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Aab Parasitology PROFICIENCY TESTING SERVICE AMERICAN ASSOCIATION OF BIOANALYSTS 205 West Levee St. Brownsville, TX 78520-5596 800-234-5315 281-436-5357 Fax 713-781-5008 PARTICIPANT STATISTICS PARASITOLOGY THIRD QUADRIMESTER 2015 Sample 1 Referees Extent 1 Extent 2 Total Code Organism Frequency % No. % No. % No. % No. 586 Fasciola hepatica/Fasciolopsis buski Few 9.1% 1 6.5% 2 9.1% 3 7.8% 5 533 Dientamoeba fragilis Few to Many 72.7% 8 0.0% 0 39.4% 13 20.3% 13 534 Giardia lamblia 3.2% 1 3.0% 1 3.1% 2 544 Endolimax nana Few 18.2% 2 0.0% 0 18.2% 6 9.4% 6 546 Entamoeba hartmanni Few 9.1% 1 3.2% 1 12.1% 4 7.8% 5 553 Cryptosporidium sp. 0.0% 0 3.0% 1 1.6% 1 524 parasite(s) found referred for ID Few to Many 9.1% 1 58.1% 18 3.0% 1 29.7% 19 525 No parasites found 9.1% 1 29.0% 9 12.1% 4 20.3% 13 Due to a lack of participant and referee consensus, Sample 1 was not evaluated this event. Intended result was Dientamoeba fragilis. Extent 1 Acceptable results are 533, 586 or 524. Extent 2 Acceptable results are to report 533 or 586. All acceptable results are 533, 586, 544 and 524. SPECIMEN 1: 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 a few Fasciola hepatica/Fasciolopsis buski eggs. These eggs cannot be differentiated on the basis of morphology; the sizes and shapes are almost identical. In actual clinical practice, these eggs also resemble very closely those of some other more rare trematodes. Only one of the referees (1/11) identified the presence of these eggs. This finding was incidental and not the main challenge for Specimen 1 (see permanent stained slide information below). The key challenge for this specimen was Dientamoeba fragilis, a protozoan flagellate that is extremely difficult to identify on the wet mount. Fasciola hepatica/Fasciolopsis buski eggs (note the open operculum on the right egg). SPECIMEN 1: PERMANENT STAINED SLIDE: This smear contains mod – many Dientamoeba fragilis trophozoites; this organism was the intended challenge. These organisms can sometimes be confused with Endolimax nana, particularly if the D. fragilis trophozoite contains only a single nucleus (see images below). Extent one acceptable responses are Dientamoeba fragilis or parasite(s) found, referred for ID. Extent 2 acceptable results are Dientamoeba fragilis. All acceptable results include Dientamoeba fragilis, Fasciola hepatica/Fasciolopsis buski, Endolimax nana, or parasite(s) found referred for ID. Since D. fragilis is considered a pathogen, it is very important that laboratories be able to identify this organism. As mentioned above, D. fragilis can sometimes be confused with E. nana; however, confusion with E. hartmanni is rare and in this specimen was not an issue. The overall morphology of E. hartmanni is very typical (see images below). The nucleus of E. hartmanni is very precise and appears like a “target” with even nuclear chromatin and a dot-like central karyosome. Confusion of D. fragilis with E. nana is possible when the D. fragilis nucleus is not completely fragmented like those seen images 1 and 2 and “clearing” appears to be in the center of the nucleus (see the E. nana image below). However, again it is important to emphasize the clinical relevance of D. fragilis – this organism is pathogenic and impacts patient care. 1 AAB 3rd Quadrimester Parasitology, 2015 1,2: Dientamoeba fragilis 3: Endolimax nana 4,5: Entamoeba hartmanni Sample 2 Referees Extent 1 Extent 2 Total Code Organism Frequency % No. % No. % No. % No. 524 parasite(s) found referred for ID 60.0% 15 0.0% 0 1.6% 1 566 Hymenolepis nana Few to Many 90.9% 10 32.0% 8 89.5% 34 567 Taenia sp. 4.0% 1 0.0% 0 580 Trichuris trichiura Few to Many 9.1% 1 0.0% 0 5.3% 2 556 Plasmodium sp.; undetermined 0.0% 0 2.6% 1 23.8% 15 541 Blastocystis hominis 0.0% 0 2.6% 1 69.8% 44 565 Hymenolepis diminuta cover slip 4.0% 1 0.0% 0 4.8% 3 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, 580 and 524. SPECIMEN 2: FORMALIN: This specimen contains few to many Hymenolepis nana eggs; (10/11) of the referees reported correctly (90.9%), as did the majority of the participants. One referee and 2 participants also reported Trichuris trichiura eggs. 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, Trichuris trichiura, 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 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 3 Referees Extent 1 Extent 2 Total Code Organism Frequency % No. % No. % No. % No. 524 parasite(s) found referred for ID 3.7% 1 2.6% 1 0.0% 0 525 No parasites found N/A 100.0% 11 92.6% 25 94.9% 37 100.0% 34 522 Invalid response 3.7% 1 2.6% 1 0.0% 0 Extent 1 flagging appears for failure to report 525. Extent 2 flagging appears for failure to report 525. There are no other codes allowed. SPECIMEN 3 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. 2 AAB 3rd Quadrimester Parasitology, 2015 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, positive specimens selected for proficiency testing tend to have moderate to many organisms that are present for identification. Flagging appears in both extents for reporting other than “No Parasites Found” – participants performed very well in the examination of Sample 3 with an overall correct response of >90% for the participants and 100% for the referees. Sample 4 Referees Extent 1 Extent 2 Total Code Organism Frequency % No. % No. % No. % No. 554 Cystoisospora belli 0.0% 0 3.7% 1 42.9% 3 524 parasite(s) found referred for ID 36.8% 7 7.4% 2 42.9% 3 553 Cryptosporidium sp. 100.0% 11 63.2% 12 88.9% 24 14.3% 1 Extent 1 flagging appears for failure to report 554 or 524. Extent 2 flagging appears for failure to report 554. Flagging also appears in both extents for reporting other than 554 and 524. SPECIMEN 4 (Digital Image): This specimen (digital image of modified acid-fast stain) contains Cryptosporidium sp. oocysts. The referees (11/11) reported correctly, identifying Cryptosporidium sp. oocysts. Extent 1 flagging appears for failure to report Cryptosporidium sp. or parasite(s) found referred for ID. Extent 2 flagging appears for failure to report Cryptosporidium sp. Flagging also appears in both extents for reporting other than Cryptosporidium sp. or parasite(s) found referred for ID. Overall, participants and referees performed very well with this challenge. When examining the permanent stained smears, (modified acid fast stain) it is important to read at least 300 fields using the oil immersion objective (100X objective) for a total magnification of X1000). When reviewing the designated regions indicated below, notice the large number of oocysts that appear in the background of the slide. It is also important to measure these organisms to arrive at the correct identification. Example 1 contains two oocysts, both of which measure less than 5 microns. Note: sporozoites can be clearly seen, particularly in the oocyst in the upper left. Example 2 contains one oocyst, which appears a bit shrunk inside the oocyst wall. The size, including the shrinkage halo, is a bit over 5 microns. Although the sporozoites are visible, they are not as clear as those seen in Example 1. Example 3 contains a single oocyst, in which the sporozoites are clearly visible. The oocyst measures approximately 5 microns. Example 4 contains one oocyst that appears to be somewhat shrunk within the oocyst wall.
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