american association of bioanalysts proficiency testing

AAB PARASITOLOGY – SECOND QUADRIMESTER, 2020

1 AAB 2nd Quadrimester Parasitology, 2020

American Association of Bioanalysts Proficiency Testing

11931 Wickchester Ln., Ste. 200 Houston, TX 77043

800-234-5315 ♦ 281-436-5357 Q2 2020 Parasitology

Sample 6 Referees Extent 1 Extent 2 Total Code - Organism Frequency % No. % No. % No. % No.

525 – No parasites found None 100.0% 10 100.0% 5 95.8% 23 96.6% 28 544 – Endolimax nana 0.0% 0 4.2% 1 3.4% 1 Extent 1 flagging appears for failure to report 525. Extent 2 flagging appears for failure to report 525. There are no other acceptable codes.

SPECIMEN 6: FORMALIN: The specimen was a fecal suspension in 10% formalin for direct wet mount examination; concentration was not necessary. The specimen was to be examined for all parasites unstained, with iodine or other acceptable wet mount stain. There are no parasites in this specimen. Artifact material and/or yeast cells can be somewhat confusing when reviewing the wet preparation using the low power and even high dry power objectives. However, there is nothing present that can be specifically identified at 100X and 400X magnifications as a parasite, either helminth or protozoan. When having trouble seeing possible internal structures and/or morphologic details, tap the coverslip and get things to move around a bit. Also, reduce the light intensity if you’re not using iodine and drop the condenser to increase contrast. Iodine can be used to provide a bit more contrast; some laboratories routinely use iodine, while others do not. Too much light for wet preparations may prevent you from seeing parasites, particularly protozoa, which might be present in the specimen. Although occasionally a formalin preparation may contain very rare organisms, specimens selected for proficiency testing tend to have moderate to many organisms that are present for identification. Flagging appears in all extents for reporting other than “No Parasites Found” – participants performed very well in the examination of Sample 1 with an overall 96.6% correct response. One participant incorrectly reported Endolimax nana. SPECIMEN 6: PERMANENT SMEAR FOR STAINING: The permanent stained smear contains no parasites. Artifact material is rarely consistent in either size or shape. This specimen contains the normal stool debris, most of which has no standard shape or size. In contrast to examination of the wet preparation, remember to use plenty of light when examining the permanent stained smears and to use the oil immersion objective (100X). You need to cover at least 300 oil immersion fields before you call the stained smear negative. Flagging appears for reporting other than No parasites found (Referees 10/10, 100%).

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Sample 7 Referees Extent 1 Extent 2 Total Code - Organism Frequency % No. % No. % No. % No.

524 - parasite(s) found referred for ID Many 10.0% 1 28.6% 2 0.0% 0 6.7% 2

545 – Entamoeba coli Few to Many 90.0% 9 57.1% 4 100.0 23 90.0% 27

547 – Entamoeba histolytica 14.3% 1 0.0% 0 3.3% 1 Extent 1 flagging appears for failure to report 545 or 524. Extent 2 flagging appears for failure to report 545. Flagging appears in both extents for reporting other than 545 or 524.

SPECIMEN 7: FORMALIN: The specimen was a fecal suspension in 10% formalin for direct wet mount examination; concentration was not necessary. The specimen was to be examined for all parasites unstained, with iodine or other acceptable wet mount stain. This specimen contains Entamoeba coli cysts. The cysts were very typical with five or more nuclei being visible; some cysts contained the full complement of eight nuclei. Also, rare cysts contained splintered chromatoidal bars. The cyst shape varied from round to a bit more oval. The referees (10/10) identified few to many E. coli. Extent one flagging appears for failure to report Entamoeba coli or parasite(s) found, referred for ID. Extent 2 flagging appears for failure to report Entamoeba coli. Flagging also appears in both extents for reporting other than Entamoeba coli or parasite(s) found, referred for ID. Overall the participants performed at a higher percentage than earlier challenges with 93.1% reporting Entamoeba coli and 6.9% reporting parasite(s) found, referred for ID.

Entamoeba coli cysts (note chromatoidal bars in last image) Sample 8 Referees Extent 1 Extent 2 Total

Code - Organism Frequency % No. % No. % No. % No. 524 - parasite(s) found referred for ID 28.6% 2 0.0% 0 6.7% 2 565 – Hymenolepis diminuta 14.3% 1 0.0% 0 3.3% 1

566 – Hymenolepis nana Few to Many 100.0% 10 42.9% 3 100.0% 23 86.7% 26

574 – Hookworm 1 0 1 Extent 1 flagging appears for failure to report 566 or 524. Extent 2 flagging appears for failure to report 566. Flagging also appears in both extents for reporting other than 566, or 524.



SPECIMEN 8 FORMALIN: This specimen contains few to many Hymenolepis nana eggs; (10/10) of the referees reported correctly (100%), as did the majority of the participants (86.7%). The H. nana egg contains a distinct six-hooked embryo (oncosphere) with polar filaments lying between the egg shell and the oncosphere (see below). Extent 1 flagging appears for failure to report Hymenolepis nana or parasite(s) found, refer for ID; Extent 2 flagging appears for failure to report Hymenolepis nana; flagging also appears in both extents for reporting other than Hymenolepis nana, or Parasites found, refer for ID.

Hymenolepis nana eggs; note the polar filaments (arrows) Hymenolepis diminuta egg The two images (left, middle) show the typical six-hooked oncosphere within the egg shell (hooklets are within the circle) and the polar filaments that lie between the egg shell (arrows) and the oncosphere. The only other egg that looks somewhat similar is that of Hymenolepis diminuta, which contains the same type of oncosphere but no polar filaments (image on the right). Sample 9 Referees Extent 1 Extent 2 Total

Code - Organism Frequency % No. % No. % No. % No. 524 - parasite(s) found referred for ID 33.3% 2 0.0% 0 8.0% 2 547 – Entamoeba histolytica 50.0% 5 50.0% 3 31.6% 6 36.0% 9 548 – Entamoeba histolytica / Entamoeba dispar 50.0% 5 0.0% 0 68.4% 13 52.0% 13 549 – Iodamoeba brutschlii 16.7% 1 0.0% 0 4.0% 1 Extent 1 flagging appears for failure to report 547, 548 or 524. Extent 2 flagging appears for failure to report 547 or 548. Flagging also appears in both extents for reporting other than 547, 548 or 524.

SPECIMEN 9 (Digital Image): This permanent stained fecal smear demonstrates excellent examples of the true pathogen Entamoeba histolytica. Since most of the organisms selected do not contain ingested RBCs within the cytoplasm, Entamoeba histolytica/E. dispar group is considered a correct response. Extent 1 flagging occurs for failure to report Entamoeba histolytica, Entamoeba histolytica/E. dispar, or parasite(s) found, referred for ID. Flagging occurs in Extent 2 for failure to report Entamoeba histolytica or Entamoeba histolytica/E. dispar. Flagging also appears in both extents for reporting other than Entamoeba histolytica, Entamoeba histolytica/E. dispar or parasite(s) found, not identified referred for ID. THE MAJORITY OF THE ORGANISMS ARE TYPICALLY ENTAMOEBA HISTOLYTIC/E. DISPAR (nuclear characteristics and precysts).

http://parasite.org.au/pugh-collection/JpegsStamped/Hymenolepis%20nana%2003.jpg



If a positive fecal immunoassay specific for the true pathogen, Entamoeba histolytica, is positive or the trophozoites on the permanent stained slide contain ingested RBCs within the cytoplasm, it is acceptable to identify any of these organisms as Entamoeba histolytica; in this specimen most of the trophozoites do not contain ingested red blood cells. However, without the confirmation of a positive appropriate immunoassay, even with ingested RBCs, some experts indicate the correct identification could also be: Entamoeba histolytica/E. dispar (or Entamoeba histolytica/E. dispar group). However, if you report with both organism names rather than Entamoeba histolytica, a comment should be added indicating ingested RBCs were seen within the organism cytoplasm. If the RBCs are present, then the true pathogen, Entamoeba histolytica, can be reported (rare in this smear and not selected as one of the numbered examples. Example 1 contains a trophozoite containing a single nucleus with even nuclear chromatin, and a central karyosome that appears neat and compact. Note the pleomorphic RBCs in the background to the left. Example 2 contains a single trophozoite with the nucleus having evenly arranged peripheral chromatin and a central, compact karyosome. No ingested RBCs were seen within the cytoplasm. Example 3 contains a very typical Entamoeba histolytica/E. dispar precyst with a single enlarged nucleus and chromatoidal bars. Example 4 contains a very pale staining trophozoite. Note the typical nucleus. Example 5 is a single precyst with the typical enlarged nucleus and chromatoidal bars. Example 6 contains a typical later precyst with an enlarged nucleus and chromatoidal bars with smooth, rounded ends. Example 7 contains a trophozoite and a typical nucleus with even chromatin and a central, compact karyosome. Example 8 contains a typical nucleus. Example 9 does not appear to contain ingested RBCs, but note the typical nucleus and RBCs in the background. Example 10 contains a very typical precyst with an enlarged nucleus and chromatoidal bars. RESULTS: The referee laboratories (100%) and participants (96%) that identified Entamoeba histolytica and Entamoeba histolytica/E. dispar as being present. Although rare trophozoites were seen containing ingested RBCs, the examples were not as good as the organisms selected. This is an actual clinical smear and demonstrates the difficulties in making the correct identification. See additional tips below: 1. Although the nuclear peripheral chromatin may not always go all the way around (Entamoeba histolytica or Entamoeba histolytica/E. dispar, it is not clumpy and uneven like that seen in Entamoeba coli. 2. While the karyosome seen in Entamoeba coli tends to be somewhat large and blot-like, karyosomes seen in Entamoeba histolytica tend to be compact and neat. NOTE THAT THE POSITION OF THE KARYOSOME (CENTRAL or ECCENTRIC) IS NOT AS IMPORTANT AS THE APPEARANCE OF THE CHROMATIN AND KARYOSOME. Note the differences in the precysts (Entamoeba histolytica/E. dispar – single nucleus; Entamoeba coli – two nuclei).



Entamoeba coli E. coli precyst Entamoeba histolytica/E. dispar Entamoeba histolytica precyst at right Ingested (RBCs) ______________________________________________________________ Sample 10 Referees Extent 1 Extent 2 Total

Code - Organism Frequency % No. % No. % No. % No. 524 - parasite(s) found referred for ID 33.3% 2 0.0% 0 8.0% 2 534 – Giardia lamblia 100.0% 10 66.7% 4 100.0% 19 92.0% 23

Extent 1 flagging appears for failure to report 537, 539 or 524. Extent 2 flagging appears for failure to report 537 or 539. Flagging also appears in both extents for reporting other than 537, 539 or 524.

SPECIMEN 10 (Digital Image): This stained stool slide demonstrates excellent examples of Giardia lamblia cysts. The overall morphology is excellent with nuclei, median bodies, and axonemes being visible in some of the organisms. Remember that these organisms are very three-dimensional; it is mandatory that when reviewing patient material, you focus up and down. You may want to increase the magnification and contrast when examining these organisms. You will note the pale staining seen in this smear; overall this appearance of the Giardia trophozoites is very typical. The referees performed well (100%), while the participants also reported 100% (Giardia lamblia or parasite(s) found referred for ID. This is a good digital image to scan for practice – see if you can find other Giardia cysts that are not boxed. NOTE: In some publications, you may see Giardia lamblia designated as either G. intestinalis or G. duodenalis. For the present, we will continue to use the species name as G. lamblia. Example 1 contains an excellent Giardia cyst. The linear axonemes and curved median bodies are visible. Example 2 contains a single cyst, in which several nuclei and linear axonemes are visible. Example 3 contains two cysts; in the upper one the nuclei, linear axonemes and curved median bodies are visible. In the cyst on the right some of the curved median bodies are seen. Example 4 Although the nuclei are merely suggestive, the linear axonemes and curved median bodies are visible. Example 5 contains a single cyst with one nucleus visible and some suggestive axonemes. Example 6 contains a typical Giardia cyst. Two of the nuclei are visible and some suggestive curved median bodies. Example 7 contains two typical cysts. In the one on the left, nuclei, axonemes, and median bodies are visible. In the one on the right excellent median bodies and axonemes are visible. Example 8 contains an excellent Giardia cyst. Note the two nuclei, linear axonemes and suggestive curved median bodies.



Giardia lamblia cysts GENERAL COMMENTS:

If you are currently using one of the stool fixatives that contains a mercuric chloride substitute (zinc sulfate, etc.), remember that the proficiency testing specimens you receive for permanent staining have been preserved in PVA using the mercuric chloride fixative base. If you use the Trichrome or iron hematoxylin staining method for your mercuric chloride substitute fixatives, you may have eliminated the 70% alcohol/iodine step and the following 70% alcohol rinse step from your method. However, when you stain the proficiency testing fecal smears, you will need to incorporate the iodine step plus the next 70% alcohol rinse back into your staining protocol prior to placing your slides into the trichrome stain or iron hematoxylin stain. These two steps are designed to remove the mercury from the smear and then to remove the iodine; therefore, when your slide is placed into the Trichrome or iron hematoxylin stain, both the mercury and iodine are no longer present in the fecal smear. If you fail to incorporate these two steps into your staining protocol, the quality of your proficiency testing stained smears will be poor. With very rare exceptions, the organisms in any of the proficiency testing (PT) specimens that you are asked to identify will be few to many in number. The presence of a very rare organism probably reflects something that was not seen in the screening process. The purpose of the PT specimen is to provide sufficient parasite numbers (few to many) so that ALL of the participants see the same organisms. It is neither realistic nor practical to expect participants to find and identify organisms that are rare or very rare in number; this is not the purpose of the program. We appreciate the fact that in a patient specimen you would indicate all organisms seen, regardless of the numbers. However, in the PT specimens, you are being tested on those organisms that are present in “few” numbers or greater. You may be asked to quantitate the organisms as a “quality control check” on the “aliquotting” process used to prepare participant vials prior to shipment. The information provides data for review related to the consistency of organism numbers throughout the aliquotting process. In a clinical setting, quantitation of most of these organisms is not relevant and this information would not be added to the patient report. We encourage participants to report Blastocystis spp; however, these organisms are much easier to identify correctly from a permanent stained smear. Blastocystis is an extremely common parasite with a worldwide distribution. It is not uncommon for it to be the most frequently isolated parasite in epidemiological surveys. Prevalence varies widely from country to country and within various communities of the same country. In general, developing countries have higher percentages of the parasite than developed countries, and this has been linked to poor hygiene, exposure to animals, and consumption of contaminated food or water. Based on PCR-based genotype classification data, there may be approximately 10 or more different subtypes within the genus. Some subtypes are pathogenic and some are non-pathogenic. If no other pathogens are found, B. hominis may be the cause of patient symptoms. Confirmation of these subtypes and their pathogenic status may also explain why some patients are asymptomatic and some have clinical symptoms. In the future, it will be recommended that these organisms be reported as Blastocystis spp. Two report comments that should be used when this organism is reported are as follows: 1. The name Blastocystis hominis contains approximately 10 different organism subtypes, none of which can be differentiated on the basis of organism morphology; some subtypes are pathogenic and some are non-



pathogenic. If no other pathogens are found, B. hominis may be the cause of patient symptoms. The proper designation is Blastocystis spp. 2. Other organisms capable of causing diarrhea should also be ruled out.

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