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Presentation of Quiz #53

The Microbiology section has just decided to incorporate immunoassays for Giardia lamblia and Cryptosporidium spp. into the laboratory test menu. What factors should be considered prior to implementation of these immunoassays? 

It is important to remember that the immunoassay tests currently available are not designed to "take the place" of the Ova and Parasite examination (O&P) (direct wet smear [fresh specimen only], concentration, and permanent stained smear). Both the O&P examination and the immunoassays for intestinal protozoa should be separate orderable, billable tests that appear on the test menu.  STOOL TESTING RECOMMENDED ORDERS

Please comment on the issues presented below.

  • Test kit options and specimen collection requirements
  • Test ordering menu
  • Test coding
  • Test report formats
  • Client education
  • Personnel expertise
  • Geographic area

What are the tests illustrated below called (name and abbreviation or short name)?

Scroll Down for Answer and Discussion

 

 

 

 

 

 

Answer and Discussion of Quiz #53

The immunoassay kits seen above can be identified as follows:

Left: Lateral flow cartridge ("Rapid");

Center: Enzyme-linked immunosorbent assay (ELISA);

Right: Fluorescent antibody test (FA)

Because there are so many variables to consider when introducing new tests into the laboratory, not every laboratory will offer the same test formats. There are very specific reasons for selection of new methods. Some of these factors include: geographic location and presence of the parasites being considered, patient populations (local, national, and/or international), size of the laboratory and number of specimens submitted, clients serviced, number of tests ordered, availability of trained personnel, trained consultants for trouble-shooting technical problems, client educational options, etc. If we examine the various issues surrounding the list presented above, this information may help confirm the benefits of the Ova and Parasite exam, as well as the fecal immunoassays.

Test kit options and specimen collection requirements: All immunoassay kits can be used with fresh or frozen specimens; however, only the kit formats that include Giardia lamblia and Cryptosporidium spp. can be performed using formalinized specimens (5% or 10% formalin, sodium acetate-formalin-acetic acid [SAF], and some of the single vial collection systems), particularly the Universal Fixative (TOTAL-FIX). If testing is performed for the Entamoeba histolytica/Entamoeba dispar group or the true pathogen, Entamoeba histolytica, fresh or frozen specimens or those in Cary Blair can be used. These kits are not compatible with fecal specimens submitted in fixatives.

NOTE:

All available commercial immunoassay kits for the detection of antigen or actual organisms (cysts and/or oocysts) have published reports of both sensitivity and specificity of approximately 95% or better. A positive or negative immunoassay result does NOT require confirmation testing by the O&P exam.

TEST             KIT OPTIONS

PROS

CONS

Enzyme Immunoassay (EIA)

Easy to use, can batch test; available for Giardia, Cryptosporidium, E. histolytica/E. dispar group, or E. histolytica

Color for low positives may be difficult to interpret if read manually, rather than using instrumentation; thorough washing mandatory

Fluorescent Antibody (FA)

Easy to read; combination reagent available for both Giardia and Cryptosporidium; visual identification of cysts and oocysts (different sizes and shapes); can batch test; perform on stool concentrates

Requires fluorescent microscope; cost may be a factor.

Cartridge, EIA or Chromatographic IA (Rapid)

Ease to use and read; combination reagents available for Giardia or Cryptosporidium; can batch test; E. histolytica/E. dispar group also available

If the cartridge contains reagents for the detection of the E. histolytica/E. dispar group, fresh or frozen stools or those in Cary Blair will be required

Test Ordering Menu:

Depending on the patient history, it may be appropriate to order immunoassays for very specific organisms. However, it may be more important to order the O&P examination, particularly if the patient has been in a geographic area where parasites other than Giardia and/or Cryptosporidium may be present. Remember, these tests should always be separate orderable, billable tests. STOOL TESTING RECOMMENDED ORDERS

Test Coding:

 It is important for billing purposes to use separate codes for the O&P examination, as well as separate codes for the EIA, FA, or cartridge immunoassays.

Test Report Formats:

When reporting results of the O&P examination, it is important to inform the physician that Cryptosporidium spp. (with rare exceptions), Cyclospora cayetanensis and the microsporidia will not be detected from the routine O&P examination; special procedures must be ordered (modified acid-fast stains for the coccidia and modified trichrome stains for the microsporidia). When reporting results of the stool immunoassays, it is important to inform the physician that these procedures are designed to detect certain organisms ONLY. Indicate that a negative report means that specific organisms (name the organisms) were not seen.

Client Education:

Since the laboratory does not have sufficient patient information on which to base laboratory orders, it is mandatory that the physician clients have appropriate and complete information related to test ordering options. They should thoroughly understand the pros and cons of the O&P examination compared with the immunoassays; specific orders for one or the other are relevant, depending on the patient history. Some of the considerations include: patient's immune status, geographic area, travel history, and a history of daycare. One of the most important considerations is the following: IF THE PATIENT BECOMES ASYMPTOMATIC AT THE POINT AT WHICH NEGATIVE LABORATORY RESULTS ARE REPORTED, NO FURTHER TESTING IS REQUIRED; IF THE PATIENT REMAINS SYMPTOMATIC, THEN ADDITIONAL TESTING IS WARRANTED.

Personnel Expertise:

The performance of stool immunoassay testing is not difficult, providing the manufacturer's directions are followed. Thorough washing during EIA testing will eliminate the possibility of incorrect results. Known positive and negative specimens should be tested as a part of the learning phase.

Geographic Area:

STOOL TEST ORDERS are based partly on the location of the laboratory and travel history of the patients. Again, it is important to provide specific guidelines on test ordering options for the physicians; adequate information will help ensure appropriate laboratory testing and clinically relevant information for quality patient care.

COMMENTS ON PERFORMANCE OF FECAL IMMUNOASSAYS: TIPS ON FECAL IMMUNOASSAYS

Some comments that may be helpful in performing various immunoassay formats are provided to assist you in test performance and/or result interpretation. It is very important to read the kit information sheet before use. Currently, fecal immunoassays are available for Giardia lamblia, the Entamoeba histolytica/E. dispar group, Entamoeba histolytica, and Cryptosporidium spp. Development of reagents is also ongoing for Dientamoeba fragilis, Blastocystis spp. and the microsporidia.

Some comments that may be helpful in performing various immunoassay formats are provided to assist you in test performance and/or result interpretation.  Some reagents are available in the research setting for Enterocytozoon bieneusi and Encephalitozoon intestinalis, but they are not FDA approved; some commercial kits are available outside of the United States. Based on the published literature, fecal immunoassays are more sensitive and specific than the routine Ova and Parasite (O&P) examination; this is particularly true for Giardia lamblia.  However, unlike the O&P exam that facilitates the recovery of many different parasites, the fecal immunoassays are limited to one or two organisms only.  The fecal immunoassays are also more sensitive than the special stains (modified acid-fast stains) for the coccidia (Cryptosporidium spp.)  

Fresh specimens can be stored at 2-8°C and should be tested within 48 h or they should be frozen at -20 to -70°C (freezing not acceptable for FA method – freeze/thaw cycle will damage organisms).  Stool specimens preserved in 10% formalin, MIF, SAF or TOTAL-FIX fixatives may be refrigerated (2-8°C) or stored at room temperature (20-25°C) and should be tested within 2 months.  Stool specimens submitted in Cary Blair transport medium (or equivalent) should be refrigerated or frozen and tested within 1 week after collection.  Fecal specimens that have been preserved in fixatives containing PVA are not acceptable for testing.

To enhance the sensitivity of the FA procedure, it is recommended that testing be performed on centrifuged stool sediment (500 X g for 10 min).  Fixatives containing mercury and/or polyvinyl alcohol/PVA adhesives are not recommended for immunoassay testing.

Enzyme immunoassays (antigen detection, no centrifugation recommended); the antigen will be found in the top fluid layer of the stool collection vial (ELISA or EIA):

Enzyme immunoassays (antigen detection, no centrifugation recommended); the antigen will be found in the top fluid layer of the stool collection vial

  • Remember to thoroughly rinse the wells according to the instructions; do not eliminate any of the rinse steps.  Make sure each well receives the total number of rinses required.
  • Make sure the stream of buffer goes directly into the wells.  Use a wash bottle with a small opening, so you have to squeeze the bottle to get the fluid to squirt directly into the wells.
  • When the directions tell you to “slap” the tray down onto some paper towels to remove the last rinse fluid, make sure you slap it several times.  Don’t be too gentle; the cups will not fall out of the holder.
  • Prior to adding the last reagents, the wells should be empty of rinse buffer (not dry, but empty of excess fluid).
  • Note:  If you shake the specimen vial prior to testing, allow the vial contents to settle out for several minutes.  Adding too much particulate stool to the wells will interfere with testing.

Fluorescence (visual identification of the organisms):

  • Since you will be looking for the actual organisms (cysts of Giardia and/or oocysts of Cryptosporidium), this test should be performed on centrifuged stool (500 X g for 10 min) to increase the sensitivity.
  • Remember to thin out the smear; it is important to make sure the slides are thoroughly dry before adding reagents.  The slides can be placed in a 35ºC incubator for about 30 minutes to an hour to make sure they are dry prior to processing.  If the material on the wells is too thick, it may not dry thoroughly and may fall off of the glass.  It is better to let them dry longer rather than too short a time.  A heat block is NOT recommended for this purpose.
  • Gently rinse the reagents from the wells; do not squirt directly into the wells, but allow the rinse fluid to flow over the wells.
  • Remember that not all clinical specimens will provide the 3+ to 4+ fluorescence that we often see in the positive control.  Also, from time to time, you may see fluorescing bacteria and/or some yeast in certain patient specimens.  This is not that common, but the shapes can be distinguished from Giardia cysts and/or Cryptosporidium oocysts.  Occasionally, you may see pale fluorescence from Giardia trophozoites (they share some cyst antigens).
  • The intensity of the fluorescence may vary, depending on the filters.  If the fluorescent microscope dual filter system is used, it demonstrates both the yellow-green fluorescence and the red-orange counterstain and both Giardia and Cryptosporidium may not appear quite as bright as when using the yellow-green filter only.  Both approaches are acceptable and may reflect personal laboratory preferences.  However, remember that when the single FITC (yellow green) filter is used alone, some artifact materials may also appear to fluoresce more brightly, while the artifact material might not be seen when both filters (FITC and counterstain) are used.  Artifact material may fluoresce a dull color without the bright outlines seen around the Giardia cysts and Cryptosporidium oocysts that can be seen using both filter systems.

Both filters = less fluorescence intensity, less visible artifacts

Single FITC filter = brighter fluorescence, more visible artifacts

  • Make sure to examine the edges of the wells.  Sometimes in a light infection, the edges may contain organisms, while in the middle of the well the organisms may be a bit more difficult to detect (thick area).

Lateral Flow Cartridges/Rapids (antigen detection, no centrifugation recommended); the antigen will be found in the top fluid layer of the stool collection vial:

  • If the stool is too thick, the addition of reagents will not thin it out enough.  If the specimen poured into the well remains too thick, the fluid will not flow up the membrane.  If your specimens arrive in fixative and there is no fluid at the top of the vial overlaying the stool, this means the vial may have been overfilled with stool. These specimens will have to be diluted with the appropriate diluent before testing.
  • It is always important to see the control line indicated as positive all the way across the membrane, not just at the edges.
  • A positive test result may be much lighter than the control line; this is normal. 
  • At the cutoff time to read the result, any color at all visible in the test area should be interpreted as a positive.
  • Do not read/interpret the results after the time indicated in the directions; you may get a false positive result.
  • Note:  If you shake the specimen vial prior to testing, allow the vial contents to settle out for several minutes.  Adding too much particulate stool to the wells will interfere with testing. The specimen will clog the filter and the test result will be invalid. 
  • IT IS EXTREMELY IMPORTANT TO USE ONLY THE LIQUID PORTION OF THE SPECIMEN; AVOID ALL PARTICULATE MATTER.

 

References:

  • Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.
  • Garcia, L.S. (ed.). 2010. Clinical Microbiology Procedures Handbook, 3rd Ed., vol. 1, 2, and 3. ASM Press, Washington, D.C.
  • Garcia, L.S. and R.Y. Shimizu.  1997.  Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens.  J. Clin. Microbiol. 35:1526-1529

3.         Garcia, LS and R.Y. Shimizu.  2000.  Detection of Giardia lamblia and Cryptosporidium parvum antigens in human fecal specimens using the ColorPAC combination rapid solid-phase qualitative immunochromatographic assay.  J. Clin. Microbiol. 38: 1267-1268.

4.         Hanson, K. L., and C. P. Cartwright. 2001. Use of an enzyme immunoassay does not eliminate the need to analyze multiple stool specimens for sensitive detection of Giardia lamblia. J. Clin. Microbiol. 39:474-477.

5.         Isenberg, H.D. (ed.), 2004.  Clinical Microbiology Procedures Handbook, 2nd ed. ASM Press, Washington, D.C., Parasitology Section in Vol 2 of 3 vols.

6.         Wilson, M., and P.M. Schantz.  2000.  Parasitic immunodiagnosis. In. Strickland, G.T. (ed), Hunter’s Tropical Medicine and Emerging Infectious Diseases, 8th ed. W.B. Saunders Co., Philadelphia, pages 1117-1122.

3.         Garcia, LS and R.Y. Shimizu.  2000.  Detection of Giardia lamblia and Cryptosporidium parvum antigens in human fecal specimens using the ColorPAC combination rapid solid-phase qualitative immunochromatographic assay.  J. Clin. Microbiol. 38: 1267-1268.

4.         Hanson, K. L., and C. P. Cartwright. 2001. Use of an enzyme immunoassay does not eliminate the need to analyze multiple stool specimens for sensitive detection of Giardia lamblia. J. Clin. Microbiol. 39:474-477.

5.         Isenberg, H.D. (ed.), 2004.  Clinical Microbiology Procedures Handbook, 2nd ed. ASM Press, Washington, D.C., Parasitology Section in Vol 2 of 3 vols.

6.         Wilson, M., and P.M. Schantz.  2000.  Parasitic immunodiagnosis. In. Strickland, G.T. (ed), Hunter’s Tropical Medicine and Emerging Infectious Diseases, 8th ed. W.B. Saunders Co., Philadelphia, pages 1117-1122.