STOOL IMMUNOASSAY OPTIONS

What are some of the immunoassay options available for stool protozoa?

Currently, there are immunoassays available for Giardia lamblia, Cryptosporidium spp., the Entamoeba histolytica/E. dispar group, and Entamoeba histolytica.  Reagents for the detection of Dientamoeba fragilis and the microsporidia are under development.

What methods are available commercially?

Direct fluorescence (DFA), enzyme immunoassay (EIA), and cartridge formats (EIA or a solid phase qualitative immunochromatographic procedure) are currently available.

Why might someone want to use a fecal immunoassay option?

If the most common organisms found in the area are Giardia, Cryptosporidium and/or the Entamoeba histolytica/E. dispar group, then fecal immunoassays are certainly options.  Specific patients/history/symptoms would suggest the use of fecal immunoassays.  Both the O&P examinations and fecal immunoassays are recommended for a laboratory test menu – both would be orderable, billable procedures.  Refer to the table in this section – stool orders.

How would stool immunoassay requests fit into a lab that also performs Ova and Parasite (O&P) examinations?

Any diagnostic laboratory performing routine parasitology testing should offer both options:  routine O&P examinations and the fecal immunoassays.

What do you mean by the “routine O&P examination”?

This test includes (fresh material- liquid or semiliquid stools) a direct wet exam, the concentration and the permanent stain; preserved specimens would require a concentration and permanent stained smear (no direct wet mount is required).

How would you fit the fecal immunoassays into your laboratory?

Per separate requests, you could offer the routine O&P examination and you could also offer on request the stool immunoassay option.  Refer to the table in this section – stool orders.

What type of educational initiatives would have to be undertaken prior to offering these options?

Physicians would need to know the pros and cons of ordering either the immunoassay or the routine O&P examination.  Specific recommendations can be found in the table in this section – stool orders.

What are some of the pros and cons for the fecal immunoassays?  (Also refer to the table on “Tips for Performing Fecal Immunoassays”

Pros:  

• Depending on the format selected, the immunoassays are fast and relatively simple to perform.
• The result can “rule in or out” very specific organisms.
• If the patient becomes asymptomatic at the time the immunoassay is negative, additional testing may not be necessary.
• May help reduce personnel costs (time to perform procedures).
• The fecal immunoassays are more sensitive than the routine O&P exams
             

Cons:  

• The fecal immunoassay kits test only for selected organisms.
• Depending on the format, they might be somewhat complex to perform.
• Test requests may not justify certain formats (cost, equipment, or training).
• It is critical that the physician realize that a negative immunoassay will not rule out all possible parasitic etiologic agents causing diarrhea.
• In the case of Giardia lamblia, it may require a fecal immunoassay on two different stool specimens in order to get a positive result.

Can the fecal immunoassays be used for duodenal fluid (giardiasis)?

First of all, the tests have not been approved/validated for this type of specimen.  Also, the duodenal fluid/aspirate would contain primarily the trophozoite form, not the cyst stage for which the reagents have been designed.  An example:  if one is using the FA (rather than EIA or cartridge) the trophs may appear to be fluorescent at a very pale 1+, while the cysts are a strong 3-4+.  So, while there may be a few antigenic sites that are shared by the cysts and trophs, the commercial tests for Giardia are picking up the cyst antigens.

You can "try" the reagents on these specimens, but if the result is negative, the result in no way has "ruled giardiasis out." I would just recommend testing stool; this approach might avoid the need for duodenal aspirate testing altogether. 

Comment on the use of the Giardia fecal immunoassay; how many specimens should be tested before assuming the patient is negative?

Since the evidence indicates that Giardia is shed sporadically, and that more than one immunoassay might be required to diagnose the infection, the recommendation is similar to that seen for stool collection for the routine O&P examination:  perform the immunoassay on two different stools (assuming the first specimen is negative), collected within no more than a 10 day period - a good collection schedule would be day one, then day three or four.  That way, one would assume one of those collections would yield a positive if the organisms (in sufficient numbers) are present.  However, you also have to remember that if the patient is a carrier with a low organism load, even the second immunoassay might be negative.

Although an FA for Cryptosporidium/Giardia was negative, the O&P concentrate showed Giardia trophs on the wet mount. Might we miss positive Giardia if only the FA is ordered? Do Also, do any of the EIA antigen tests detect both trophs and cysts and should we switch to one of those?

Although the antibody in the immunoassay kits is to the cyst antigen (primarily, but it is a polyclonal reagent), in some of the kits the trophs do fluoresce, but much less intense (around a 1+, maybe even a 2+, thus indicating some shared epitopes).  In almost all patients, there will be a combination of cysts and trophs, unless the patient has active diarrhea and is passing only trophs (no time for cysts to form with rapid passage through the GI tract).  However, most patients will have cysts, as well, and the results (if above the test limits for sensitivity) will be positive.  The situation you describe can happen, but it's probably not that common.  However, with ONLY trophs present, you may get a negative result.  Some of the kits tend to provide a bit higher fluorescence with the troph, but it varies. 

After the patient has been treated, how long will the Giardia antigen test remain positive?

It has been recommended that you test about 7 days after therapy, hopefully to avoid picking up residual antigen.  However, if you wait too long (several weeks), you always run the risk of picking up antigen from a possible reinfection.  Some also feel that low antigen levels can be found for up to about 2 weeks.  A good time frame for retesting would probably be about 7-10 days after therapy.  If the first specimen at 7 days is still positive, then you could retest at 10-12 days.  Also, we know that the immunoassays (for diagnosis) may not pick up low antigen loads (organism shedding issues), thus the recommendation for diagnostic immunoassay testing for giardiasis has been changed and recommends performing immunoassay testing on one additional stool specimen (if the first one is negative).  The testing on two different stool specimens should be performed within about 3-5 days.

Why do fecal immunoassay kits that test for either Entamoeba histolytica or the Entamoeba histolytica/E. dispar group require fresh or frozen stools?

Unfortunately, at the present time, these reagents will not function properly on preserved fecal specimens.  Although the manufacturers are trying to develop such kits, they are not yet available.

How long will antigen survive in fresh stools? 

It is recommended that fresh stools be tested within 24 h of collection; they can be stored overnight in the refrigerator.  They can also be frozen or preserved in 10% formalin prior to testing (both freezing and/or formalin preservation methods will preserve antigen for long periods of time – even years).