DIAGNOSTIC PARASITOLOGY THURSDAY 4 -6 PM SPRING 2007

DIAGNOSTIC PARASITOLOGY THURSDAY 4 -6 PM SPRING 2007 1

WHY PERFORM THIS TYPE OF TESTING § § § 1. 2. 3. 4. 5. Travel Control Issues Epidemiologic Considerations Compromised Patients Approach to Therapy 2

WHO SHOULD PERFORM TESTING § 1. Laboratory Personnel § a. CLIA 88 -High- complexity procedures § b. Few procedures are automated § c. Organism identification relies on § morphologic characteristics § 2. Nonlaboratory Personnel § a. CLIA 88 -Waived tests § b. Wet mount examinations § c. No over-the-counter testing methods 3

Where should testing be performed § 1. Inpatient Setting § a. Hospital or offsite location § b. Stat procedures: § Blood smears for malaria § CSF for free-living amebae § 2. Outpatient or Referral Setting § a. Routine testing § b. Batch tested 4

Where should testing be performed § 3. Decentralized, Physician Office Labs, § Over-the-Counter (Home Care) § a. Usually not considered appropriate § for diagnostic parasitology tesing § 5

What Factors Should Precipitate Testing § § § § 1. 2. 3. 4. 5. 6. 7. 8. Travel History Immune Status of the Patient Clinical Symptoms Documented Previous Infection Contact with Infected Individuals Screening – outbreak situations Occupational Screening – food handlers Therapeutic Failure – retested after therapy 6

What Testing Should Be Performed § 1. Screening Test § a. Wide range in both sensitivity and § specificity § b. O&P Exam § c. Monoclonal antibody based test § d. Tests on “on request” 7

What Testing Should Be Performed § 2. Routine Methods § a. May be screening methods: O&P § b. Blood smears for malaria § c. Pinworm tapes § d. Occult blood tests § e. Specimens from other body sites § (urine, sputum, duodenal aspirates) 8

What Testing Should Be Performed § 3. Special Testing § a. Parasite culture § b. Serologic Testing for parasitic § disease § 4. Other (Nonmicrobiological) Testing § a. Routine urinalysis § b. Hematology – CBC § c. Chemistry profiles 9

PARASITE CLASSIFICATION § SIX MAJOR DIVISIONS: § § § 1. PROTOZOA (INTESTINAL) A. AMEBAE 1. SINGLE CELLED 2. PSEUDOPODS (MOTILITY) 3. TROPHOZOITE STAGE 4. CYST STAGE 5. FECAL-ORAL TRANSMISSION 6. MOUTH-MOUTH (ENTAMOEBA GINGIVALIS) 7. ENTAMOEBA HISTOLYTICA – SIGNIFICANT ORGANISM 10

PARASITE CLASSIFICATION § PROTOZOA § § § § § 2. FLAGELLATES A. FLAGELLA B. FECAL-ORAL TRANSMISSION C. TROPHOZOITE & CYST STAGES D. DIENTAMOEBA FRAGILIS & TRICHOMONAS (TROPHOZOITE) E. GIARDIA LAMBLIA MOST COMMON PATHOGEN 11

PARASITE CLASSIFICATION § PROTOZOA § § § 3. CILIATES A. SINGLE-CELLED B. MOVE BY CILIA C. FECAL-ORAL TRANSMISSION D. BALANTIDIUM COLI – ONLY HUMAN PATHOGEN E. TROPHOZOITE & CYST STAGES 12

PARASITE CLASSIFICATION § PROTOZOA 4. COCCIDIA A. INGESTION OF MEAT B. FECAL-ORAL TRANSMISSION VIA CONTAMINATED FOOD AND/OR WATER C. INFECTIVE STAGE – OOCYST D. CRYPTOSPORIDIUM, CYCLOSPORA, ISOPORA, SARCOCYSTIS 13

PARASITE CLASSIFICATION § PROTOZOA § § § § 5. MICROSPORIDIA A. RANGE 1 TO 2. 5 UM B. INFECTIVE FORM – SPORE C. MODIFIED TRICHROME STAINS D. INFECTIONS THRU INGESTION INHALATION, OR DIRECT INOCULATION FROM ENVIRONMENT 14

PARASITE CLASSIFICATION § PROTOZOA (OTHER BODY SITES) § § § § 1. AMEBAE A. PATHOGENIC, FREE LIVING ORGANISMS ASSOCIATED WITH WARM, FRESHWATER ENVIRONMENTS. B. FOUND IN CNS, EYES AND OTHER BODY SITES. C. NAEGLERIA, ACANTHAMOEBA, BALAMUTHIA § 15

PARASITE CLASSIFICATION § PROTOZOA (OTHER BODY SITES) § § § § 2. FLAGELLATES A. TRICHOMONAS VAGINALIS IN THE GENITOURINARY SYSTEM, ACQUIRED BY SEXUAL TRANSMISSION B. TRICHOMONAS TENAX FOUND IN MOUTH, NONPATHOGENIC 16

PARASITE CLASSIFICATION § 3. PROTOZOA (OTHER BODY SITES) § § § § 1. COCCIDIA A. IMPORTANT IN THE COMPROMISED PATIENT B. DISSEMINATE FROM THE INTESTINAL TRACT TO OTHER BODY SITES C. CRYPTOSPORIDIUM 17

PARASITE CLASSIFICATION § PROTOZOA (OTHER BODY SITES) § § § 4. MICROSPORIDIA A. MICROSPORIDIA (1 TO 2. 5 UM) B. INTESTINE TO OTHER BODY SITES C. MODIFIED TRICHOME STAINS TO DETECT 18

PARASITE CLASSIFICATION § PROTOZOA (BLOOD AND TISSUE) § § § 1. SPOROZA A. ALL ARE ARTHROPOD BORNE B. PLASMODIUM (MOSQUITOES) C. BABESIA (TICK BORNE) D. EXAM BOTH THICK AND THIN BLOOD FILMS § 19

PARASITE CLASSIFICATION § FLAGELLATES § § § § 1. LEISHMANIAE A. RECOVERY AND ID RELATED TO BODY SITE. B. AMASTIGOTES LIMITED TO THE SITE OF THE LESION C. CUTANEOUS, MUCOCUTANEOUS, VISCERAL D. SAND FLY § 20

PARASITE CLASSIFICATION § FLAGELLATES § § § § 2. TRYPANOSOMES A. ID TO THE SPECIES LEVEL BASED ON GEOGRAPHIC EXPOSURE HISTORY AND CLINICAL SYMPTOMS B. TRYPOMASTIGOTE FORM C. AFRICAN SLEEPING SICKNESS 21

PARASITE CLASSIFICATION § NEMATODES (INTESTINAL) § § § 1. ROUNDWORMS A. ELONGATE-CYLINDRICAL B. ACQUIRED BY INGESTION OF EGGS OR PENETRATION OF THE SKIN BY LARVAL FORMS FROM THE SOIL § 22

PARASITE CLASSIFICATION § NEMATODES (TISSUE) § § § § 1. ROUNDWORMS A. RARELY SEEN IN THE U. S. B. TRICHINELLA SPIRALIS C. ACQUIRED BY INGESTION OF OF INFECTIVE RAW OR POORLY COOK MEAT (PORK, BEAR, WALRUS) 23

PARASITE CLASSIFICATION § NEMATODES (BLOOD AND TISSUE) § § § 1. FILARIAL WORMS A. ARTHROPOD BORN B. ID BY LARVAL WORMS (MICROFILARIAE) IN BLOOD, OTHER BODY FLUIDS, OR SKIN 24

PARASITE CLASSIFICATION § CESTODES (INTESTINAL) § § § § § 1. TAPEWORMS A. INGESTION OF LARVAL FORMS IN POORLY COOKED OR RAW MEATS OR FRESHWATER FISH, INFECTED BEETLES B. TAPEWORM CONSISTS OF A CHAIN OF EGG-PRODUCING UNITS CALLED PROGLOTTIDS 25

PARASITE CLASSIFICATION § CESTODES (TISSUE) § § § 1. TAPEWORMS A. TISSUE INFECTION B. TAENIA SOLIUM C. ECHINOCOCCUS GRANULOSUS 26

PARASITE CLASSIFICATION § TREMATODES (INTESTINAL) § 1. FLATWORMS § A. FLAT, WITH ORAL AND VENTRAL § SUCKERS § B. REQUIRE A FRESHWATER § SNAIL TO SERVE AS AN § INTERMEDIATE HOST § C. INFECTIONS ARE FOODBORNE § (FRESHWATER FISH, MOLLUCKS, § FRESHWATER PLANTS) 27

PARASITE CLASSIFICATION § TREMATODES (LIVER AND LUNG) § 1. FLATWORMS § A. REQUIRE A FRESHWATER SNAIL § TO SERVE AS INTERMEDIATE § HOST § B. INFECTIONS ARE FOODBORNE § (FRESHWATER FISH, CRAYFISH, § CRABS, OR PLANTS) § C. CLONORCHIS, PARAGONIMUS EXAMPLES § 28

PARASITE CLASSIFICATION § TREMATODES (BLOOD) § 1. FLATWORMS § A. SEXES ARE SEPARATE § B. INFECTION IS ACQUIRED BY § SKIN PENETRATION BY THE § CERCARIAL FORMS THAT ARE § RELEASED FROM FRESHWATER § SNAILS. § C. WORMS RESIDE IN THE BLOOD VESSELS § OVER THE SMALL & LARGE INTESTINE, OR § THE BLADDER. § D. SCHISTOSOMES 29

PARASITE CLASSIFICATION § PENTASTOMIDS § 1. TONGUE WORMS 30

PARASITE CLASSIFICATION § ACANTHOCEPHALA § 1. THORNY-HEAD WORMS 31

COLLECTION OPTIONS 32

1. SAFETY § A. HANDLE ALL FRESH SPECIMENS § CAREFULLY § B. USE UNIVERSAL PRECAUTIONS: § GLOVES, PROPER CONTAINERS, § SAFETY CABINETS, DISCARD § POLICIES 33

2. FRESH STOOL SPECIMENS § A. INTERFERING SUBSTANCES § SHOULD BE AVOIDED WHEN STOOL § SPECIMENS ARE COLLECTED § § a. Collect before barium is used b. Mineral oil, bismuth, antibotics, antimalarial agents. c. Collection delayed 5 to 10 days 34

COLLECTION METHOD § 1. CLEAN CONTAINERS OR FIXATIVE § VIALS. § 2. CONTAMINATION WITH URINE OR § WATER SHOULD BE AVOIDED. § 3. LABELED WITH PATIENT NAME, TIME § AND DATE OF COLLECTION 35

NUMBER OF SPECIMENS § 1. NORMAL EXAMINATION FOR STOOL § PARASITES BEFORE THERAPY § INCLUDE 3 SPECIMENS. § 2. THREE SPECIMENS POOLED – § CONTROVERSIAL § 3. POSTTHERAPY SPECIMENS NOT § COLLECTED UNLESS PATIENT BECOMES § SYMPTOMATIC AGAIN. 36

COLLECTION TIMES § 1. THREE SPECIMENS COLLECTED ON § SEPARATE DAYS. § 2. WITHIN NO MORE THAN 10 DAYS. § 3. EXCEPTION: PATIENT WITH SEVERE, § WATERY DIARRHEA, ORGANISMS § MIGHT BE MISSED BECAUSE OF § TREMEDOUS DILUTION FACTOR RELATED § TO FLUID LOSS. 37

SPECIMEN TYPE, STABILITY, AND PRESERVATION § 1. FRESH SPECIMENS MANDATORY § FOR RECOVERY OF MOTILE § TROPHOZOITES. § 2. LIQUID STOOL SHOULD BE § EXAMINED OR PRESERVED WITHIN § 30 MINUTES OF PASSAGE (TROPS) § 3. SOFT STOOL EXAM OR PRESERVED § WITHIN 1 HOUR OF PASSAGE (TROPS & § CYSTS) DIENTAMOEBA FRAGILIS TROPHOZOITES § 4. FORMED STOOL SHOULD BE EXAMINED OR PRESERVED § WITHIN 24 HOUR OF PASSAGE. 38

PRESERVATION OF STOOL SPECIMENS § § 1. DELAY IN RECEIVING STOOL SPECIMENS, PLACE IN FIXATIVES § § 2. FORMALIN, SODIUM ACETATEACETIC ACID-FORMALIN (SAF), SCHAUDINN’S FLUID, AND POLYVINYL ALCOHOL (PVA) § § 3. KEEP IN MIND, A PERMANENT STAINED SMEAR IS MANDATORY. § § 4. MAY WANT TO PERFORM SCREENING METHODS: FLUORESCENT-ANTIBODY (FA) OR ENZYME IMMUNOASSAY (EIA). MAKE SURE THE FIXATIVE IS COMPATIBLE WITH THE KIT YOU ARE USING. § § 5. DISPOSAL REGULATIONS FOR COMPOUNDS CONTAINING MERCURY ARE BECOMING MORE STRICT 39

FOMALIN FIXATIVE § FORMALIN § § § § § 1. ALL-PURPOSE FIXATIVE FOR HELMINTH EGGS AND LARVAE, AND PROTOZOAN CYSTS. § 3. 2. 5% AND 10% CONCENTRATIONS A. 5% FOR PRESERVATION OF PROTOZOAN CYSTS B. 10% FOR PRESERVATION OF HELMINTH EGGS AND LARVAE 40

FORMALIN § PROS § CONS § GOOD OVERALL FIXATIVE FOR STOOL CONCENTRATE § DOES NOT PRESERVE TROPHOZOITES WELL § EASY TO PREPARE, LONG SHELF LIFE § DOES NOT ADEQUATELY PRESERVE ORGANISM MORPHOLOGY FOR A GOOD § PERMANENT STAINED SMEAR § CONCENTRATED SEDIMENT CAN BE USED WITH THE NEW IMMUNOASSAY METHODS 41

SODIUM ACETATE-ACETIC ACIDFORMALIN (SAF) § PROS § CONS § CAN BE USED FOR CONCENTRATION AND PERMANENT STAINED SMEARS § POOR ADHESIVE PROPERTIES, ALBUMIN-COATED SLIDES RECOMMENDED § CONTAINS NO MERCURY COMPOUNDS § EASY TO PREPARE, LONG SHELF LIFE § PROTOZOAN MORPHOLOGY BETTER IF IRON HEMATOXYLIN STAINS USED FOR PERMANENT STAINED SMEARS (TRICHROME NOT AS GOOD. § CONCENTRATED SEDIMENT CAN BE USED WITH THE NEW IMMUNOASSAY METHODS § MAY BE MORE DIFFICULT TO USE THAN PVA FIXATIVE, HOWEVER, THIS IS REALLY NOT A LIMITING FACTOR. 42

SCHAUDINN’S FLUID § PRO § CONS § FIXATIVE FOR SMEARS PREPARED FROM FRESH FECAL SPECIMENS OR SAMPLES FROM THE INTESTINAL MUCOSAL SURFACES § NOT GENERALLY RECOMMENDED FOR USE IN CONCENTRATION PROCEDURES § PROVIDES EXCELLENT PRESERVATION OF PROTOZOAN TROPHOZOITES AND CYSTS § CONTAINS MERCURIC CHLORIDE, WHICH IS A DISPOSAL PROBLEM § POOR ADHESIVE QUALITIES WITH LIQUID OR MUCOID SPECIMENS 43

POLYVINYL ALCOHOL (PVA) § 1. PVA IS A PLASTIC RESIN THAT IS § INCORPRORATED INTO § SCHAUDINN’S FIXATIVE. § 2. PVA POWDER SERVES AS AN § ADHESIVE FOR THE STOOL § MATERIAL, WHEN THE STOOL-PVA § MIXTURE IS SPREAD ONTO THE GLASS § SLIDE. § 3. FIXATION IS STILL ACCOMPLISHED BY § SCHAUDINN’S FLUID ITSELF. 44

POLYVINYL ALCOHOL (PVA) § § PRO CAN PREPARE PERMANENT STAINED SMEARS AND PERFORM CONCENTRATION TECHNIQUES (LESS COMMON) § CONS § § PROVIDES EXCELLENT PRESERVATION FO PROTOZOAN TROPHOZOITES AND CYSTS § TRICHURIS EGGS AND GIARDIA CYSTS ARE NOT CONCENTRATED AS EASILY. STRONGYLOIDES LARVAL MORPHOLOGY IS POOR. ISOSPORA OOCYSTS MAY NOT BE VISIBLE IN PVA-PRESERVED MATERIAL. § LONG SHELF LIFE IN TIGHTLY SEALED CONTAINERS AT ROOM TEMPERATURE. § ALLOWS SPECIMENS TO BE SHIPPED TO LABORATORY FOR SUBSEQUENT EXAMINATION. § CONTAINS MERCURY COMPOUNDS (SCHAUDINN’S FLUID) § MAY TURN WHITE AND GELATINOUS WHEN IT BEGINS TO DEHYDRATE OR WHEN REFRIGERATED. § DIFFICULT TO PREPARE IN THE LAB. § SPECIMENS CONTAINING PVA CANNOT BE USED WITH THE IMMUNOASSAY DETECTION KITS. 45

MODIFIED PVA § PRO § CONS § CAN BE USED FOR PERMANENT STAINED SMEARS AND CONCENTRATION TECHNIQUES. § OVERALL PROTOZOAN MORPHOLOGY OF TROPS AND CYSTS IS POOR WHEN PRESERVED IN THE COPPER SULFATE-BASED FIXATIVE. § MANY WORKERS PERFER THE ZINC SUBSTITUTES OVER THOSE PREPARED WITH COPPER SULFATE. § ZINC-BASED FIXATIVES APPEAR TO BE SOME OF THE BETTER ALTERNTIVE. § STAINING CHARACTERISTICS OF PROTOZOA NOT CONSISTENT. § SMALL PROTOZOAN CYSTS (SUCH AS ENDOLIMAX NANA) IDENTIFICATION MAY BE DIFFICULT. § DOES NOT CONTAIN MERCURY COMPOUNDS. 46

SINGLE-VIAL COLLECTION SYSTEMS § PRO § CONS § CAN PREPARE PERMANENT STAINED SMEARS AND PERFORM CONCENTRATION TECHNIQUES. § OVERALL PROTOZOAN MORPHOLOGY OF TROPHOZOITES AND CYSTS IS NOT AS GOOD AS THAT WITH MERCURIC CHLORIDE-BASED FIXATIVES. § CAN PERFORM IMMUNOASSAY PROCEDURES. § DO NOT CONTAIN MERCURY COMPOUNDS. § STAINING CHARACTERISTICS OF PROTOZOA NOT CONSISTENT. § UNLESS ORGANISM NUMBERS ARE SMALL, ACCEPTABLE ORGANISM RECOVERY AND ID IS POSSIBLE. 47

PROCEDURE NOTES § § § 1. MOST OF THE COMMERCIALLY AVAILABLE KITS HAVE A “FILL TO” LINE ON THE VIAL LABEL. § § § 2. TW 0 VIAL SYSTEM (ONE OF 5 OR 10% FORMALIN [CONCENTRATION] AND ONE VIAL OF PVA [PERMANENT STAINED SMEAR] HAS BEEN THE GOOD STANDARD. § § § 3. CHANGE IN SELECTION OF FIXATIVES: A. DISPOSAL OF MERCURY-BASED FIXATIVES B. COST OF TWO-VIAL SYSTEM COMPARED WITH ONE VIAL. C. SELECTION OF SPECIFIC STAINS TO USE WITH SPECIFIC FIXATIVES. D. CAN IMMUNOASSAY KITS BE USED WITH A PARTICULAR FIXATIVE. 48

PROCEDURE LIMITATIONS § 1. ADEQUATE FIXATION: § A. MEETING RECOMMENDED TIME LIMITS FOR LAG TIME BETWEEN PASSAGE OF THE SPECIMEN AND FIXATION. B. CORRECT RATIO OF FIXATIVE TO SPECIMEN. C. THOROUGH MIXING OF THE FIXATIVE & SPECIMEN. D. APPROPRIATE STAIN USED WITH EACH FIXATIVE. 49

COLLECTION OF BLOOD PARASITES: PLASMODIUM, BABESIA, TRYPANOSOMA, LEISHMANIA, AND MICROFILARIAE. 1. 2. 3. REQUEST FRESH BLOOD (EDTA ANTICOAGULANT) SMEARS PREPARED WITHIN 1 HOUR AFTER THE SPECIMEN IS DRAWN. ONE NEGATIVE SPECIMEN DOES NOT RULE OUT THE POSSIBILITY OF A PARASITIC INFECTION. 50