2013 PARASITOLOGY WORKSHOP LYNNE S. GARCIA, MS, FAAM, CLS, BLM Diagnostic Medical Parasitology Workshop 2013 UPDATE – PART 1: METHODS SPONSORED BY MEDICAL CHEMICAL CORPORATION www.med-chem.com

Lynne S. Garcia, MS, MT, CLS, BLM, FAAM Director, LSG & Associates LYNNE S. GARCIA – CONTACT EMAIL: Lynnegarcia2@verizon.net Lynne S. Garcia, MS, MT, CLS, BLM, FAAM Director, LSG & Associates Santa Monica, CA 90402-2908 PHONE (310) 393-5059 FAX (310) 899-9722

WORKSHOP OBJECTIVES Discuss stool parasite orders; educational initiatives required for clients in relation to patient care and test orders Review STATS vs. routines testing vs. send outs Discuss ordering : O&P vs IAs vs Special Stains Discuss collection and Universal Fixatives Discuss laboratory reporting, importance of report comments Discuss various protozoa, helminths, blood parasites: identification, tests, reporting, etc.

PARASITOLOGY 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 Stains Possible Send outs: Specimens for culture and serologies; majority of requests performed in large reference centers

DIAGNOSTIC PARASITOLOGY: TESTING OPTIONS Microscopy: O&P, Blood Films, Arthropod ID, Fluids/Tissues, Cultures Requires visual review, morphological assessment Organism vs artifact, size, morphology Geographic area, collection/processing options Other: Immunoassays, serologies (expertise, reagents, interpretation) DFA, ELISA, rapids: setups easy, interpretation Most diagnostic methods are categorized as high complexity (training, judgment, interpretation)

PARASITOLOGY TESTING: WHAT YOU NEED TO KNOW Minimum: Specimen acceptability, collection, processing, test method, reporting format Relevant 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 findings Risk Management: STAT testing (CSF, brain tissue, blood films)

STOOL SPECIMENS Ordering, Specimen Options: Method options, number and type of specimen Collection Options: Fresh or fixed (Universal Fixative), immunoassays, method pros and cons Potential 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

STOOL PRESERVATIVES and TESTING OPTIONS: O&P O&P Examination (Fresh or Preserved Stool Specimens) Direct Wet Smear (Motility): NO if in preservative Concentration: YES, performed for all O&Ps Permanent Stained Smear: Yes, performed for all O&Ps If O&P ordered, concentration AND permanent stained smear must be performed (CAP, NCCLS/CLSI) Fecal Immunoassays (Fresh, Frozen, Formalin) EIA: Performed on unspun specimens FA: Concentrated specimen (500 Xg for 10 min) Cartridge Systems: Unspun specimens

STOOL FIXATIVES Formalin: concentration, immunoassays Fixative with PVA: Polyvinyl Alcohol (glue) Mercury-based fixatives: phased out for environmental restrictions Copper or Zinc-based fixatives: zinc-based best morphology – being routinely used, including PT Universal Fixatives: (1) Concentration, (2) permanent stained smear, (3) special stains for coccidia/microsporidia, (4) fecal immunoassays, (5) PCR (TOTAL-FIX)

UNIVERSAL FIXATIVES OPTIONS: (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 FORMALIN TOTAL-FIX: NO PVA; NO MERCURY, NO FORMALIN Critical to make sure stool smears are TOTALLY DRY Drying in 37ºC incubator (on a tray); 30 min to 1 h IF THE SMEARS ARE TOTALLY DRY, THE STOOL MATERIAL WILL ADHERE TO THE SMEAR WITHOUT USING PVA OR ALBUMIN

STOOL 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. 37:2408-11, 1999) First stool: 75.9% detection Second stool: 92% detection Third stool: May not be cost-effective Data: Hanson and Cartwright (J. Clin. Microbiol. 39:474-8, 1993) Two specimens by either EIA or O&P revealed >90% detection Third Stool: May not be cost-effective

STOOL 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 cathartic ROUTINE: 3 stools collected in preservative (complete O&P) Data: Nazar (Br. J. Clin. Prac. 47:76-8, 1993) First stool: 58.3% of population tested Second 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 histolytica Yield increased 11.3%: Giardia lamblia Yield increased 31.1%: Dientamoeba fragilis

STOOL COLLECTION SUMMARY (O&P) Fresh or Preserved Stool Specimens Personal preference Consider ALL orders (O&P, IA, special stains) RECOMMENDATION: Fixatives (lag time problems) Number of specimens to Collect Two specimens is acceptable; three is better RECOMMENDATION: Three, but two acceptable Testing O&P, Immunoassays, Special Staining Separate, orderable, billable tests (CPT codes)

O&P EXAMINATION

Fresh or Preserved Stool Specimens Personal preference Consider ALL testing being ordered (O&P, IA, special stains) RECOMMENDATION: Fixatives eliminate lag time problems Number of specimens to Collect Two specimens is acceptable Three is better RECOMMENDATION: Three, but two acceptable Testing O&P, Immunoassays, Special Testing 11 IV. Cyclospora Autofluorescence Special stains STOOL ORDER RECOMMENDATIONS

ORGANISMS: O&P EXAM WET MOUNTS (SALINE, IODINE)

ORGANISMS: O&P EXAM PERMANENT STAINED SMEARS

OPTION: FECAL IMMUNOASSAYS INTRODUCTION If 1st stool for Giardia NEG, perform IA on one more stool before reporting NEG! Not required for Cryptosporidium testing.

FECAL IMMUNOASSAYS - ANTIGEN

ENZYME IMMUNOASSAY

ENZYME 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, specificity Color judgment - interpretation if manually read False negatives may result due to low organism numbers (asymptomatic carriers)

ENZYME IMMUNOASSAY – TIPS Use Unspun Specimen - Fluid If vial is mixed, let settle for 5+ min before testing Thoroughly rinse wells, don’t cut any rinse steps Each well MUST receive total number of rinses Squirt buffer directly into wells; squeeze bottle When you “slap” trays onto paper towels, do so several times; don’t be gentle; cups won’t fall out Prior to adding last reagents, wells should be empty (not dry, but empty of excess fluid)

FLUORESCENCE IMMUNOASSAY (FA) Organism Detection and Differentiation Limited to certain organisms (Cryptosporidium, Giardia cyst) – generally 2+ to 4+ (faint trophs) All kits have comparable sensitivity, specificity Single, batch testing; fluorescent microscope Requires color judgment and interpretation False negatives may result due to low organism numbers (asymptomatic carriers) – centrifugation!

GIARDIA, CRYPTOSPORIDIUM Combination FA Immunoassay Two filters (FITC, background) Giardia lamblia cyst Cryptosporidium spp. oocysts Immunofluorescence (FA scope) One filter (FITC only) Garcia 24

FLUORESCENCE IMMUNOASSAY USE SEDIMENT FOR TESTING Provides Organism Detection and Differentiation Limited to certain organisms (Cryptosporidium, Giardia) – generally 2+ to 4+ All kits have comparable sensitivity, specificity Requires fluorescent microscope (cost issue) Requires color judgment and interpretation False negatives may result due to low organism numbers (asymptomatic carriers) – perform centrifugation – use sediment

FLUORESCENCE – 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 30-60 min); if not dry, stool may fall off; do NOT use heat GENTLY RINSE; allow fluid to flow over wells Organisms may not always fluoresce at 3+ to 4+; may see pale fluorescing bacteria/yeast; may also see very pale Giardia trophs; examine well edges Fluorescence filters; yellow-green = more intense fluorescence; both filters = a bit less intense

IMMUNOCHROMATOGRAPHIC 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.

“3 ORGANISM” Cartridge IA CPT Codes: 87328 + 87329 + 87336 Top three lines = Controls Middle three lines = Tests Bottom line = Negative Control Results: POSITIVE GIARDIA NOTE: EHIST = Entamoeba histolytica/E. dispar group NOT Entamoeba histolytica (true pathogen)

CARTRIDGE IMMUNOASSAYS Multiple products – antigen detection (membrane flow) – possible well clogging with specimen  All = comparable sensitivity and specificity Excellent; simple to use; clogging  Single and/or batch testing options Set up for the detection and identification of multiple organisms; note control line color

LATERAL 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 flow Do NOT mix vial, but use fluid at top of vial; if vial mixed, settle for 5 min+ before testing fluid Control line must be visible all the way across Positive test line is almost always less intense than control; any color should be interpreted as positive Do NOT read after time indicated in directions – may get a false positive.

KEY QUESTIONS Why 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) ???

OPTION – SPECIAL STAINS (Coccidia, Microsporidia) Cryptosporidium spp. (C. hominis, C. parvum) Modified AFB, fluorescent stains Cyclospora cayetanensis Modified AFB, autofluorescence Microsporidia Modified trichrome, Calcofluor /DNA DAPI fluorochrome dye Fresh or preserved specimens: concentrated sediment (500 xg for 10 min); smears allowed to air dry

SPECIAL STAINS (Less Sensitive than Immunoassays) PROS: Rapid, simple, moderately specific/sensitive, defined patient situation test orders, patient should fit profiles; low supply costs CONS: Limited to coccidia or microsporidia, Modified Trichrome preps difficult to examine, high labor costs, orders may be inappropriate, requires client education Organism numbers will impact diagnosis; if suspect false negative, retest in days (coccidia) to 1-2 weeks (microsporidia)

SPECIAL STAINS – TIPS Use Centrifuged Specimen Sediment Modified acid-fast (coccidia); destain step critical Destain: 1% sulfuric acid recommended; good Cryptosporidium, Cyclospora, Cystoisospora Avoid thick smears; thin preparations best Microsporidia; modified trichrome – thin smears helpful; look for horizontal or diagonal line (polar filament) within the microsporidial spores

CRYPTOSPORIDIUM SPP. C. hominis, C CRYPTOSPORIDIUM SPP. C. hominis, C. parvum (Stool Morphology will not ID species) Modified Acid-fast stain: Sporozoites within oocysts CPT Codes 87015 + 87207 Organisms at edge of intestinal surface; EM required for species ID

CYCLOSPORA CAYETANENSIS (Suspected Food Borne Outbreak) 36 Modified acid-fast stain Autofluorescence Acid-fast variable Often 1+ to 3+

CRYPTOSPORIDIUM SPP. CYCLOSPORA CAYETANENSIS Cryptosporidium spp.: Modified acid-fast stain, note sporozoites, infectious; 4-6 microns Cyclospora sp: Lower power; 8-10 microns Modified acid-fast stain, no sporozoites, not infectious

MICROSPORIDIA in GI TRACT (Enterocytozoon, Encephalitozoon) Intestinal Tissue FA Urine: Calcofluor White CPT Codes: 87015 + 87206 Spores of E. intestinalis

MICROSPORIDIAN SPORES Ryan Blue Trichrome Gram Stain Weber Green Trichrome Note: horizontal “stripes” (polar tubule) CPT Codes: 87015 (concentration) + 87207 (stain)

Note: horizontal “stripes” (polar tubule) MICROSPORIDIA Giemsa stain (eye) Ryan Blue Trichrome Note: horizontal “stripes” (polar tubule)

ORDERING OPTIONS (DIAGNOSTIC PARASITOLOGY) Clinical Relevance: Patient’s clinical condition Geographic Location: Parasites seen, travel, population types, metropolitan or other areas Cost of Supplies: Slides, reagents, kits, labor Utilization of Personnel: Licensure vs. lab assistants Physician Education: Correct ordering options Use of Algorithms: Regulatory, education issues Proper Billing/Coding/Compliance: Critical

ORDERING OPTIONS: WHAT’S IMPORTANT AND WHY PATIENT Important 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. ORDER Specific tests are designed to provide specific information: O&P, fecal immunoassays, special stains; physician must order tests, not laboratory NOTE If the test ordered is negative AND the patient becomes asymptomatic, additional testing may not be required.

ORDERING OPTIONS PATIENT ORDER Immunocompromised patient with diarrhea Potential waterborne outbreak (municipal) ORDER Cryptosporidium or Giardia/Crypto IA Negative immunoassay / symptoms remain Order O&Ps, microsporidia, Cyclospora

ORDERING OPTIONS PATIENT ORDER Diarrhea (day care, camper, backpacker) Potential waterborne outbreak (resort) ORDER Giardia or Giardia/Cryptosporidium IA Negative immunoassay / symptoms remain Order O&Ps, microsporidia, Cyclospora

ORDERING OPTIONS PATIENT ORDER Diarrhea, travel history outside of U.S. Past, present resident of developing country ORDER O&P exams Negative O&Ps / patient still symptomatic Order Cryptosporidium, microsporidia, Cyclospora

ORDERING OPTIONS PATIENT ORDER Diarrhea, area within U.S. where multiple parasites are seen on a routine basis (large metropolitan areas – NY, LA, DC, etc.) ORDER O&P exams Negative O&Ps / patient still symptomatic Order Cryptosporidium, microsporidia, Cyclospora

ORDERING 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. ORDER Giardia or combination immunoassay Negative immunoassay / symptoms remain Order O&Ps, Cryptosporidium, Cyclospora, microsporidia

ORDERING OPTIONS PATIENT ORDER Diarrhea (may or may not be present) Eosinophilia, unexplained Do not intentionally immunosuppress a patient until this issue is resolved! ORDER O&P exams, Strongyloides stercoralis Negative O&Ps / symptoms remain Order Cryptosporidium, microsporidia, Cyclospora

ORDERING OPTIONS PATIENT ORDER Diarrhea present Suspected food borne outbreak (group activity) Produce (berries, basil, mesclun, snow peas) ORDER Special stain (modified acid-fast) for Cyclospora cayetanensis Negative stains/autofluorescence / patient still symptomatic Order O&Ps, immunoassays

ORDERING 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 Microsporidia Orders often depend on immune state of the patient. Must be clear to physicians that O&P will not capture these organisms.

RESULT REPORTING LIMITATIONS IMPORTANCE: Understanding of diagnostic tests; physicians don’t understand test pros and cons O&P: Understanding of possible results, organism names (pathogenicity), limitations of procedure (coccidia and microsporidia IMMUNOASSAYS: Understanding of limitations, number of tests to order, specific organism options, collection limitations SPECIAL STAINS: Per organisms; difficulties in test interpretation; specific patients

RESULT REPORTING O&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 belli IMMUNOASSAY: 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

REPORTING ORGANISMS ORGANISM 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)

REPORTING ARTIFACTS ORGANISM NAMES: Human cells, crystals, yeast (consult only); PMNs, macrophages, eosinophils, Charcot-Leyden crystals QUANTITATION: General – rare, few, moderate, many, packed

ARTIFACTS SEEN IN O&P EXAM REPORT DO NOT REPORT

STOOL RECOMMENDATIONS Do not perform O&Ps if patient has been in- house for >3 days If you call test O&P, must include concentration, permanent stain (CAP) Examination of 2 specimens = most IDs If lot of mucus, don’t add ethyl acetate Centrifugation speed/time = 500 xg, 10 min CAP Check List Requirements: Permanent stain mandatory for O&P

IMPORTANT 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.

PROFICIENCY TESTING Specimen Quality, etc. WET MOUNTS: Shake, allow fluid to settle, remove material –don’t stir up sediment PERMANENT 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.)

ISSUES – REVIEW STOOL PARASITOLOGY STOOL: Ordering, collection, processing, examination, reporting O&P: Fresh vs preserved, required steps FECAL IMMUNOASSAYS: Orders, pros/cons of methods, pitfalls SPECIAL STAINS: Coccidia, microsporidia, interpretation, pitfalls REPORTING: Consistency, artifacts, comments PROFICIENCY TESTING: Practicality, tips

THANKS – QUESTIONS?