2013 PARASITOLOGY WORKSHOP

LYNNE S. GARCIA, MS, FAAM, CLS, BLM

Diagnostic Medical Parasitology Workshop

2013 UPDATE – PART 1: METHODS

SPONSORED BY

MEDICAL CHEMICAL CORPORATIONwww.med-chem.com

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LYNNE S. GARCIA – CONTACTEMAIL: Lynnegarcia2@verizon.net

Lynne S. Garcia, MS, MT, CLS, BLM, FAAM

Director, LSG & AssociatesSanta Monica, CA 90402-2908

PHONE (310) 393-5059FAX (310) 899-9722

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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.3

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

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DIAGNOSTIC PARASITOLOGY:TESTING OPTIONS

¨ Microscopy: O&P, Blood Films, Arthropod ID, Fluids/Tissues, Cultures Requires visual review, morphological assessmentOrganism vs artifact, size, morphologyGeographic 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) 5

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)

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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 7

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 8

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) 9

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 10

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

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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

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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) 13

O&P EXAMINATION

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¨ 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

11STOOL ORDER

RECOMMENDATIONS

IV. CyclosporaAutofluorescenceSpecial stains

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

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ORGANISMS: O&P EXAMPERMANENT STAINED SMEARS

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OPTION: FECAL IMMUNOASSAYSINTRODUCTION

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

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FECAL IMMUNOASSAYS - ANTIGEN

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ENZYME IMMUNOASSAY

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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)

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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)

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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! 23

GIARDIA, CRYPTOSPORIDIUMCombination FA Immunoassay

Giardia lamblia cystCryptosporidium spp.

oocysts

Immunofluorescence(FA scope)

One filter (FITC only)Garcia 24

Two filters (FITC, background)

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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

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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

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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.

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“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 NOTEntamoeba histolytica (true pathogen) 28

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

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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. 30

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) ???31

OPTION – SPECIAL STAINS

(Coccidia, Microsporidia)

¨Cryptosporidium spp. (C. hominis, C. parvum)Modified AFB, fluorescent stains

¨Cyclospora cayetanensisModified AFB, autofluorescence

¨MicrosporidiaModified trichrome, Calcofluor /DNA DAPI

fluorochrome dye

Fresh or preserved specimens: concentrated sediment (500 xg for 10 min); smears allowed to air dry 32

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)

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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

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CRYPTOSPORIDIUM SPP.C. hominis, C. parvum

(Stool Morphology will not ID species)

Modified Acid-fast stain: Sporozoites within oocystsCPT Codes 87015 + 87207

Organisms at edge of intestinal surface; EM required for species ID 35

CYCLOSPORA CAYETANENSIS(Suspected Food Borne Outbreak)

Modified acid-fast stain AutofluorescenceAcid-fast variable Often 1+ to 3+

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CRYPTOSPORIDIUM SPP.CYCLOSPORA CAYETANENSIS

Cryptosporidium spp.: Modified acid-fast stain, note sporozoites, infectious; 4-6 microns

Cyclospora sp: Lower power; 8-10 micronsModified acid-fast stain, no sporozoites, not infectious

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MICROSPORIDIA in GI TRACT(Enterocytozoon, Encephalitozoon)

Intestinal Tissue FA Urine: Calcofluor White CPT Codes: 87015 + 87206

Spores of E. intestinalis 38

MICROSPORIDIAN SPORES

Ryan Blue Trichrome Gram Stain Weber Green TrichromeNote: horizontal “stripes” (polar tubule)

CPT Codes: 87015 (concentration) + 87207 (stain)

oo o39

MICROSPORIDIA

Giemsa stain (eye) Ryan Blue TrichromeNote: horizontal “stripes” (polar tubule)

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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

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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. 42

ORDERING OPTIONS

¨ PATIENTImmunocompromised patient with diarrhea

Potential waterborne outbreak (municipal)

¨ ORDERCryptosporidium or Giardia/Crypto IA

Negative immunoassay / symptoms remain

Order O&Ps, microsporidia, Cyclospora43

ORDERING OPTIONS

¨ PATIENTDiarrhea (day care, camper, backpacker)Potential waterborne outbreak (resort)

¨ ORDERGiardia or Giardia/Cryptosporidium IANegative immunoassay / symptoms remainOrder O&Ps, microsporidia, Cyclospora

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ORDERING OPTIONS

¨ PATIENTDiarrhea, travel history outside of U.S.Past, present resident of developing country

¨ ORDERO&P examsNegative O&Ps / patient still symptomaticOrder Cryptosporidium, microsporidia, Cyclospora 45

ORDERING OPTIONS

¨ PATIENTDiarrhea, 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

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ORDERING OPTIONS

¨ PATIENTMany 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 immunoassay Negative immunoassay / symptoms remainOrder O&Ps, Cryptosporidium, Cyclospora, microsporidia 47

ORDERING OPTIONS

¨ PATIENTDiarrhea (may or may not be present)

Eosinophilia, unexplainedDo not intentionally immunosuppress a

patient until this issue is resolved!¨ ORDER

O&P exams, Strongyloides stercoralisNegative O&Ps / symptoms remainOrder Cryptosporidium, microsporidia, Cyclospora 48

ORDERING OPTIONS

¨ PATIENTDiarrhea 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

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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.

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RESULT REPORTINGLIMITATIONS

¨ 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 51

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 52

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)

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REPORTING ARTIFACTS

¨ ORGANISM NAMES: Human cells, crystals, yeast (consult only); PMNs, macrophages, eosinophils, Charcot-Leyden crystals

¨ QUANTITATION: General – rare, few, moderate, many, packed

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ARTIFACTS SEEN IN O&P EXAM

REPORT DO NOT REPORT

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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 56

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. 57

PROFICIENCY TESTINGSpecimen 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.)

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ISSUES – REVIEWSTOOL 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

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THANKS – QUESTIONS?

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