american association of bioanalysts parasitolo… · preparation. if you create a bubble, allow the...

AAB PARASITOLOGY – SECOND QUADRIMESTER, 2019

1 AAB 2nd Quadrimester Parasitology, 2019

American Association of Bioanalysts

5615 Kirby Drive, Suite 870 Houston, TX 77005

800-234-5315 ♦ 281-436-5357 Q2 2019 Parasitology

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

534 - Giardia lamblia 100.0% 11 25.0% 4 84.8% 28 65.3% 32 524 - parasite(s) found referred for ID 68.8% 11 0.0% 0 22.4% 11 541 - Blastocystis hominis 0.0% 0 12.1% 4 8.2% 4 525 - No parasites found 6.3% 1 0.0% 0 2.0% 1 545 - Entamoeba coli 0.0% 0 3.0% 1 2.0% 1 Extent 1 flagging appears for failure to report 534 or 524. Extent 2 flagging appears for failure to report 534. Flagging also appears in both extents for reporting other than 534, 541 or 524

SPECIMEN 6: FORMALIN: Specimen 6A 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 Giardia lamblia cysts and rare trophozoites. Blastocystis spp. is also present; however, the organism morphology is much more defined in the permanent stained smear. Although the overall morphology of Giardia was best seen in the permanent stained smear, the wet mount morphology is typical. SPECIMEN 6: PERMANENT SMEAR FOR STAINING: Specimen 6B was the permanent stained smear. This specimen contains 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. There were some rare trophozoites present. Note – in some publications, you may see Giardia lamblia designated as either G. intestinalis or G. duodenalis. For the present, we will continue to the species name as G. lamblia. There were also few to many Blastocystis spp. (formerly Blastocystis hominis) central body forms present. Currently this organism is classified as an unusual anaerobic, single-celled stramenopile (examples are brown algae such as kelp, diatoms, slime nets, and water molds). Blastocystis comprises a multitude of subtypes (or species – not yet decided) many of which have been identified only recently; molecular epidemiological studies have revealed a significant difference in the distribution of subtypes across host species and geographical regions. Also, approximately half of the 9 subtypes are pathogenic, while the others are considered nonpathogenic. This explains over the years the controversy concerning pathogenicity and symptomatic vs. asymptomatic patients. The subtypes ST1-ST4 account for probably more than 90% of human carriage, while the remaining approximately 10% are colonized by isolates belonging to ST5-ST9. All of these STs, apart from ST9 have also been found in various non-human hosts. Unfortunately, the pathogenic subtypes cannot be differentiated from those that are nonpathogenic based on microscopic morphology. Report comments can be helpful when Blastocystis spp. is reported (see report comments above). Referee laboratories (11/11 – 100%) reported few to many G. lamblia, while one referee reported few to many Blastocystis spp. Most participants performed well on this specimen (87.7%). Extent 1 flagging appears for failure to report Giardia lamblia, or Parasites found, referred for ID. Extent 2 flagging appears for failure to report Giardia



lamblia. Flagging also appears in both extents for reporting other than Giardia lamblia, Blastocystis spp. or Parasites found, referred for ID.

Very high magnification G. lamblia cyst G. lamblia trophozoite G. lamblia cyst G. lamblia trophozoite Wet Mounts Permanent Stained Smear Sample 7 Referees Extent 1 Extent 2 Total

Code - Organism Frequency % No. % No. % No. % No. 563 - Diphyllobothrium latum Few to Many 81.8% 9 12.5% 2 83.9% 26 57.1% 28 524 - parasite(s) found referred for ID 75.0% 12 0.0% 0 24.5% 12 574 - Hookworm 9.1% 1 12.5% 2 3.2% 1 6.1% 3 544 - Endolimax nana 0.0% 0 3.2% 1 2.0% 1 571 - Ascaris lumbricoides 0.0% 0 3.2% 1 2.0% 1 546 - Entamoeba hartmanni 0.0% 0 3.2% 1 2.0% 1 525 - No parasites found 9.1% 1 0.0% 0 6.3% 1 6.3% 1

Extent 1 flagging appears for failure to report 563 or 524. Extent 2 flagging appears for failure to report 563. Flagging also appears in both extents for reporting other than 563 or 524

SPECIMEN 7 FORMALIN: The referees correctly identified this specimen as positive for few to many Diphyllobothrium latum eggs. They are very typical with an operculum and no opercular shoulders. The eggs measure 58-75 by 40-50 microns. The specimen was graded on the basis of the direct wet mount examination; identification of Diphyllobothrium latum or Parasite(s) found, referred for ID was considered a correct response. Two of the referee laboratories reported Endolimax nana; however, these organisms are normally identified on the permanent stained smear, not the wet mount. 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 (81.6%) correctly reported the specimen as containing Diphyllobothrium latum eggs or Parasite(s) found, refer for ID. Flagging appears in Extent 1 for failure to report Diphyllobothrium latum. or parasite(s) found, referred for ID. In Extent 2, flagging appears for failure to report Diphyllobothrium latum. Flagging also appears in both extents for reporting other than Diphyllobothrium latum, parasite(s) found, referred for ID and Endolimax nana. Very few participants reported other organisms seen; however, on extensive pre-examination of this specimen, no other organisms were confirmed.



Note the typical Diphyllobothrium latum eggs below (operculated with no opercular shoulders). In the image on the left, the opercula are open. When reviewing a wet mount, if you tap on the coverslip, you may get the operculum of this egg to pop open.

Sample 8 Referees Extent 1 Extent 2 Total

Code - Organism Frequency % No. % No. % No. % No. 525 - No parasites found Few to Many 90.9% 10 100.0% 17 96.3% 26 97.7% 43 563 - Diphyllobothrium latum 9.1% 1 0.0% 0 3.7% 1 2.3% 1 Extent 1 flagging appears for failure to report 525.

SPECIMEN 8 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, 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 Sample 3 with an overall 97.7% correct response. One participant reported Diphyllobothrium latum; however, we suspect this was an accidental carryover from the previous specimen.



SPECIMEN 9: Permanent Stained Smear (Trichrome): Digital Image, Stool, Maximum Magnification is 1000X

Sample 9 Referees Extent 1 Extent 2 Total

Code - Organism Frequency % No. % No. % No. % No. 547 - Entamoeba histolytica 81.8% 9 53.3% 8 75.0% 18 66.7% 26 548 - Entamoeba histolytica / Entamoeba dispar 18.2% 2 13.3% 2 16.7% 4 35.3% 6 524 - parasite(s) found referred for ID 20.0% 3 4.2% 1 23.5% 4 545 - Entamoeba coli 0.0% 0 4.2% 1 5.9% 1 534 - Giardia lamblia 6.7% 1 0.0% 0 5.9% 1 525 - No parasites found 6.7% 1 0.0% 0 5.9% 1 Extent 1 flagging appears for failure to report 547 or 524. Extent 2 flagging appears for failure to report or 547. Flagging also appears in both extents for reporting other than 547 or 524.

SPECIMEN 9 (Digital Image): This permanent stained fecal smear demonstrates excellent examples of the true pathogen Entamoeba histolytica. Extent 1 flagging occurs for failure to report Entamoeba histolytica, or parasite(s) found, referred for ID. Flagging occurs in Extent 2 for failure to report Entamoeba histolytica. Flagging also appears in both extents for reporting other than Entamoeba histolytica or parasite(s) found, not identified referred for ID. THESE ORGANISMS ARE TYPICALLY ENTAMOEBA HISTOLYTICA. The majority of participants (90.2%) correctly reported the specimen as containing Entamoeba histolytica or Parasite(s) found, refer for ID.

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 the trophozoites 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. Example 1 contains a trophozoite containing ingested RBCs, has a single nucleus with even nuclear chromatin, and a central karyosome that appears neat and compact. Note the pleomorphic RBCs in the background. The outline of the trophozoite is not that clear, primarily due to the ingested RBCs (note lower half of the organism). Example 2 contains a single trophozoite with the nucleus having evenly arranged peripheral chromatin and a central, compact karyosome (very typical nucleus). Note: as the RBCs are digested, they become more round and smaller (see right side of the trophozoite). Example 3 contains ingested RBCs in the cytoplasm (small and almost digested – see right side of the trophozoite), and has a single nucleus with even chromatin and a compact central karyosome. Again, note the numerous RBCs in the background.



Example 4 contains a very pale staining trophozoite. Note the nucleus does not look that typical (somewhat out of focus), and the single ingested RBC is somewhat elongate. Example 5 is a single trophozoite containing ingested RBCs and a typical nucleus with fairly even chromatin and a compact central karyosome. Example 6 contains ingested RBCs and a single typical nucleus (at the bottom of the trophozoite). Example 7 contains a typical nucleus (somewhat pale and difficult to see the karyosome) even chromatin and a central, compact karyosome. Although no recently ingested RBCs are seen in the cytoplasm, the overall morphology seen within the context of this smear confirms Entamoeba histolytica. Although some might report Entamoeba histolytica/E. dispar group, within the context of this smear it is not clinically relevant (presence of many true pathogen Entamoeba histolytica present). Example 8 contains numerous ingested RBCs (note they are small and round indicating digestion is occurring) and a typical nucleus. Again, note the many RBCs in the background. Example 9 Note the typical nucleus and digested RBCs (lower half of the organism). Example 10 contains several small ingested RBCs in the cytoplasm and a very typical nucleus. RESULTS: Congratulations to the referee laboratories (81.8%) and participants (90.2%) that identified Entamoeba histolytica or Parasite(s) found, refer for ID as being present. See additional tips below: 1. Note that the ingested RBCs are being digested within cytoplasmic vacuoles, so they will often appear smaller than the RBCs found in the background of the fecal smear. 2. 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. 3. 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.

Entamoeba coli E. coli Entamoeba histolytica/E. dispar Entamoeba histolytica Ingested (RBCs) SPECIMEN 10: Permanent Stained Smear (Blood Stain): Digital Image, Blood, 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. _______________________________________________________________ Sample 10 Referees Extent 1 Extent 2 Total

Code - Organism Frequency % No. % No. % No. % No. 534 - Giardia lamblia 100.0% 11 66.7% 10 100.0% 25 59.6% 35 524 - parasite(s) found referred for ID 26.7% 4 0.0% 0 23.5% 4 547 - Entamoeba histolytica 6.7% 1 0.0% 0 5.9% 1 Extent 1 flagging appears for failure to report 534 or 524. Extent 2 flagging appears for failure to report 534. Flagging also appears in both extents for reporting other than 534 or 524.



SPECIMEN 10 (Digital Image): This stained stool slide demonstrates excellent examples of Giardia lamblia trophozoites. 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 reported 83.1% (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 trophozoites 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. Also, if the trophozoite is lying on the side, it can appear like the curved portion of a spoon.

Note the shape is somewhat like the curved portion of a spoon. Example 1 contains an excellent Giardia trophozoite (teardrop shaped). The organism is somewhat pale (typical staining for trophozoites) and both nuclei are seen. Example 2 contains a single pale-staining trophozoite, in which both nuclei are visible. The linear axonemes are seen, but pale (left side of the organism). Example 3 contains one trophozoite; the teardrop shape is visible, as well as the two nuclei Example 4 contains an excellent Giardia trophozoite. The nuclei are visible with the organism being somewhat teardrop shaped. Example 5 contains a single trophozoite; note the “face/eyes” looking back at you and the teardrop shape. Example 6 contains three typical Giardia trophozoites. Note the karyosomes are not visible in the top right organism. Example 7 contains two very typical trophozoites; note the nuclei, overall shape, and dark karyosomes. The linear axonemes are also visible, but pale (see upper organism). Often the pointed end tends to stain more pale. Example 8 contains two excellent Giardia trophozoites. Note the teardrop shape and typical two nuclei. In the organism at the top of the frame, the nuclear cytoplasm is visible, but the karyosomes are not seen. Example 9 contains a typical trophozoite with the teardrop shape, two nuclei, and median bodies (the median bodies look more linear than curved; look right below the nuclei). Example 10 contains a typical Giardia trophozoite; the nuclei are very clear, and the shape is typical (often the end of the organism stains very pale). The overall shape is somewhat blurred in this example).

Giardia lamblia trophozoites



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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