool .It is named Giardia after proffesor Giard of paris & Lamblia after proffesor Lambl of prague . PowerPoint Presentation: BACK GROUND— Sample collection– Collection should be done in a clean & wide mouth container. Sample should not be contaminated with water & urine,because water contain free living organisms which can be mistaken for human parasite, urine may destroy the motile parasite & organism. Every specimen should be identified with the information of patient’s name& lab no. Sample collection always be done before barium is used for radiological investigation for any cause ,because intestinal Protozoa may be undetectable, for 5-10 days after barium is given.Other substances that also can interfare with the detection of parasite are mineral oil,antibiotic agent,so it’s better that collection should be delayed for 5-10 days. PowerPoint Presentation: NO. OF SAMPLE::To Be Collected It is recommended that normal examination of stool before & after therapy include 3 specimen’s. 2 from normal bowel movements. 1 after the use of cathertic (Mgso4)– proven to have flushing action in GIT obtaining more parasite. If intestinal Amoebiasis 6 sample should be collected90% parasite detect.(Rarely asked) TIMINING OF EXAMINATION:: Liquid specimen( Trophozoite ) with in 30 minutes of passage. Soft( Trophozoite & cyst) with in 1 hour of passage. Formed(Cyst) Any time with in 24 hours ,there is no loss. PowerPoint Presentation: SOME IMPORTANT POINTS: IF well formed stool 20-40 gram is required. watery sample 5-6 T.S.F. is required. Stool should not be collected from lavatory pan. Stool should not be collected from floor. Specimen must reach with in 30 minutes of passing stool because motile stage of parasite may die. Try to keep the sample in warm temperature(Hot room). Drying of specimen should be prevented(Wet chamber). As discussed if 3 stool specimen or a series of 6 stool specimen if asked should be examined every alternate day because intestinal protozoa don’t appear in stool in daily basis. PowerPoint Presentation: PRESERVATIVE: When there is a time lag - from the time of specimen passage until reaching to the lab. Done mainly to preserve the morphology , motility( wetmount ) of the parasite. PVA(Polyvinyl alcohol)-- Trophozoite stage of protozoa . MIF( Merthiolate Iodine Formalin)---> For helminth’s egg, Larvae,Protozoan cyst, oocysts . SAF(sodium acetate acetic acid formalin)---> For helminth’s eggs,larvae , protozoan trophozoite,cyst,oocyst Microsporidia. PowerPoint Presentation: MACROSCOPIC APEARANCE Colour - Dark Stool- Bleeding from GI tract Fresh blood in stoolbleeding from GI tract Consistency- Formed (cyst) soft (trophozoite & cyst) Liquid (trophozoite) Coccidia oocyst & microsporidium spores can be found in any type of stool Surface Presence of adult warms or segments of adult worms may be seen in or on the surface of stool. (Pin warm , Ascaris lumbricoides ) Bottom of container Tape worm proglottid may be found. Other things Mucus, pus , blood, also be present in certain parasitic infection. PowerPoint Presentation: MICROSCOPIC EXAMINATION: Direct wet smear examination(Saline mount) Iodine mount Both are done mainly by direct examination from fresh sample or from the sample obtain from concentration method. Direct saline mount (.85% NaCl ) -- NaCl .85g.+100ml DW is use. Iodine mount D’Antoni’s iodine -KI(1g)+Iodine crystal(1.5 g)+100ml DW Lugol’s iodine - KI(10 g)+Iodine crystal(5g)+ 100 ml DW Procedure 1 drop saline(.85%Nacl) lt side, 1 drop of iodine rt side Take a small amount of stool & emulsify the stool firstly in saline and then in iodine. place 22mm cover slip in each suspension PowerPoint Presentation: FORMS OF PARASITES FOUND IN STOOL Protozoa CESTODE TREMATODE (cyst/ Trophozoite ) ( Eggs) ( Eggs) Diphyllobothrium latum . Schistosoma mansoni . Entamoeba histolytica . Taenia solium . S. japanocium . Giardia lamblia . Taenia saginata . Fasciolopsis busci . Balantidium coli. Hymenolepis nana . Fasciola hepatica. Sarcocystis hominis . Dipylidium caninum . Clonorchis sinensis . Isospora belly . Opisthorcsis spp. Cyclospora cayetanensis . Watsonius watsoni . Cryptosporidium parvum . Gastrodiscoides hominis . . PowerPoint Presentation: Cont………………….. NEMATODE ADULT WARMS CESTODE NEMATODE (Eggs) Taenia solium Ascaris lumbricoides Ascaris lumbricoides . T.saginata Ancylostoma deodenale Trichuris trichura . Diphyllobothrium latum Enterobius vermicularis Necator americanas . Necator americanas Ancylostoma deodenale . Enterobius vermicularis . LARVAE-- Strongyloides stercoralis. PowerPoint Presentation: LIST OF BILE STAIN & NON BILE STAIN EGGS— Bile stained Non bile stained Ascaris lumbricoides Hook worm Trichuris trichiura Enterobius vermicularis . Taenia spp. Hymenolepis nana. Diphyllobothrium latum . S. Mansoni S. Japonicum S. Hamatodium Fasciola hepatica Fasciolepsis nana Bile stained egg of Ascaris Non bile stained egg of E. vermicularis PowerPoint Presentation: Cont…… QUANTIFICATION OF WORM BURDEN….. Direct smear egg coun t - 2mg of stool sample is mixed with normal saline & examine under low power & no of eggs is counted in 2mg of sample, then no of eggs per gram of faeces is estimated. Stoll’s method - 4 gram of sample is mixed with 56ml of N/10 NaOH in a flask to make a uniform suspension .075 ml of suspension is removed by a measuring pipette on a glass slide & see under low power. No of eggs / gram= count x 200 Total egg production=no of eggs / gram x 24 hrs sample. PowerPoint Presentation: METHODS DETECT LESS NO OF PARASITE( Concetration) Eggs ,cyst & trophozoites are often in less in nos that they are difficult to detect in direct smear therefore should perform.. Sedimantation (low specific gravity) Flotation (high specific gravity) Formalin ether sedimantation technique is use. Ethyl acetate use as an extractor of debris & fat from faeces. Formalin is use for fixation of parasite Principle – In sedimantation technique faeces is suspended in a solution of low specific gravity so that eggs & cysts get sedimented at the bottom either spontaneously or centrifugation. PROCEDURE 1-2mg stool+10mlDW,mix well strained through gauzefiltered centrifuged..2000rpm* 2min. discard the supernatant+ 7ml 10% formal saline soln mix well stand for 10 min +3 ml 0f ether shake Cetrifuged 2000*2-3 min allowed stand 5min- Ether, Plug of debris ,clear formalsaline,Sediment All 3 layers discard small drop of sediment is look under direct&iodine mount ADVANTAGE – Helminthes egg& larvae(Ascaris lumbricoides, larvae of hookworm) coccidian oocyst(Isospora belly). Protozoan cyst&trophozoite(Entamoiba coli). LIMITATION – Small parasite difficult to detect. G. lamblia, hook warm eggs may not concentrate . Isospora belly oocyst routinely missed. If gauze > 2 layers cant let pass Cryptosporidium oocyst . PowerPoint Presentation: FLOTATION METHOD…. P rinciple -the faeces is suspended in a sol’n of high specific gravity so that parasitic eggs & cysts float up & concentrated at the surface. Satured salt flotation technique- NaCl (Sp gravity 1.2) 2ml salt soln+1gm faeces emulsified in flat bottomed tube container filled with the salt soln &slide placed on the container that keep in contact with the surface of solnstand 20-30 min-slide is removed reversed it to bring the wet surface on top and examine under microscopy ADVANTAGE- Eggs of round worm except( unfertilised eggs), Hook worm , whipworm. LIMITATION- Not applicable for tape worms,trematodes,Protozoan cyst . zinc sulphate centrifugal flotation technique ZnSo4 ,33%,1.18 SpG ,. 1 gm faeces in 10 ml of water strain through gauze—filtrate 2500RPM*1min discard supernatant Add water,again cetrifuged till supernatant is clear pour off the supernatant + ZnSo4-……2500RPM* 1 min take sample by the wire loop from the surface examine under microscope. ADVANTAGE -Protozoan cyst & eggs of nematodes &small tape warms. LIMITATION - Not detect unfertilised round worm eggs ,nematode larvae,large tapewarms . PowerPoint Presentation: PERMANANT STAINING … …WHY The smallest parasite are easily identified Morphology is beautifully appreciated. Use for detection and correct identification of parasite. Help to maintain of permanent record of the parasite identified of our interest. Used for consultation for unusual morphological character. GENARAL PRINCIPLE - purpose is to provide contrasting colours for the backgroud debris & the parasite.It allows examination & recognition of detailed parasitic morphology under oil immersion(100X) STOOL EXAMINATION Permanent Stained smears: STOOL EXAMINATION Permanent Stained smears Iron haematoxylin stain Trichrome stain Modified Ziehl Neelsen stain (Crypto.) PowerPoint Presentation: PREPARATION…. For fresh sample- prepare slide with applicator sticks place in Schaudinn’s fixative* 30 min placed in 70% alcohol to remove the excess fixative Lequid sample add with 3-4 drops PVA Allow to dry in 37*C or in incubatotor . For preserved sample- In PVA- put PVA + stool mixture in a paper towel allow to absorb & than from this material make slide & allow them to dry At 37*c. Dry slide than place in iodine alcohol. In SAF - first filtered& than centrifugation is done. After that smear is made from the sediment . Than 1 drop of sediment + 1 drop mayer”s albumin is added air dry at room temperature. And staining is made. In MIF- MIF+Stool sample allow to sattel for 1hour . Take 1 drop of sedimenton the Mayer”s albumin coated slide. Keep it for dry& examine. Mayer’s albumin- it’s a fixative. Glycerin +fresh egg white. Stored at 4*C. . PowerPoint Presentation: PROCEDURE; Prepare slide with Schaudin’s fixative-put in 70% ETHANOL(5MIN)-put 70% ethanol+iodine (1min)-place in 70 % ethanol for 5min & 3 min (different container) - place in Trichome stain (10min)-place in 90% ethanol +acetic acd (1-3s)-dip the slide 2-3 times in 100% ethanol-place it in Xyline - allow the smear to dry and examine in 100X. If sample comes in preservative other than PVA iodine alcohol step is not requred . How to identify-- Background is usally stained green. Protozoan trophozoite blue green Cyst appear slightly purple, Chromatid bodie’s appear red. RESULT - Trophozoite of E. histolytica, trophozoite of Giardia,Other trophozoite & cyst, Others str eg . Macrophages , PMN’s, RBC also can identified. LIMITATION: Not recommended for helminth’s eggs& larvae because it appear too dark. Cryptosporidium parvum & Cyclospora oocyst will generally not be recognised . PowerPoint Presentation: Trophozoite of Entamoeba histolytica Trophozoite & cyst of Giardia lamblia PowerPoint Presentation: IRON HEMATOXYLIN stain Is one of the number of stains that allows one to make permanent stained slide for detecting &quantating parasitic organism. Morphology can be well appreciated Two method – Spencer –Monroe Method & Topkin’s Miller method Costituents- Hematoxylin(10gm)+Ethanol (100ml) Ferrus & ferric ammonium sulphate(10gm)+HCl(10ml)+DW(1000ml) PowerPoint Presentation: PROCEDURE Spencer-monroe method Prepare the slide for staining- put in70% ethanol--*5 min-place iodine+alcohol soln *5min- place again in 70% ethanol *5min- wash slide- place in iron hematoxylin* 4-5min-wash again –place the slide in 70%,95%,100% ethanol *5min each- - see under oil immersion. For non mercury based fixative iodine alcohol step is not done. HOW TO IDENTIFY - Cytoplasm of protozoan trophozoite appear blue grey,Cyst looks darker,Nuclei &Charcot layden crystal looks blue, Background stains pale grey. Topkin’s Miller method- A method that destained the protozoa by the use of phosphotungstic acid. After the iodine alcohol step &after washing properly phosphotungstic acid is added. RESULT - Trophozoite of Entamoibacoli ,dientamoeba fragilis .Yeast cell pseudohyphae seen WBC,Macrophages’ Rbc can be identified. Modified Iron hematoxylin stain - it screens Acid fast organism along with parasite. Corbolfuschin is incorporated in this method. Cryptosporidium parvum, Cyclospora cayatanensis, isospora belly can idetified. PowerPoint Presentation: PROCEDURE Spencer-monroe method Prepare the slide for staining- put in70% ethanol--*5 min-place iodine+alcohol soln *5min- place again in 70% ethanol *5min- wash slide- place in iron hematoxylin* 4-5min-wash again –place the slide in 70%,95%,100% ethanol *5min each- - see under oil immersion. For non mercury based fixative iodine alcohol step is not done. HOW TO IDENTIFY - Cytoplasm of protozoan trophozoite appear blue grey,Cyst looks darker,Nuclei &Charcot layden crystal looks blue, Background stains pale grey. Topkin’s Miller method- A method that destained the protozoa by the use of phosphotungstic acid. After the iodine alcohol step &after washing properly phosphotungstic acid is added. RESULT - Trophozoite of Entamoibacoli ,dientamoeba fragilis .Yeast cell pseudohyphae seen WBC,Macrophages’ Rbc can be identified. Modified Iron hematoxylin stain - it screens Acid fast organism along with parasite. Corbolfuschin is incorporated in this method. Cryptosporidium parvum, Cyclospora cayatanensis, isospora belly can idetified. PowerPoint Presentation: Egg of E vermicularis iron hematoxylin eosine stain PowerPoint Presentation: SPECIAL STAIN: Modified Kinyoun’s Acid fast stain(cold method) It’s a specialised stain to identify cryptosporidium parvum , isospora belley(acid fast) cause of severe diarrhoea in immunocompromised patient Also can identify Microsporidial spore which are also acid fast. Unlike ZN stain this staining procedure not required heating of reagent Kinyoun’s carbol fuschin is used.(4gram basic fuschin in 20ml of 95% ethanol. Decolourizing is 1% sulphuric acid Counter stain is methyline blue. In this the back ground will stain blue. The oocyst of cryptosporidium parvum, Isospora belley,stain pink to red to deep purpel The stain intensity will depend on thickness of smear. LIMITATION Small no of oocyst can be missed. PowerPoint Presentation: MODIFIED Zeihl Neelson Stain Stain to identify cryptosporidium parvum , isospora belley (acid fast) cause of severe diarrhoea in immunocompromised & immunocompetant patient . oocyst is very difficult to identify by this method In this carbol fuschin is heated & milder decolouriser is used allow the organism to retain the pink red colour . Constituent Carbol fuschin is used .3gm in 10 ml of 95% ethanol. Sulphuric acid used as a decolouriser (5%) counter stain is Metheline blue. Carbol fuschin is heated by a bunsen burner . In this the back ground will stain blue. The oocyst of cryptosporidium parvum , Isospora belley,stain pink to red to deep purpel The stain intensity will depend on thickness of smear. LIMITATION Small no of oocyst can be missed. Multiple specimen is requre to examine. PowerPoint Presentation: Oocyst of cyclospora modified ZN staining PowerPoint Presentation: MODIFICATION OF TRICHOME STAIN. WEBER- GREEN MODIFIED TRICHOME STAIN Useful of diagnosis of intestinal Microsporiodiosis ( Encephalitozoon intestinalis,Enterocytozoon bieneusi ) Dye content of chromotrope 2R is greter (6.0 gram) than routinely used fact that stain penetration of the microsporidial spore isdifficult . Background stain green in colour . S pores look ovoid,&refractile,spore wall looks bright pinkish red in colour . RYAN-BLUE MODIFIED TRICHOME STAIN Attempt to improve contrast colour of spore& back ground. Aniline blue is used. Background stain Blue in colour . Spores look ovoid,&refractile,spore wall looks bright pinkish red in colour . Modified trichome staining (KOKOSKIN-HOT METHOD) Useful for microsprodia,stain done in 50* c, time 10 min. PowerPoint Presentation: spores of Enterocystozoon bienusi in modified trichome staining. PowerPoint Presentation: ACID FAST TRICHOME STAINING useful for Microsporidia & Cryptosporidium. It is important because dual infection is common in AIDS patient. Here Trichome stain & carbol fuschin solution both are used. Background stain Blue in colour. Spores look ovoid,&refractile,spore wall looks bright pinkish red in colour. Spore being clear or perhaps shows a diagonal stripe which represent the polar tube. LIMITATION The range of stain intensity & small size of spore cause difficulty to identify. PowerPoint Presentation: Oocyst of cyclospora in acid fast trichome staining. PowerPoint Presentation: STOOL CULTURE FOR PARASITE…. Culture not employed routinely For the cultivation of Entamoeba histolytica . Balamuth’s aques egg yolk infusion medium-- commonly used for cultivation of amoebae,& other intestinal parasite.This are mainly polyxenic midia . in all this media it is necessary to include such as enteric bacteria or the flagelates (T. cruzi ), aswell as starch or rice flour. composition – phosphate buffer,whole liver concentrate soln , egg yolk soln. Boecand drbohlav’s locke egg serum( diphasic medium) - Eggs slant base with an isotonic overlay. Diamond(1965) - First describe axenic cultivation for Entamoeba histolytica. For the cultivation of larval stage of Nematode — Harado -Mori filter paper & charcoal culture is use. USES Commonly used for cultivation of amoebae,& other intestinal parasite Culture increases the no of identifiable cases. Morphological studies can be done. Useful to see the efficacy of antiprotozoal drugs. Preparation of protozoal antigens for diagnostic use.