2013 PARASITOLOGY WORKSHOP

1. 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

2. LYNNE S. GARCIA – CONTACTEMAIL: 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

3. 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.

4. 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

5. 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)

6. 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)

7. 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

8. 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

9. 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)

10. 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

11. 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

12. 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

13. 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)

14. O&P EXAMINATION

15. 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

16. ORGANISMS: O&P EXAMWET MOUNTS (SALINE, IODINE)

17. ORGANISMS: O&P EXAMPERMANENT STAINED SMEARS

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

19. FECAL IMMUNOASSAYS - ANTIGEN

20. ENZYME IMMUNOASSAY

21. 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)

22. ENZYME IMMUNOASSAY – TIPSUse 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)

23. 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!

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

25. 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

26. 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

27. IMMUNOCHROMATOGRAPHICASSAY – 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 IACPT 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)

29. 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

30. LATERAL FLOW CARTRIDGE – TIPSUse 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.

31. 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) ???

32. 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

33. 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)

34. SPECIAL STAINS – TIPSUse 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

35. 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

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

37. 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

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

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

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

41. 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

42. 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.

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

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

45. ORDERING OPTIONS • PATIENT 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

46. ORDERING OPTIONS • PATIENT 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

47. ORDERING OPTIONS • PATIENT 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

48. ORDERING OPTIONS • PATIENT 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

49. ORDERING OPTIONS • PATIENT 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

50. ORDERING OPTIONS: REVIEWWHAT’S IMPORTANT AND WHY • O&P 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.