American Association of Bioanalysts 3Q2019.pdf · 546 - Entamoeba hartmanni Rare 18.2% 2 0 7.4% 2 4.5% 2 Extent 1 flagging appears for failure to report 544, 526 or 524. Extent 2

AAB PARASITOLOGY – THIRD QUADRIMESTER, 2019

1 AAB 3rd Quadrimester Parasitology, 2019

American Association of Bioanalysts

5615 Kirby Drive, Suite 870 Houston, TX 77005

800-234-5315 ♦ 281-436-5357 Q3 2019 Parasitology

Sample 11 Referees Extent 1 Extent 2 Total Code - Organism Frequency % No. % No. % No. % No.

525 - No parasites found 18.2% 2 47.0% 8 48.1% 13 47.7% 21 544 - Endolimax nana Few to Many 72.7% 8 11.8% 2 44.4% 12 31.8% 14 524 - parasite(s) found referred for ID 41.2% 7 0.0% 0 15.9% 7 546 - Entamoeba hartmanni Rare 18.2% 2 0 7.4% 2 4.5% 2 Extent 1 flagging appears for failure to report 544, 526 or 524. Extent 2 flagging appears for failure to report 544 or 546. Flagging also appears in both extents for reporting other than 544, 546 or 524

SPECIMEN 11: 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 Endolimax nana cysts and trophozoites. Entamoeba hartmanni was also reported. Although the overall morphology of E. nana was best seen in the permanent stained smear, the wet mount morphology is typical. SPECIMEN 11: PERMANENT SMEAR FOR STAINING: Specimen 1B was the permanent stained smear. This specimen contains Endolimax nana. The E. nana cyst morphology was very typical with a round to oval shape and the presence of four karyosomes. 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. There were some rare trophozoites present. E. hartmanni was also present (see images below). Referee laboratories (8/11 – 72.7%) reported few to many E. nana, while two referees reported rare E. hartmanni (18.2%). Some participants failed to actually identify E. nana, while others reported the specimen as a negative (47.7%). Extent 1 flagging appears for failure to report Endolimax nana, Entamoeba hartmanni, or Parasites found, referred for ID. Extent 2 flagging appears for failure to report Endolimax nana or Entamoeba hartmanni. Flagging also appears in both extents for reporting other than Endolimax nana, Entamoeba hartmanni. or Parasites found, referred for ID.

Very high magnification

E. nana trophozoite E. nana cyst E. hartmanni trophozoite E. hartmanni cyst




530 - Balantidium coli Few to Many 90.9% 10 25.0% 4 78.6% 22 59.1% 26 524 - parasite(s) found referred for ID 68.8% 11 3.6% 1 27.3% 12 563 - Diphyllobothrium latum Few 9.1% 1 0.0% 0 10.7% 3 6.8% 3 571 - Ascaris lumbricoides 0.0% 0 3.6% 1 2.3% 1 525 - No parasites found 6.3% 1 0.0% 0 2.3% 1 566 - Hymenolepis nana 0.0% 0 3.6% 1 2.3% 1

Extent 1 flagging appears for failure to report 530 or 524. Extent 2 flagging appears for failure to report 530. Flagging also appears in both extents for reporting other than 530, 567 or 524

SPECIMEN 12: FORMALIN: The referees (10/11, 90.9%) correctly identified this specimen as positive for few to many Balantidium coli. These organisms are quite large and can often be misidentified as helminth eggs. The trophozoites measure 50-100 by 40-70 microns. The specimen was graded on the basis of the direct wet mount examination; identification of Balantidium coli or Parasite(s) found, referred for ID was considered a correct response. One of the referee laboratories reported Diphyllobothrium latum; while these organisms were reported by 6.8% of the participants. METHOD REMINDER: Allow the vial contents to settle out for at least 5 min. Then, WITHOUT CREATING A BUBBLE WITH THE PIPETTE (WILL STIR UP SEDIMENT AGAIN), carefully remove material at the bottom of the vial. This approach will help ensure that organisms present will be visible on the wet coverslip preparation. If you create a bubble, allow the vial contents to resettle again before taking the specimen for examination. The participants (59.1%%) correctly reported the specimen as containing Balantidium coli or Parasite(s) found, refer for ID. Flagging appears in Extent 1 for failure to report Balantidium coli or parasite(s) found, referred for ID. In Extent 2, flagging appears for failure to report Balantidium coli. Flagging also appears in both extents for reporting other than Balantidium coli, parasite(s) found, referred for ID and Diphyllobothrium latum. Very few participants reported other organisms seen; however, on extensive pre-examination of this specimen, no other organisms were confirmed.




567 - Taenia sp. Few to Many 90.9% 10 17.6% 3 85.7% 24 60.0% 27

524 - parasite(s) found referred for ID 70.6% 12 0.0% 0 26.7% 12 525 - No parasites found 11.8% 2 7.1% 2 8.9% 4 568 - Taenia saginata proglottid Few 90.9% 1 0 3.6% 1 2.2% 1 569 - Taenia solium proglottid 0 3.6% 1 2.2% 1 Extent 1 flagging appears for failure to report 567, 568, 569 or 524. Extent 2 flagging appears for failure to report 567, 568 or 569. Flagging also appears in both extents for reporting other than 567, 568, 569 or 524.

SPECIMEN 13: FORMALIN: The referees (10/11, 90.9% correctly identified this specimen as positive for Taenia spp. eggs. The specimen was graded on the basis of the direct wet mount examination; identification of Taenia sp. or Parasite(s) found, referred for ID was considered a correct response. One of the referee laboratories reported Taenia saginata proglottid; however, we suspect this was an incorrect selection/marking. METHOD REMINDER: Allow the vial contents to settle out for at least 5 min. Then, WITHOUT CREATING A BUBBLE WITH THE PIPETTE (WILL STIR UP SEDIMENT AGAIN), carefully remove material at the bottom of the vial. This approach will help ensure that organisms present will be visible on the wet coverslip preparation. If you create a bubble, allow the vial contents to resettle again before taking the specimen for examination. The majority of participants (86.7%) correctly reported the specimen as containing Taenia spp. eggs or Parasite(s) found, refer for ID. Identification of adult worms to the species level is not possible from the eggs because T. solium (pork) and T. saginata (beef) eggs look identical. Identification is usually based on the recovery and examination of the gravid proglottids, in which the main uterine lateral branches on one side are counted after India ink injection staining or some other clearing mechanism (7 to 13 for T. solium and 15 to 20 for T. saginata). If the scolex is recovered after therapy (purgation may be necessary to recover the intact attachment organ), then the presence of the four suckers and the armed rostellum with hooks (T. solium) will differentiate the worm from T. saginata. There are some other differences in the mature proglottids; however, these require staining and clearing of the proglottids and may be much more difficult to see. Taenia spp. eggs are round or slightly oval (31 to 43 µm), have a thick, striated shell, and contain a six-hooked embryo or oncosphere. These eggs may remain viable in the soil for weeks. In the case of T. solium, if the eggs are accidentally ingested, cysticercosis can occur. The adult tapeworms are acquired from the ingestion of raw and/or undercooked pork or beef. Flagging appears in Extent 1 for failure to report Taenia sp. or parasite(s) found, referred for ID. In Extent 2, flagging appears for failure to report Taenia sp. Flagging also appears in both extents for reporting other than Taenia sp., or parasite(s) found, referred for ID. Note the typical Taenia spp. eggs below (thick, striated shell, six-hooked oncosphere within the egg shell).




545 - Entamoeba coli 90.9% 10 62.5% 10 66.7% 14 64.9% 24 524 - parasite(s) found referred for ID 37.5% 6 0.0% 0 16.2% 6 547 - Entamoeba histolytica 0.0% 0 14.3% 3 8.1% 3 548 - Entamoeba histolytica / Entamoeba dispar 0.0% 0 14.3% 3 8.1% 3 546 - Entamoeba hartmanni 0.0% 0 4.8% 1 2.7% 1 Extent 1 flagging appears for failure to report 545 or 524. Extent 2 flagging appears for failure to report or 545. Flagging also appears in both extents for reporting other than 545 or 524.

SPECIMEN 14 (Digital Image): This permanent stained fecal smear demonstrates excellent examples of Entamoeba coli. Extent flagging occurs for failure to report Entamoeba coli or parasite(s) found referred for ID. Flagging occurs in Extent 2 for failure to report Entamoeba coli. Flagging also appears in both extents for reporting other than Entamoeba coli, or parasite(s) found, not identified referred for ID.

Unless a positive fecal immunoassay for the true pathogen, Entamoeba histolytica, is positive or the trophozoites on the permanent stained slide contain ingested RBCs within the cytoplasm, it is incorrect to identify any of these organisms as Entamoeba histolytica; in this specimen none of the trophozoites contain ingested red blood cells! Without ingested RBCs or a positive fecal immunoassay for Entamoeba histolytica, the correct identification would be: Entamoeba histolytica/E. dispar. Differentiation between the true pathogen Entamoeba histolytica and the nonpathogen E. dispar cannot be made based on morphology unless RBCs are present within the trophozoite cytoplasm (and in the background of the smear). If the RBCs are present, then the true pathogen, Entamoeba histolytica, can be reported. Overall, the Entamoeba coli in this smear are very typical. The presence of cysts with five or more nuclei (see Examples 8, 12) confirm the presence of E. coli. In general, the overall nuclear appearance is darker, denser, and contains a much more blot-like, eccentric karyosome in E. coli, while the nuclear characteristics of Entamoeba histolytica/E. dispar tend to be more delicate, less dense, with a central karyosome that is more compact. Make sure to use the brightness tool to reveal the typical nuclear characteristics. The background may appear washed out, but the organisms will demonstrate key characteristics when the overall brightness is increased. NOTE: The compactness or blot-like characteristics of the karyosome ARE MORE IMPORTANT THAN THE POSITION (central or eccentric) for differentiating Entamoeba histolytica/E. dispar vs E. coli. Example 1 measures 14 microns, contains ingested debris has uneven nuclear chromatin and an eccentric karyosome that appears somewhat blot-like (Entamoeba coli) Example 2 measures 12.4 microns, and contains an eccentric karyosome that appears more blot-like (E. coli) Example 3 measures 13.3 microns; The nucleus is typical with an eccentric karyosome (large/blot-like). Example 4 measures 17.9 microns, and the nucleus has uneven chromatin and an eccentric karyosome. Note the highly vacuolated cytoplasm.



Example 5 measures 16.6 microns; the nuclear chromatin is uneven and the karyosome is blot-like and eccentric. Example 6 measures 15.2 microns; the nuclear chromatin is uneven, cytoplasm is heavily vacuolated, and the karyosome is large and blot-like. In this example, the large karyosome appears to be in the center (this is not unusual). Example 7 measures 20.3 microns, and the karyosome is eccentric and very blot-like. Also note the nuclear chromatin is unevenly arranged (E. coli) and the evidence of multiple vacuoles containing debris. Example 8 measures 13.1 microns. With enhanced light, the presence of five nuclei is confirmed, thus the ID is an E. coli cyst. Example 9 measures 13.6 microns. The nuclear chromatin is somewhat uneven, the karyosome is somewhat eccentric and spread out/blot-like. (E. coli) Example 10 measures 16.4 microns, is vacuolated containing a lot of debris, and contains irregular nuclear chromatin and an eccentric karyosome. Example 11 measures 23 microns. Note the nuclear chromatin is very blot-like, (not compact) (E. coli), and the nuclear chromatin appears to be uneven. This is often seen with trichrome staining and E. coli. Example 12 measures 9.5 microns, is a cyst, and contains five nuclei, (E. coli). This configuration is typical of E. coli. Although the measurement is small (cyst is shrunk), the presence of five nuclei ID the organism as E. coli. RESULTS: The referee laboratories reported correctly – E. coli (90.9%). Those of you who indicated that Entamoeba histolytica were present were incorrect – review all comments above. Participants reported E. coli (64.9%), while 3 laboratories incorrectly reported Entamoeba histolytica.

Entamoeba coli E. coli Entamoeba histolytica/E. dispar Entamoeba histolytica Ingested (RBCs) _______________________________________________________________ Sample 15 Referees Extent 1 Extent 2 Total

Code - Organism Frequency % No. % No. % No. % No. 546 - Entamoeba hartmanni 90.9% 10 62.5% 10 85.7% 18 75.7% 28 524 - parasite(s) found referred for ID 37.5% 6 0.0% 0 35.3% 6 545 - Entamoeba coli 0.0% 0 2.7% 1 9.1% 1 549 - Iodamoeba brutschlii 0.0% 0 2.7% 1 9.1% 1 547 - Entamoeba histolytica 0.0% 0 2.7% 1 9.1% 1 Extent 1 flagging appears for failure to report 546 or 524. Extent 2 flagging appears for failure to report 546. Flagging also appears in both extents for reporting other than 546 or 524.

SPECIMEN 15 (Digital Image): This specimen contains Entamoeba hartmanni trophozoites and cysts (very few cysts, but they are present). Accepting all correct responses, 90.9% (10/11) of the referees and 75.7% of the participants reported the presence of E. hartmanni. E. hartmanni is a nonpathogen, and the organisms were very typical, with the trophozoites having the characteristic nucleus (evenly arranged nuclear chromatin with central compact karyosome – resembles a “bull’s eye”); with rare exceptions the trophozoites measure <12 microns. The cysts have four nuclei, but frequently cysts containing only two nuclei are seen. Also, these cysts often contain



numerous chromatoidal bars (smooth, rounded ends); the cysts measure <10 microns. Although these organisms resemble Entamoeba histolytica/E. dispar, both the trophozoites and cysts of E. hartmanni are smaller. Remember when measuring the cysts on permanent stained smears, one should measure the total distance, including the clear “halo” that represents shrinkage during staining. No cysts were included in the 10 identification options. Extent 1 flagging appears for failure to report Entamoeba hartmanni or Parasite(s) found, referred for ID. Extent 2 flagging appears for failure to report Entamoeba hartmanni. Flagging also appears in both extents for reporting other than Entamoeba hartmanni or Parasite(s) found, not identified referred for ID. 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). Typical E. hartmanni parasites can be seen below:

Note: The nucleus is very typical with evenly arranged chromatin and the central, compact karyosome (resembling Entamoeba histolytica/E. dispar, but smaller); the trophozoites measure <12 microns, while the cysts measure <10 microns. Often, cysts are seen that may contain only two nuclei, rather than the four that are found in the mature cyst. Also, it is common to see multiple, small chromatoidal bars within the cysts.

It is incorrect to identify any of these organisms as Entamoeba histolytica or Entamoeba histolytica/E. dispar group/complex; none of the trophozoites contain ingested red blood cells and the overall size is too small! Make sure to use the brightness tool to reveal the typical nuclear characteristics. The background may appear washed out, but the organisms will demonstrate key characteristics when the overall brightness is increased. Example 1 contains one trophozoite, in which the nuclear chromatin is evenly arranged – the karyosome is a bit eccentric (approximately 7.6 microns). The size and morphology are very typical and characteristic of E. hartmanni. Example 2 also contains one trophozoite (somewhat round), with typical morphology; the “target” nucleus is clearly seen with the central, compact karyosome. This troph measures approximately 6 microns. In Example 3 the trophozoite measures approximately 6.5 microns); the target or bull’s eye nucleus is very typical. The overall shape and morphology are “textbook” – perfect representation of written description. In Example 4 the trophozoite displays very typical morphology (evenly arranged nuclear chromatin, central karyosome). The organism measures approximately 9.1 microns). The cytoplasm is heavily vacuolated, a characteristic that may be seen. In Example 5 the trophozoite is typical and measures approximately 8.8 microns, and the overall morphology is that of E. hartmanni. Although one side of the nuclear chromatin is a bit darker, the target nuclear shape is typical. In Example 6, the trophozoite measures approximately 6.5 microns and demonstrates a good “target” nucleus.



In Example 7 the trophozoite measures approximately 7.3 microns and has an excellent “bull’s eye” nucleus with the central karyosome. The overall shape and morphology are very typical. In Example 8, the trophozoite measures approximately 5.5 microns; the nuclear chromatin is evenly arranged, and the karyosome is centrally located. The overall morphology is very typical for E. hartmanni. Example 9 measures 9.4 microns, and the nucleus appears like the typical bull’s eye. The overall size and morphology are definitely that of E. hartmanni. In Example 10, the trophozoite measures approximately 6.3 microns and has a good target nucleus configuration, typical for E. hartmanni. The other dots in the cytoplasm represent debris. SPECIMEN 14: Permanent Stained Smear (Trichrome): Digital Image, Stool, Maximum Magnification is 1000X SPECIMEN 15: Permanent Stained Smear (Trichrome): Digital Image, Stool, Maximum Magnification is 1000X Use Micrometer embedded in viewer to measure organism. Images photographed using 10X oculars and the 100X oil immersion objective for a total magnification of 1000X. 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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American Association of Bioanalysts 3Q2019.pdf · 546 - Entamoeba hartmanni Rare 18.2% 2 0 7.4% 2 4.5% 2 Extent 1 flagging appears for failure to report 544, 526 or 524. Extent 2