4.3 FECAL IMMUNOASSAYS FOR INTESTINAL PROTOZOA

Fecal immunoassays for antigen detection are generally simple to perform and allow a large number of tests to be performed at one time, thereby reducing overall costs. A major disadvantage of antigen detection in stool specimens is that the method can detect only one or two pathogens at a time. The routine ova-and-parasite (O&P) examination must be performed in order to detect other parasitic pathogens. The current commercially available antigen tests have excellent sensitivity and specificity compared with those of routine microscopy.

Current formats include the ELISA, the fluorescence assay (FA), and the rapid membrane flow immunochromatographic cartridges. Sensitivity and specificity are comparable among the various formats and diagnostic kits currently available. The selection of any particular immunoassay format will often depend on the work flow options within the laboratory; different laboratories may select different formats for the rapid tests available. The methods are different, but the results are comparable

For diagnosis of amebiasis, antigen-based fecal immunoassays have several significant advantages over other methods currently used: (i) some of the assays can differentiate the true pathogen Entamoeba histolytica from the nonpathogen Entamoeba dispar, (ii) they have excellent sensitivity and specificity, (iii) they are readily usable by most laboratory personnel, and (iv) the assays have potential use in situations such as waterborne outbreaks. Because there are distinct genetic differences between E. dispar and E. histolytica, commercial kits have been developed to detect their presence and differentiate them in clinical samples. However, current antigen detection tests require the examination of fresh or frozen (not preserved) stool specimens, while many laboratories have switched to stool collection methods using various preservatives.

A number of commercially available immunoassay kits are available for Cryptosporidium spp. and are more sensitive and specific than routine microscopic examination of smears stained by modified acid-fast staining. Stool specimens may be fresh, frozen, or fixed; however, polyvinyl alcohol (PVA)-fixed specimens are unacceptable for use in fecal immunoassays.

For Giardia duodenalis (G. lamblia, G. intestinalis), antigen detection tests are reliable and more sensitive and specific than the routine O&P examination. Commercial immunoassay kits are readily available. Users have to evaluate which kit format will be most useful for their own laboratories. Some of the kits may not detect both trophozoites and cysts of Giardia but may be selective for only one life cycle stage; most antibodies used for testing are designed to detect the cyst form. Because there are some shared antigens between trophozoites and cysts, occasionally positive reactions may be obtained for specimens primarily containing trophozoites. However, this situation could also lead to a false-negative result; fluorescence for the trophozoites tends to be very pale.

Although not currently available commercially within the United States, several reagents are in various developmental phases. These include antigen detection kits for Dientamoeba fragilis, Blastocystis spp., Cyclospora cayetanensis, and various species of the microsporidia.

Some comments that may be helpful in performing various immunoassay formats are provided in Table 9.10.2–A10 to assist 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 duodenalis (G. lamblia, G. intestinalis), the Entamoeba histolytica/E. dispar group, Entamoeba histolytica, and Cryptosporidium spp. Based on the published literature, fecal immunoassays are more sensitive and specific than the routine O&P examination; this is particularly true for Giardia duodenalis (G. lamblia, G. intestinalis). However, unlike the O&P examination, which 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 to 8ºC and should be tested within 48 h, or they should be frozen at −20 to −70ºC (freezing is not acceptable for the FA method—the freeze-thaw cycle will damage organisms). Stool specimens preserved in 10% formalin, merthiolate-formalin, sodium acetate-acetic acid-formalin, or single-vial fixatives may be refrigerated (2 to 8ºC) or stored at room temperature (20 to 25ºC) 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.

REFERENCE

1. Garcia LS. 2016. Diagnostic Medical Parasitology, 6th ed. ASM Press, Washington, DC.

Fecal antigen detection method options
EIAs	Fluorescence assays	Rapid cartridge assays (lateral flow membrane)
EIAs (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. Do not 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 [microscopy] 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 sediment (500 × g for 10 min) to increase the sensitivity. Remember to thin out the smear; make sure the slides are thoroughly dry before adding reagents. The slides can be placed in a 35°C incubator for about 30 min 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 the glass. It is better to let the slides 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. Not all clinical specimens will provide the 3+ to 4+ fluorescence that is often seen in the positive control. Also, from time to time, you may see fluorescing bacteria and/or some yeasts in certain patient specimens. This is not that common, but the shapes can be distinguished from Giardia cysts and/or Cryptosporidium oocysts. Rarely, one might see the faint outlines of a Giardia trophozoite (they do not fluoresce as brightly as the cysts)If the trophozoite is seen, the result can be interpreted as “positive”. 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 FITCa (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 around the Giardia cysts and Cryptosporidium oocysts that can be seen using both filter systems. Both filters = less fluorescence intensity, fewer 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 (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 the 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 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.

a FITC, fluorescein isothiocyanate.

SUPPLEMENTAL READING

Garcia LS, Shimizu RY. 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.

Garcia LS, Shimizu RY. 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.

Hanson KL, Cartwright CP. 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.

Isenberg HD (ed). 2004. Clinical Microbiology Procedures Handbook, 2nd ed, vol 2, p 9.0.1– 9.10.8.3. ASM Press, Washington, DC.

Wilson M, Schantz PM. 2000. Parasitic immunodiagnosis, p 1117–1122. In Strickland GT (ed), Hunter’s Tropical Medicine and Emerging Infectious Diseases, 8th ed. WB Saunders Co, Philadelphia, PA.