Quiz 46
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)
- 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. 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.
- 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.
- 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)
- 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.
- 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.
- 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)
- 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 is generally 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.
STOOL TESTING RECOMMENDED ORDERS
References:
- Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.
- Garcia, L.S. 2009. Practical Guide to Diagnostic Parasitology, 2nd Ed., ASM Press, Washington, D.C.
Quizzes
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.
- Quiz 1
- Quiz 2
- Quiz 3
- Quiz 4
- Quiz 5
- Quiz 6
- Quiz 7
- Quiz 8
- Quiz 9
- Quiz 10
- Quiz 11
- Quiz 12
- Quiz 13
- Quiz 14
- Quiz 15
- Quiz 16
- Quiz 17
- Quiz 18
- Quiz 19
- Quiz 20
- Quiz 21
- Quiz 22
- Quiz 23
- Quiz 24
- Quiz 25
- Quiz 26
- Quiz 27
- Quiz 28
- Quiz 29
- Quiz 30
- Quiz 31
- Quiz 32
- Quiz 33
- Quiz 34
- Quiz 35
- Quiz 36
- Quiz 37
- Quiz 38
- Quiz 39
- Quiz 40
- Quiz 41
- Quiz 42
- Quiz 43
- Quiz 44
- Quiz 45
- Quiz 46
- Quiz 47
- Quiz 48
- Quiz 49
- Quiz 50
- Quiz 51
- Quiz 52
- Quiz 53
- Quiz 54
- Quiz 55
- Quiz 56
- Quiz 57
- Quiz 58
- Quiz 59
- Quiz 60
- Quiz 61
- Quiz 62
- Quiz 63
- Quiz 64
- Quiz 65
- Quiz 66
- Quiz 67
- Quiz 68
- Quiz 69
- Quiz 70
- Quiz 71
- Quiz 72
- Quiz 73
- Quiz 74
- Quiz 75
- Quiz 76
- Quiz 77
- Quiz 78
- Quiz 79
- Quiz 80
- Quiz 81
- Quiz 82
- Quiz 83
- Quiz 84
- Quiz 85
- Quiz 86
- Quiz 87
- Quiz 88
- Quiz 89
