3WORKSHOP OBJECTIVESDiscuss stool parasite orders; educational initiatives required for clients in relation to patient care and test ordersReview STATS vs. routines testing vs. send outsDiscuss ordering : O&P vs IAs vs Special StainsDiscuss collection and Universal FixativesDiscuss laboratory reporting, importance of report commentsDiscuss various protozoa, helminths, blood parasites: identification, tests, reporting, etc.
4PARASITOLOGY TESTS THAT EVERY LAB SHOULD BE ABLE TO PERFORM True STATS:Thick and thin blood film (preparation, exam)CSF exam for free-living amebae (wet, stain)(Naegleria, Acanthamoeba, Balamuthia, Sappinia)Big 3: O&P, Immunoassays, Special StainsPossible Send outs:Specimens for culture and serologies; majority of requests performed in large reference centers
6PARASITOLOGY TESTING: WHAT YOU NEED TO KNOW Minimum: Specimen acceptability, collection, processing, test method, reporting formatRelevant Information: Collection/test, specimen acceptability, method, result (make sense?), report comments, method limitations, clinical disease, disease mimics, geographic endemic areas, optimal methods, correlation of life cycles and diagnostic findingsRisk Management: STAT testing (CSF, brain tissue, blood films)
7STOOL SPECIMENSOrdering, Specimen Options: Method options, number and type of specimenCollection Options: Fresh or fixed (Universal Fixative), immunoassays, method pros and consPotential Problems: Poor specimen; inadequate collection; inappropriate processing; wrong test selection; failure to recognize potential problems (collection, processing, testing, and/or reporting)Physician/Laboratory Problems: Lack of complete ordering and specimen collection guidelines
8STOOL PRESERVATIVES and TESTING OPTIONS: O&P O&P Examination (Fresh or Preserved Stool Specimens)Direct Wet Smear (Motility): NO if in preservativeConcentration: YES, performed for all O&PsPermanent Stained Smear: Yes, performed for all O&PsIf O&P ordered, concentration AND permanent stained smear must be performed (CAP, NCCLS/CLSI)Fecal Immunoassays (Fresh, Frozen, Formalin)EIA: Performed on unspun specimensFA: Concentrated specimen (500 Xg for 10 min)Cartridge Systems: Unspun specimens
9STOOL FIXATIVES Formalin: concentration, immunoassays Fixative with PVA: Polyvinyl Alcohol (glue)Mercury-based fixatives: phased out for environmental restrictionsCopper or Zinc-based fixatives: zinc-based best morphology – being routinely used, including PTUniversal Fixatives: (1) Concentration, (2) permanent stained smear, (3) special stains for coccidia/microsporidia, (4) fecal immunoassays, (5) PCR (TOTAL-FIX)
10UNIVERSAL FIXATIVESOPTIONS: (1) Concentration, (2) permanent stained smear, (3) special stains for coccidia/microsporidia, (4) fecal immunoassays, (5) molecular testing (PCR)SAF: iron-hematoxylin stain (a bit more difficult/picky);albumin used as glue; no PVA, BUT CONTAINS FORMALINTOTAL-FIX: NO PVA; NO MERCURY, NO FORMALINCritical to make sure stool smears are TOTALLY DRYDrying in 37ºC incubator (on a tray); 30 min to 1 hIF THE SMEARS ARE TOTALLY DRY, THE STOOL MATERIAL WILL ADHERE TO THE SMEAR WITHOUT USING PVA OR ALBUMIN
11STOOL COLLECTION 2 SPECIMENS (O&P) 2 Specimens (Fresh or Preserved Stool Specimens)Every other day or every day, but not all in same day (within 10 days)If no diarrhea, 1 from normal movement, 1 using cathartic (UNCOMMON)ROUTINE: 2 stools collected in preservative (complete O&P)Data: Cartwright (J. Clin. Microbiol. 37: , 1999)First stool: 75.9% detectionSecond stool: 92% detectionThird stool: May not be cost-effectiveData: Hanson and Cartwright (J. Clin. Microbiol. 39:474-8, 1993)Two specimens by either EIA or O&P revealed >90% detectionThird Stool: May not be cost-effective
12STOOL COLLECTION 3 SPECIMENS (O&P) 3 Specimens (Fresh or Preserved Stool Specimens)Every other day or every day, but not all in same day (within 10 days)If no diarrhea, 2 from normal movements, 1 using catharticROUTINE: 3 stools collected in preservative (complete O&P)Data: Nazar (Br. J. Clin. Prac. 47:76-8, 1993)First stool: 58.3% of population testedSecond stool: Added 20.6%Third stool: Added another 21.1%Data: Hiatt, et al. (Am. J. Trop. Med. Hyg. 53:36-9, 1995)Yield increased 22.7%: Entamoeba histolyticaYield increased 11.3%: Giardia lambliaYield increased 31.1%: Dientamoeba fragilis
13STOOL COLLECTION SUMMARY (O&P) Fresh or Preserved Stool SpecimensPersonal preferenceConsider ALL orders (O&P, IA, special stains)RECOMMENDATION: Fixatives (lag time problems)Number of specimens to CollectTwo specimens is acceptable; three is betterRECOMMENDATION: Three, but two acceptableTestingO&P, Immunoassays, Special StainingSeparate, orderable, billable tests (CPT codes)
15Fresh or Preserved Stool Specimens Personal preferenceConsider ALL testing being ordered (O&P, IA, special stains)RECOMMENDATION: Fixatives eliminate lag time problemsNumber of specimens to CollectTwo specimens is acceptableThree is betterRECOMMENDATION: Three, but two acceptableTestingO&P, Immunoassays, Special Testing11IV. CyclosporaAutofluorescenceSpecial stainsSTOOL ORDERRECOMMENDATIONS
21ENZYME IMMUNOASSAY (EIA) Antigen Detection (Single or batch testing)Limited to certain organisms [Cryptosporidium, Giardia, (Entamoeba histolytica/E. dispar group), E. histolytica] (Dientamoeba, Blastocystis under development)All kits have comparable sensitivity, specificityColor judgment - interpretation if manually readFalse negatives may result due to low organism numbers (asymptomatic carriers)
22ENZYME IMMUNOASSAY – TIPS Use Unspun Specimen - Fluid If vial is mixed, let settle for 5+ min before testingThoroughly rinse wells, don’t cut any rinse stepsEach well MUST receive total number of rinsesSquirt buffer directly into wells; squeeze bottleWhen you “slap” trays onto paper towels, do so several times; don’t be gentle; cups won’t fall outPrior to adding last reagents, wells should be empty (not dry, but empty of excess fluid)
23FLUORESCENCE IMMUNOASSAY (FA) Organism Detection and DifferentiationLimited to certain organisms (Cryptosporidium, Giardia cyst) – generally 2+ to 4+ (faint trophs)All kits have comparable sensitivity, specificitySingle, batch testing; fluorescent microscopeRequires color judgment and interpretationFalse negatives may result due to low organism numbers (asymptomatic carriers) – centrifugation!
24GIARDIA, CRYPTOSPORIDIUM Combination FA Immunoassay Two filters (FITC, background)Giardia lamblia cystCryptosporidium spp.oocystsImmunofluorescence(FA scope)One filter (FITC only)Garcia 24
25FLUORESCENCE IMMUNOASSAY USE SEDIMENT FOR TESTING Provides Organism Detection and DifferentiationLimited to certain organisms (Cryptosporidium, Giardia) – generally 2+ to 4+All kits have comparable sensitivity, specificityRequires fluorescent microscope (cost issue)Requires color judgment and interpretationFalse negatives may result due to low organism numbers (asymptomatic carriers) – perform centrifugation – use sediment
26FLUORESCENCE – TIPS Use Specimen Sediment Looking for cysts & oocysts, not antigen detection; centrifuged sediment (500 xg – 10 min)Prepare thin smears, dry slides (35°C for min); if not dry, stool may fall off; do NOT use heatGENTLY RINSE; allow fluid to flow over wellsOrganisms may not always fluoresce at 3+ to 4+; may see pale fluorescing bacteria/yeast; may also see very pale Giardia trophs; examine well edgesFluorescence filters; yellow-green = more intense fluorescence; both filters = a bit less intense
27IMMUNOCHROMATOGRAPHIC ASSAY – CARTRIDGE/STRIP Test line will USUALLY be lighter than control line.Too much stool can clog the sample well.If shake vial, allow to stand 5+ min before testing fluid at top.
28“3 ORGANISM” Cartridge IA CPT Codes: 87328 + 87329 + 87336 Top three lines = ControlsMiddle three lines = TestsBottom line = Negative ControlResults: POSITIVE GIARDIANOTE: EHIST = Entamoeba histolytica/E. dispar group NOTEntamoeba histolytica (true pathogen)
29CARTRIDGE IMMUNOASSAYS Multiple products – antigen detection (membrane flow) – possible well clogging with specimen All = comparable sensitivity and specificityExcellent; simple to use; clogging Single and/or batch testing optionsSet up for the detection and identification of multiple organisms; note control line color
30LATERAL FLOW CARTRIDGE – TIPS Use Unspun Specimen - Fluid If stool is too thick, reagents will not thin it out enough; if mixture is too thick, fluid will not flowDo NOT mix vial, but use fluid at top of vial; if vial mixed, settle for 5 min+ before testing fluidControl line must be visible all the way acrossPositive test line is almost always less intense than control; any color should be interpreted as positiveDo NOT read after time indicated in directions – may get a false positive.
31KEY QUESTIONSWhy not just substitute fecal immunoassays for the O&P examination? (see ordering table)Time savings, cost, personnel ???Right test for the right purpose ???What about fecal immunoassays, then O&P (depending on IA results) ???
32OPTION – SPECIAL STAINS (Coccidia, Microsporidia) Cryptosporidium spp. (C. hominis, C. parvum)Modified AFB, fluorescent stainsCyclospora cayetanensisModified AFB, autofluorescenceMicrosporidiaModified trichrome, Calcofluor /DNA DAPI fluorochrome dyeFresh or preserved specimens: concentrated sediment (500 xg for 10 min); smears allowed to air dry
33SPECIAL STAINS (Less Sensitive than Immunoassays) PROS: Rapid, simple, moderately specific/sensitive, defined patient situation test orders, patient should fit profiles; low supply costsCONS: Limited to coccidia or microsporidia, Modified Trichrome preps difficult to examine, high labor costs, orders may be inappropriate, requires client educationOrganism numbers will impact diagnosis; if suspect false negative, retest in days (coccidia) to 1-2 weeks (microsporidia)
34SPECIAL STAINS – TIPS Use Centrifuged Specimen Sediment Modified acid-fast (coccidia); destain step criticalDestain: 1% sulfuric acid recommended; good Cryptosporidium, Cyclospora, CystoisosporaAvoid thick smears; thin preparations bestMicrosporidia; modified trichrome – thin smears helpful; look for horizontal or diagonal line (polar filament) within the microsporidial spores
35CRYPTOSPORIDIUM SPP. C. hominis, C CRYPTOSPORIDIUM SPP. C. hominis, C. parvum (Stool Morphology will not ID species)Modified Acid-fast stain: Sporozoites within oocystsCPT CodesOrganisms at edge of intestinal surface; EM required for species ID
36CYCLOSPORA CAYETANENSIS (Suspected Food Borne Outbreak) 36Modified acid-fast stain AutofluorescenceAcid-fast variable Often 1+ to 3+
38MICROSPORIDIA in GI TRACT (Enterocytozoon, Encephalitozoon) Intestinal Tissue FA Urine: Calcofluor WhiteCPT Codes: Spores of E. intestinalis
39MICROSPORIDIAN SPORES Ryan Blue Trichrome Gram Stain Weber Green TrichromeNote: horizontal “stripes” (polar tubule)CPT Codes: (concentration) (stain)
40Note: horizontal “stripes” (polar tubule) MICROSPORIDIAGiemsa stain (eye) Ryan Blue TrichromeNote: horizontal “stripes” (polar tubule)
41ORDERING OPTIONS (DIAGNOSTIC PARASITOLOGY) Clinical Relevance: Patient’s clinical conditionGeographic Location: Parasites seen, travel, population types, metropolitan or other areasCost of Supplies: Slides, reagents, kits, laborUtilization of Personnel: Licensure vs. lab assistantsPhysician Education: Correct ordering optionsUse of Algorithms: Regulatory, education issuesProper Billing/Coding/Compliance: Critical
42ORDERING OPTIONS: WHAT’S IMPORTANT AND WHY PATIENTImportant for clinicians to use / understand ordering guidelines; approach provides the most clinically relevant information as well as appropriate test menu names, CPT codes, and billing.ORDERSpecific tests are designed to provide specific information: O&P, fecal immunoassays, special stains; physician must order tests, not laboratoryNOTEIf the test ordered is negative AND the patient becomes asymptomatic, additional testing may not be required.
43ORDERING OPTIONS PATIENT ORDER Immunocompromised patient with diarrheaPotential waterborne outbreak (municipal)ORDERCryptosporidium or Giardia/Crypto IANegative immunoassay / symptoms remainOrder O&Ps, microsporidia, Cyclospora
44ORDERING OPTIONS PATIENT ORDER Diarrhea (day care, camper, backpacker)Potential waterborne outbreak (resort)ORDERGiardia or Giardia/Cryptosporidium IANegative immunoassay / symptoms remainOrder O&Ps, microsporidia, Cyclospora
45ORDERING OPTIONS PATIENT ORDER Diarrhea, travel history outside of U.S.Past, present resident of developing countryORDERO&P examsNegative O&Ps / patient still symptomaticOrder Cryptosporidium, microsporidia, Cyclospora
46ORDERING OPTIONS PATIENT ORDER Diarrhea, area within U.S. where multiple parasites are seen on a routine basis (large metropolitan areas – NY, LA, DC, etc.)ORDERO&P examsNegative O&Ps / patient still symptomaticOrder Cryptosporidium, microsporidia, Cyclospora
47ORDERING OPTIONS PATIENT ORDER Many patients will not have traveled outside of the U.S. and may live in an area within the U.S. where Giardia is the most common parasite found.ORDERGiardia or combination immunoassayNegative immunoassay / symptoms remainOrder O&Ps, Cryptosporidium, Cyclospora, microsporidia
48ORDERING OPTIONS PATIENT ORDER Diarrhea (may or may not be present) Eosinophilia, unexplainedDo not intentionally immunosuppress a patient until this issue is resolved!ORDERO&P exams, Strongyloides stercoralisNegative O&Ps / symptoms remainOrder Cryptosporidium, microsporidia, Cyclospora
49ORDERING OPTIONS PATIENT ORDER Diarrhea present Suspected food borne outbreak (group activity)Produce (berries, basil, mesclun, snow peas)ORDERSpecial stain (modified acid-fast) for Cyclospora cayetanensisNegative stains/autofluorescence / patient still symptomaticOrder O&Ps, immunoassays
50ORDERING OPTIONS: REVIEW WHAT’S IMPORTANT AND WHY Will allow recovery and identification of majority of parasites. Will not be as sensitive/specific as fecal immunoassays. Consider multiple specimens over time.♦ Fecal Immunoassays (IAs)Specific for certain organisms; more sensitive than O&P.Giardia requires minimum of 2 IAs due to shedding issues.Special Stains for Coccidia or MicrosporidiaOrders often depend on immune state of the patient Must be clear to physicians that O&P will not capture these organisms.
51RESULT REPORTING LIMITATIONS IMPORTANCE: Understanding of diagnostic tests; physicians don’t understand test pros and consO&P: Understanding of possible results, organism names (pathogenicity), limitations of procedure (coccidia and microsporidiaIMMUNOASSAYS: Understanding of limitations, number of tests to order, specific organism options, collection limitationsSPECIAL STAINS: Per organisms; difficulties in test interpretation; specific patients
52RESULT REPORTINGO&P: Indicate test does NOT allow ID of Cryptosporidium, Cyclospora, or microsporidia (there are always some exceptions)-iron-hematoxylin stain with carbol fuchsin step; concentration and Cystoisospora belliIMMUNOASSAY: Indicate method tests for very limited and specific organisms only (name each organism on the report)SPECIAL STAINS: Remember to name organisms on the report – both pos/neg
53REPORTING ORGANISMSORGANISM NAMES: List all names using genus/species/stage (trophozoites, cysts, oocysts, spores, eggs, larvae, etc.)QUANTITATION: Very few parasites are quantitated: Blastocystis spp., some helminth eggs (Trichuris trichiura), viability of helminth eggs (Schistosoma spp.)NON PATHOGENS: These organisms must also be reported (same route for infections)
56STOOL RECOMMENDATIONS Do not perform O&Ps if patient has been in- house for >3 daysIf you call test O&P, must include concentration, permanent stain (CAP)Examination of 2 specimens = most IDsIf lot of mucus, don’t add ethyl acetateCentrifugation speed/time = 500 xg, 10 minCAP Check List Requirements: Permanent stain mandatory for O&P
57IMPORTANT REMINDERS!Not all laboratories use the same approach for stool testing. Fecal immunoassays and O&P examinations should be set up as separate, orderable tests. There is no “right or wrong” way, just different approaches, depending on variables we have discussed today!Discuss test orders and methods with your physician clients before making changes in test menus.
58PROFICIENCY TESTING Specimen Quality, etc. WET MOUNTS: Shake, allow fluid to settle, remove material –don’t stir up sedimentPERMANENT STAINED SMEARS: Read at least 300 oil immersion fields; potential grading problems with rare organisms!FECAL IMMUNOASSAYS: Remember to perform the test EXACTLY according to directions (sediment, rinses, etc.)