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Presentation of Quiz #46

The Microbiology section has just decided to incorporate immunoassays for Giardia lamblia and Cryptosporidium spp. into the laboratory test menu. The laboratory is also considering the addition of tests for the Entamoeba histolytica/Entamoeba dispar group and the true pathogen, Entamoeba histolytica. 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 and should be available to your clients on your test menu.

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

Scroll Down for Answer and Discussion

 

 

 

 

 

 

Answer and Discussion of Quiz #46

Because there are so many variables to consider when introducing new tests into the laboratory, it is reasonable to assume that not every laboratory will offer the same test menu, even if some of the tests are sent to a reference laboratory. There are very specific reasons for selection and/or nonselection 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 you in assessing whether or not your laboratory should introduce newer testing options for some of the intestinal protozoa.

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 stool submitted in ANY of the 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. Positive or negative immunoassay results do NOT require confirmation testing by the O&P exam.  However, when testing for Giardia, if the first test is negative, order a second stool before calling the patient negative.  This is due to the variability in shedding seen in Giardia infections (not necessary with Cryptosporidium spp.).

Test Kit Options

Pros

Cons

Enzyme Immunoassay (EIA)

Easy to use, can batch test

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

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

If the cartridge contains reagents for the detection of the E. histolytica/E. dispar group or E. histolytica, 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 TESTING RECOMMENDED 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.

We hope this discussion (Quiz #46) has been helpful. Don't hesitate to contact us if you have questions and/or suggestions: LynneGarcia2@verizon.net

An ordering table can also be sent if requested from Lynne Garcia (via Email address). Specific recommendations for the performance of fecal immunoassays can be seen below.

Enzyme immunoassays (antigen detection, no centrifugation recommended)

  1. 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.
  2. Make sure the stream of buffer goes directly into the wells. Also, make sure you 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.
  3. When the directions tell you to "slap" the tray down onto some paper towels to remove all the fluid, make sure you slap it several times. Don't be too gentle; the cups will not fall out of the holder.
  4. Prior to adding the last reagents, the wells should be empty of rinse buffer (not dry, but empty of excess fluid).

Fluorescence (visual identification of the organisms)

  1. Since you will be looking for the actual organisms (cysts of Giardia and/or oocysts of Cryptosporidium), it is recommended that this test be performed on centrifuged stool (500 X g for 10 min) to increase the sensitivity of the test.
  2. 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.
  3. Gently rinse the reagents from the wells; do not squirt directly into the wells, but allow the rinse fluid to flow over the wells.
  4. 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.
  5. 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 (antigen detection, no centrifugation recommended)

  1. 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.
  2. It is always important to see the control line indicated as positive all the way across the membrane, not just at the edges.
  3. A positive test result is generally much lighter than the control line; this is normal.
  4. At the cutoff time to read the result, any color at all visible in the test area should be interpreted as a positive.
  5. Do not read/interpret the results after the time indicated in the directions; you may get a false positive result.

STOOL TESTING RECOMMENDED ORDERS

FECAL FIXATIVES

STOOL COLLECTION OPTIONS

REPORT COMMENTS

TIPS ON FECAL IMMUNOASSAYS

References:

  1. Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.
  2. Garcia, L.S. 2009. Practical Guide to Diagnostic Parasitology, 2nd Ed., ASM Press, Washington, D.C.