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 / 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.

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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 nana Few to Many 90.9% 10 32.0% 8 89.5% 34 567 Taenia sp. 4.0% 1 0.0% 0 580 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 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. The oocyst including the halo measures a bit over 5 microns. Some of the sporozoites are visible, but not that clear.  Example 5 contains one oocyst; some sporozoites are visible in the oocyst, but the morphology is not that clear.  Example 6 contains one oocyst that is darkly stained; no individual sporozoites are visible. Including the halo, the oocyst measures approximately 5 microns.  Example 7 contains a single oocyst measuring approximately 5 microns; the sporozoites are not that clear.  Example 8 contains a single oocyst. Including the shrinkage halo, the oocyst measures approximately 5 microns and the sporozoites are clearly visible.  Example 9 contains one oocyst, in which the individual sporozoites are clearly visible. The oocyst measures approximately 4.6 microns.  Example 10 contains a single oocyst. The sporozoites are clearly visible, and this oocyst exhibits very typical morphology. The size is approximately 5 microns.

Cryptosporidium sp. oocysts, some containing sporozoites (all oocysts measure 4-6 microns.

3 AAB 3rd Quadrimester Parasitology, 2015 Sample 5 Referees Extent 1 Extent 2 Total Code Organism Frequency % No. % No. % No. % No. 556 Plasmodium sp.; undetermined 18.2% 2 4.3% 1 19.0% 4 59.6% 31 555 Plasmodium sp.;refer for ID to spec level 54.5% 6 47.8% 11 47.6% 10 16.7% 8 524 parasite(s) found referred for ID 26.1% 6 0.0% 0 16.7% 8 558 Plasmodium malariae 27.3% 3 21.7% 5 33.3% 7 2.1% 1 Extent 1 flagging appears for failure to report 556, 555, 558 or 524. Extent 2 flagging appears for failure to report 556, 555 or 558.

Flagging also appears in both extents for reporting other than 556, 555, 558 and 524.

SPECIMEN 5 (Digital Image): This specimen contains Plasmodium malariae blood stages as the challenge organisms. The referees performed well with 11/11 (100%) reporting correctly, while the participants reported correctly. HOWEVER, it is unfortunate that only 3/11 of the participants and 7/21 of the participants actually reported the most accurate response: Plasmodium malariae. Extent 1 flagging appears for failure to report Plasmodium sp, undetermined, Plasmodium sp, refer for identification to species level, parasite(s) found, referred for ID, or Plasmodium malariae.  Example 1 contains one developing trophozoite/ring form; note the organism is stretched across the normal-sized RBC as a band form, very typical for P. malariae.  Example 2 also contains one trophozoite, with typical morphology as the band form; again note the small to normal-sized RBC. P. malariae tend to infect the older RBCs, while Plasmodium vivax and Plasmodium ovale tend to infect the young RBCS (they tend to be larger).  In Example 3 the trophozoite/band form is within a relatively small RBC.  In Example 4 the trophozoite displays very typical morphology as a band form with the red parasite nucleus at the bottom of the organism.  In Example 5 the developing schizont is visible, as are the developing merozoites within the parasite; note the small RBC.  In Example 6, the trophozoite/band form is very typical and is within a small RBC. Note the red nucleus.  In Example 7 the mature schizont is seen and contains the typical 8 merozoites. Although the merozoites are not arranged in the typical “daisy” shape, the number of 8 merozoites allows the identification as P. malariae.  In Example 8, the trophozoite/band form is very typical; it lies at one side of the RBC.  Example 9 demonstrates a developing gametocyte; again note the small RBC size.  In Example 10, the young trophozoite/band form is very typical; the parasite is within a normal sized RBC.  Example 11 demonstrates a developing trophozoite/band form; again note the RBC size and red/somewhat elongate parasite nucleus.  Example 12 demonstrates a developing band form at one side of the RBC; note the platelets in the background to the right.  In Example 13 the developing schizont is visible, as are the developing merozoites within the parasite; note the small RBC. The merozoites are not quite mature (do not yet appear as separate merozoites, each having its own nucleus and cytoplasm).  In Example 14 the developing schizont is more mature than the example in #13 and is clearly visible, as are the developing merozoites within the parasite; note the small RBC. The merozoites are not quite mature (appear as separate merozoites, each having its own nucleus and cytoplasm). However, from the parasite nuclei, it is clear there are 8 developing merozoites within the organism.

Plasmodium malariae (quartan malaria) 1. 72-hour cycle (long incubation period) 2. Tends to infect old cells 3. Normal to small size RBCs 4. No stippling 5. Thick ring, large nucleus 6. Trophozoite tends to form "bands" across the cell 7. Mature schizont contains 6-12 merozoites (typical number is 8)

Plasmodium malariae band form; P. malariae mature schizont (daisy configuration) Note the small RBCs.

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

4 AAB 3rd Quadrimester Parasitology, 2015 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 , 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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