AAB PARASITOLOGY – FIRST QUADRIMESTER, 2021

American Association of Bioanalysts Proficiency Testing 11931 Wickchester Ln., Ste. 200 Houston, TX 77043 800-234-5315 ♦ 281-436-5357

Q1 2021 Parasitology

Sample 1 Referees Extent 1 Extent 2 Total Code - Organism Frequency % No. % No. % No. % No. 525 – No parasites found None 50% 5 11.1% 1 44.4% 8 33.3% 9 544 – Endolimax nana 44.4% 4 11.1% 2 22.2% 6 533 – Few 40.0% 4 0.0% 0 27.8% 5 18.5% 5 546 – Entamoeba hartmanii Few 10.0% 1 11.1% 1 11.1% 2 11.1% 3 524 – parasite(s) found referred for ID 33.3% 3 0.0% 0 11.1% 3 553 – Cryptosporidium sp. 0.0% 0 5.6% 1 3.7% 1 Due to a lack of consensus, Sample 1 was not evaluated this event. The intended organism was 533-Dientamoeba fragilis.

SPECIMEN 1: FORMALIN: Specimen 1A 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. The specimen contains Dientamoeba fragilis trophozoites. Typically, it is very difficult if not impossible to identify these organisms from a wet mount examination.

SPECIMEN 1: PERMANENT SMEAR FOR STAINING: Specimen 1B was the smear to be stained and examined. This specimen contains Dientamoeba fragilis. Due to a lack of participant consensus, Specimen 1 was not evaluated for this event. However, 40% of the Referees correctly reported Dientamoeba fragilis. Many Referees (50%) and Participants (33.3%) reported the specimen as negative.

Entamoeba coli trophozoites Entamoeba coli cysts (shrunk and normal) Sample 2 Referees Extent 1 Extent 2 Total Code - Organism Frequency % No. % No. % No. % No. 534 – Gardia lamblia Few to Many 100.0% 10 80.0% 8 89.5% 17 86.2% 25 541 – Blastocystis hominis Few 10.0% 1 10.0% 1 5.3% 1 6.9% 2 545 – Entamoeba coli 0.0% 0 5.3% 1 3.4% 1 524 – parasite(s) found referred for ID 10.0% 1 0.0% 0 3.4% 1

Extent 1 flagging appears for failure to report 534 or 524. Extent 2 flagging appears for failure to report 534.

Flagging appears in both extents for reporting other than 534, 541 or 524.

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SPECIMEN 2: 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 contained Giardia lamblia. The referees reported 100% Giardia lamblia. Flagging occurs in Extent 1 for failure to report Giardia lamblia or Parasite(s) found, referred for ID. Extent 2 flagging occurs for failure to report Giardia lamblia. Flagging also appears in both extents for reporting other than Giardia lamblia, Blastocystis spp. or Parasite(s) found, referred for ID. Participants reported 86.2% Giardia lamblia. The G. lamblia cyst morphology was very typical with a round to oval shape and the presence of multiple nuclei, curved median bodies, and linear axonemes. Representative images can be seen below. The internal structures often appear very refractile in the wet preparation examination. Although some of the cysts are shrunk within the cyst wall, there are plenty of organisms that can be easily identified.

Very high magnification G. lamblia cyst G. lamblia trophozoite G. lamblia cyst G. lamblia trophozoite Wet Mounts Permanent Stained Smear Not used in this challenge.

The Blastocystis spp. central body forms were very typical with a round to somewhat ovoid shape, the presence of a central area with multiple small nuclei arranged around the central body area within the cell membrane. See comparative images below. The Referees (10%) and the Participants (6.9%) also correctly reported Blastocystis.

Sample 3 Referees Extent 1 Extent 2 Total Code - Organism Frequency % No. % No. % No. % No. 525 – No parasites found None 100.0% 10 88.9% 8 100.0% 17 96.2% 25 524 – parasite(s) found referred for ID 11.1% 1 0.0% 0 3.8% 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.

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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. 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 , 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 Seen” – participants performed very well in the examination of this specimen with an overall 96.2% correct response.

Sample 4 Referees Extent 1 Extent 2 Total Code - Organism Frequency % No. % No. % No. % No. 549 – Iodamoeba butschlii 100.0% 10 66.7% 4 100.0% 15 90.5% 19 524 – parasite(s) found referred for ID 33.3% 2 0.0% 0 9.5% 2

Extent 1 flagging appears for failure to report 549 or 524. Extent 2 flagging appears for failure to report 549.

Flagging also appears in both extents for reporting other than 549 or 524.

SPECIMEN 4 (Digital Image): This specimen (digital image of routine trichrome stain) contains Iodamoeba bütschlii trophozoites and cysts. The referees (10/10) reported correctly, identifying Iodamoeba bütschlii. Extent 1 flagging appears for failure to report Iodamoeba bütschlii or parasite(s) found referred for ID. Extent 2 flagging appears for failure to report Iodamoeba bütschlii. Flagging also appears in both extents for reporting other than Iodamoeba bütschlii or parasite(s) found referred for ID. The participants performed well with an overall percent of 90.5%. Because the trophozoite can resemble that of Endolimax nana, most identifications of Iodamoeba bütschlii will be based on the typical cyst morphology.

When examining the permanent stained smears, it is important to read at least 300 fields using the oil immersion objective (100X objective) for a total magnification of X1000). This examination is in contrast to the concentration sediment wet preparation, for which at least 1/3 to ½ of the coverslip should be examined using the high dry objective (40X) and the entire 22x22 mm coverslip should be examined using the low power objective (10X).

NOTE: MANY OF THE CYSTS CONTAIN A “BASKET NUCLEUS” – THIS MAY NOT BE TYPICAL FOR CLINICAL SPECIMENS (VARIES TREMENDOUSLY AMONG CYSTS).

 Example 1 contains a single cyst, which has the typical large karyosome. Note the fine granules at the bottom of the karyosome; this represents the “basket” nucleus, which can also occur in the trophozoite although is more commonly seen in the cyst form. Because the trophozoite can often resemble that of Endolimax nana, identification of Iodamoeba bütschlii is normally based on the cyst form. In this cyst, the vacuole (left) is somewhat obscured by debris.  Example 2 contains one typical cyst; note the large red/purple karyosome and the pale glycogen vacuole (bottom of image).  Example 3 contains one typical cyst with the clear glycogen vacuole lying to the left of the karyosome and large red/purple karyosome (granules to the left of the karyosome represent the “basket” nucleus).  Example 4 contains one cyst; note that the pale glycogen vacuole is distorted and appears collapsed and the large karyosome with many granules at the top (“basket nucleus”).

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 Example 5 contains a single cyst, which has a very large karyosome and typical glycogen vacuole. The nucleus is a typical “basket” nucleus with extra pale granules at the top of the karyosome.  Example 6 contains one typical cyst with a glycogen vacuole; note the basket nucleus (extra chromatin is at the bottom of the karyosome).  Example 7 contains a single cyst (glycogen vacuole is somewhat out of focus – left of karyosome). The karyosome is very typical; note the large number of granules right below/left of the karyosome (“basket nucleus”).  Example 8 contains a typical trophozoite (random shape). The large karyosome is typical.  Example 9 contains one cyst with a glycogen vacuole and typical large karyosome; no vacuole.  Example 10 contains a single cyst with an excellent karyosome and large glycogen vacuole (somewhat distorted).  Example 11 contains a single trophozoite containing much debris in the cytoplasm; the karyosome is large and typical. Note the trophozoite outline is somewhat difficult to see.  Example 12 contains a typical cyst with a large karyosome and glycogen vacuole.  Example 13 contains one cyst with a typical glycogen vacuole and typical large karyosome. Note the granules above the karyosome (“basket nucleus”).  Example 14 contains a single cyst with an excellent karyosome and large glycogen vacuole (somewhat pale and out of focus). There are numerous granules to the left of the karyosome (“basket nucleus”)

Iodamoeba bütschlii cysts Iodamoeba bütschlii trophozoite

______Sample 5 Referees Extent 1 Extent 2 Total Code - Organism Frequency % No. % No. % No. % No. 537 – sp. 60.0% 6 75.0% 3 53.3% 8 57.9% 11 538 – Trypanosoma cruzi 40.0% 4 0.0% 0 40.0% 6 31.6% 6 524 – parasite(s) found referred for ID 25.0% 1 0.0% 0 5.3% 1 557 – Plasmodium falciparum 0.0% 0 6.7% 1 5.3% 1

Extent 1 flagging appears for failure to report 537, 538 or 524. Extent 2 flagging appears for failure to report 537 or 538. Flagging also appears in both extents for reporting other than 537, 538 or 524.

SPECIMEN 5 (Digital Image): This stained thin blood films contains Trypanosoma cruzi trypomastigotes. They are very characteristic and have the undulating membrane, the central nucleus and the large from which the undulating membrane originates at the posterior end and protrudes beyond the body at the anterior end. Although not everyone identified the organisms to the species level, they were identified as Trypanosoma sp. or Trypanosoma cruzi. The typical “C” trypomastigote shape was evident in some of the organisms, as was the undulating membrane. The nucleus is situated in the center of the body, with a large oval kinetoplast located at the posterior extremity. The kinetoplast consists of a small blepharoplast and a large oval parabasal body. A flagellum arises from the blepharoplast and extends along the outer edge of an undulating membrane until it reaches the anterior end of the body, where it projects as a free flagellum. Chagas’ disease is a caused by the protozoan hemoflagellate Trypanosoma cruzi, which is transmitted to humans mainly by “kissing bugs” (: Reduviidae) throughout skin contact and mucous

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membranes with feces and other secretions of these insects. Other ways of transmission are oral, congenital, blood transfusion, organ transplant and laboratory accidents. Trypanosoma cruzi multiply as amastigotes in tissue, while the African trypanosomes (Trypanosoma gambiense/T. rhodesiense) multiply in the blood. Trypomastigotes may be detected in the blood in young children; however, in chronic disease, this stage is rare or absent except during fever episodes. They may be detected by using thin and thick blood films or buffy coat concentration techniques (most sensitive). Any blood stains can be used for trypomastigote and amastigote stages. T. rangeli trypomastigote may have to be differentiated from those with T. cruzi. T. cruzi trypomastigotes are usually ‘‘C’’ or ‘‘U’’ shaped on fixed blood films and have a large oval kinetoplast at the posterior end. T. rangeli trypomastigotes have a much smaller kinetoplast near the posterior end Referee laboratories (6/10) correctly reported the presence of Trypanosoma cruzi and (4/10) reported Trypanosoma sp. for a total of 100%. Participants performed very well, with correct responses being 89.5%. Extent 1 flagging appears for failure to report Trypanosoma sp., Trypanosoma cruzi or Parasites found, No ID (Participants: 94.8%). Extent 2 flagging appears for failure to report Trypanosoma sp. or Trypanosoma cruzi.

ALTHOUGH THE UNDULATING MEMBRANE IS NOT VISIBLE IN THESE ORGANISMS, THE OVERALL MORPHOLOGY (SHAPE, NUCLEUS, LARGE KINETOPLAST) CONFIRMS THE CORRECT IDENTIFICATION.

 Example 1 contains a typical trypomastigote (trypanosome) with the large kinetoplast at the posterior end (left). The structure at the right is the nucleus.  Example 2 contains one trypomastigote with the typical large kinetoplast at the right end. This trypomastigote exhibits the curved shape that is more commonly associated with the African trypomastigotes.  Example 3 contains three trypomastigotes showing the typical characteristics. However, the overall staining is pale.  Example 4 contains one trypomastigote. In this case the nucleus is visible at the top, while the kinetoplast is seen at the bottom.  Example 5 contains one trypomastigote; the nucleus (top) and kinetoplast (bottom) are clearly visible.  Example 6 contains one trypomastigote with the nucleus at the top and the kinetoplast at the bottom. Note the large number of organisms in the background.  Example 7 contains one trypomastigote with the “C” shape; the nucleus and kinetoplast are visible.  Example 8 contains a single trypomastigote with the typical kinetoplast and nucleus. Note the kinetoplast appears to be protruding from the organism; this is often seen in T. cruzi trypomastigotes.  Example 9 contains two typical trypomastigotes with characteristic morphology.  Example 10 contains two trypomastigotes that exhibit typical morphology.  Example 11 contains one trypomastigote with the nuclear color appearing to be spread out; the kinetoplast is clearly seen at the right.  Example 12 contains one trypomastigote with the “C” shape; the nucleus and kinetoplast are visible.  Example 13 contains a single trypomastigote with the typical kinetoplast (bottom) and nucleus (top).  Example 14 contains one trypomastigote with characteristic morphology; the kinetoplast appears to be a bit smaller in this organism.  Example 15 contains a single trypomastigote that exhibits typical morphology.

Trypanosoma gambiense/rhodesiense Trypanosoma cruzi* *Note the large kinetoplast (circle) in Trypanosoma cruzi trypomastigote. Also note the undulating membrane in the organism on the right (arrows).

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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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