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.
What are the tests illustrated below called (name and abbreviation or short name)?
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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): Fluorescence (visual identification of the organisms): Both filters = less fluorescence intensity, less visible artifacts
Single FITC filter = brighter fluorescence, more visible artifacts References: 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. Each Quiz has a two section format: the first section will present the Quiz topic and the second section will provide a discussion of the answer and/or various options in response to the Quiz situation presented to the user. In some situations, there may be more than one correct response.
The content within this site is made possible through the extensive contribution of Lynne S. Garcia, M.S., MT(ASCP), CLS(NCA), BLM(AAB), F(AAM), Director, Consultantation and Training Services (Diagnostic Medical Parasitology and Health Care Administration). For additional information, she can be contacted at LynneGarcia2@verizon.net. Reference: Garcia, L.S. 2015. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, D.C.
Enzyme immunoassays (antigen detection, no centrifugation recommended); the antigen will be found in the top fluid layer of the stool collection vial
Lateral Flow Cartridges/Rapids (antigen detection, no centrifugation recommended); the antigen will be found in the top fluid layer of the stool collection vial:
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