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DIAGNOSTIC PARASITOLOGY

DIAGNOSTIC PARASITOLOGY. THURSDAY 4-6 PM SPRING 2007. WHY PERFORM THIS TYPE OF TESTING. 1. Travel 2. Control Issues 3. Epidemiologic Considerations 4. Compromised Patients 5. Approach to Therapy. WHO SHOULD PERFORM TESTING. 1. Laboratory Personnel

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DIAGNOSTIC PARASITOLOGY

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  1. DIAGNOSTIC PARASITOLOGY THURSDAY 4-6 PM SPRING 2007

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

  3. 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

  4. 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

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

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

  7. 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”

  8. 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)

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  21. 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

  22. 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

  23. 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)

  24. 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

  25. 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

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

  27. 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)

  28. 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

  29. 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

  30. PARASITE CLASSIFICATION • PENTASTOMIDS • 1. TONGUE WORMS

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

  32. COLLECTION OPTIONS

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

  34. 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

  35. 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

  36. 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.

  37. 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.

  38. 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.

  39. PRESERVATION OF STOOL SPECIMENS • 1. DELAY IN RECEIVING STOOL • SPECIMENS, PLACE IN FIXATIVES • 2. FORMALIN, SODIUM ACETATE- • ACETIC 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

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

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

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

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

  44. 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.

  45. PRO CAN PREPARE PERMANENT STAINED SMEARS AND PERFORM CONCENTRATION TECHNIQUES (LESS COMMON) PROVIDES EXCELLENT PRESERVATION FO PROTOZOAN TROPHOZOITES AND CYSTS LONG SHELF LIFE IN TIGHTLY SEALED CONTAINERS AT ROOM TEMPERATURE. ALLOWS SPECIMENS TO BE SHIPPED TO LABORATORY FOR SUBSEQUENT EXAMINATION. CONS 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. 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. POLYVINYL ALCOHOL (PVA)

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

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

  48. PROCEDURE NOTES • 1. MOST OF THE COMMERCIALLY • AVAILABLE KITS HAVE A “FILL TO” • LINE ON THE VIAL LABEL. • 2. TW0 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.

  49. 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.

  50. COLLECTION OF BLOOD PARASITES: PLASMODIUM, BABESIA, TRYPANOSOMA, LEISHMANIA, AND MICROFILARIAE. • 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.

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