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

2. Lynne S. Garcia, MS, MT, CLS, BLM, FAAM Director, LSG & Associates
LYNNE S. GARCIA – CONTACT
Lynne S. Garcia, MS, MT, CLS, BLM, FAAM
Director, LSG & Associates
Santa Monica, CA
PHONE (310)
FAX (310)

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: , 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 EXAM WET MOUNTS (SALINE, IODINE)

17. ORGANISMS: O&P EXAM PERMANENT STAINED SMEARS

18. OPTION: FECAL IMMUNOASSAYS INTRODUCTION
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 – 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)

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, CRYPTOSPORIDIUM Combination 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 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. 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.

28. “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)

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 – 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.

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 – 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

35. 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
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: Spores of E. intestinalis

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

40. Note: horizontal “stripes” (polar tubule)
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 ORDER
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 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

45. 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

46. 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

47. 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

48. 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

49. 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

50. 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.

51. 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

52. 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

53. 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)

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

55. ARTIFACTS SEEN IN O&P EXAM
REPORT
DO NOT REPORT

56. 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

57. 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.

58. 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.)

59. 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

60. THANKS – QUESTIONS?