Q3 2018 Parasitology American Association of Bioanalysts 5615 Kirby Drive, Suite 870 Houston, TX 77005 800-234-5315 281-436-5357 Specimen 1 Referees Extent 1 Extent 2 Total Few to 534 Giardia lamblia Many 100.0% 11 26.3% 5 93.1% 27 66.7% 32 referred for ID 68.4% 13 0.0% 0 27.1% 13 541 Blastocystis hominis Few to Many 9.1% 1 5.3% 1 0.0% 0 2.1% 1 546 Entamoeba hartmanni 0.0% 0 3.4% 1 2.1% 1 545 Entamoeba coli 0.0% 0 3.4% 1 2.1% 1 Extent 1 flagging appears for failure to report 534 or. Extent 2 flagging appears for failure to report 534. Flagging also appears in both extents for reporting other than 534, 541 or Total Population 48 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 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 1: PERMANENT SMEAR FOR STAINING: Specimen 1B 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). 1 AAB 3rd Quadrimester Parasitology, 2018
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 (1/11-93.8%). 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 Blastocystis spp. central body forms Specimen 2 Referees Extent 1 Extent 2 Total 525 No parasites found 100.0% 11 88.9% 16 92.3% 24 83.3% 40 544 Endolimax nana 5.6% 1 3.8% 1 4.2% 2 referred for ID 5.6% 1 3.8% 1 4.2% 2 Total Population 44 Extent 1 flagging appears for failure to report 525. Extent 2 flagging appears for failure to report 525. Flagging also appears in both extents for reporting other than 525 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. 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 2 AAB 3rd Quadrimester Parasitology, 2018
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, specimens selected for proficiency testing tend to have moderate to many organisms that are present for identification. Flagging appears in all extents for reporting other than No Parasites Found participants performed very well in the examination of Sample 2 with an overall 83.3% correct response. Note: The referees found no other organisms present. Sample 3 Referees Extent 1 Extent 2 Total Few to 545 Entamoeba coli Many 100.0% 11 21.7% 5 50.0% 26 64.6% 31 referred for ID 64.7% 11 3.6% 1 26.7% 12 525 No parasites found 5.9% 1 0.0% 0 2.2% 1 547 Entamoeba histolytica 0.0% 0 3.6% 1 2.2% 1 Total Population 45 Extent 1 flagging appears for failure to report 545 or. Extent 2 flagging appears for failure to report 545. Flagging also appears in both extents for reporting other than 545 or 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. This specimen contains Entamoeba coli cysts. The cysts were very typical with five or more nuclei being visible; some cysts contained the full complement of eight nuclei. Also, rare cysts contained splintered chromatoidal bars. The cyst shape varied from round to a bit more oval. The referees (11/11) identified few to many E. coli. Extent one flagging appears for failure to report Entamoeba coli or, referred for ID. Extent 2 flagging appears for failure to report Entamoeba coli. Flagging also appears in both extents for reporting other than Entamoeba coli or, referred for ID. Overall the participants performed at a lower percentage than earlier challenges with 64.6% reporting Entamoeba coli and 26.7% reporting, referred for ID. Entamoeba coli cysts 3 AAB 3rd Quadrimester Parasitology, 2018
SPECIMEN 4: Permanent Stained Smear: Digital Image, Stool, Maximum Magnification is 1000X (Modified Acid-Fast) Specimen 4 Referees Extent 1 Extent 2 Total 552 Cyclospora cayetanensis 81.8% 9 53.8% 7 75.0% 15 66.7% 22 referred for ID 38.5% 5 5.0% 1 35.3% 6 553 Cryptosporidium sp. 18.2% 2 7.7% 1 20.0% 4 29.4% 5 Extent 1 flagging appears for failure to report 552 or. Extent 2 flagging appears for failure to report 552. Flagging also appears in both extents for reporting other than 552 or Total Population 33 This permanent stained fecal smear (modified acid-fast stain) demonstrates excellent examples of Cyclospora cayetanensis oocysts. These coccidian oocysts measure approximately 8-10 µm and are generally round in shape. Extent 1 flagging occurs for failure to report Cyclospora cayetanensis or, referred for ID. Flagging occurs in Extent 2 for failure to report Cyclospora cayetanensis. Flagging also appears in both extents for reporting other than Cyclospora cayetanensis or, not identified referred for ID. Note that in all examples seen below, there are no internal structures clearly visible; the oocysts are not mature (also not infective) when they are passed. The referees reported 81.8% correct and the participants reported 66.7% Cyclospora and 35.3% Parasites found, referred for ID. Although rare Cryptosporidium spp. was reported, the measurements for these oocysts are generally 4-6 µm. Example 1 contains an oocyst with no specific internal details. Note the stain is somewhat variable; unstained areas are also seen (acid-fast variable). This oocyst measures ~8 µm. Example 2 contains a single oocyst; the morphology is similar to that seen in example 1 (8.3 µm). Example 3 contains an oocyst with no specific internal details. Note the stain is somewhat variable; unstained areas are also seen (acid-fast variable). This oocyst measures ~8 µm. This oocyst is at the lower margin of measurement. Example 4 contains a single oocyst that is somewhat shrunk; also note the variable staining. Although this oocyst is small, it does not fall into the range for Cryptosporidium spp. (4-6 µm). Example 5 contains a single oocyst; the morphology is similar to that seen in example 1 (7.9 µm). Example 6 contains a single oocyst (pale, mottled color); it measures ~ 8.3 µm. Example 7 contains an oocyst that measures ~8.4 µm. It is important to measure the entire oocyst, not just the more darkly stained area. Example 8 contains a more darkly stained oocyst that measures 9 µm. Example 9 contains a single oocyst that is pale and acid-fast variable; it measures ~ 9 µm. Example 10 contains a large oocyst that did not stain (acid-fast variable). This image is not that unusual; it measures >9 µm. These unstained oocysts are often described as looking like wrinkled cellophane. Example 11 contains a relatively pale oocyst that measures ~ 8 µm. Example 12 contains a typical oocyst that measures > 8 µm; again, note the mottled appearance. 4 AAB 3rd Quadrimester Parasitology, 2018
Cyclospora cayetanensis oocysts (modified acid-fast variable, 8-10 µm) SPECIMEN 5: Permanent Stained Smear: Digital Image, Stool, Maximum Magnification is 1000X (Trichrome) 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. Specimen 5 Referees Extent 1 Extent 2 Total 544 Endolimax nana 90.9% 10 53.3% 8 81.0% 17 59.6% 25 referred for ID 40.0% 6 0.0% 0 35.3% 6 545 Entamoeba coli 9.1% 1 0.0% 0 14.3% 3 17.6% 3 556 Plasmodium sp.; undetermined 6.7% 1 0.0% 0 5.9% 1 546 Entamoeba hartmanii 0.0% 0 4.8% 1 5.9% 1 Total Population 36 Extent 1 flagging appears for failure to report 544 or. Extent 2 flagging appears for failure to report 544. Flagging also appears in both extents for reporting other than 544 or This specimen (digital image of routine trichrome stain) contains Endolimax nana trophozoites and cysts. The referees (11/11) reported correctly, identifying Endolimax nana (90.9%). NOTE: Endolimax nana trophozoites can often mimic Entamoeba hartmanni or Dientamoeba fragilis, particularly when the D. fragilis trophozoite contains only a single nucleus. Extent 1 flagging appears for failure to report Endolimax nana or referred for ID. Extent 2 flagging appears for failure to report Endolimax nana. Flagging also appears in both extents for reporting other than Endolimax nana or referred for ID. Overall, participants performed very well with this challenge. 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). Example 1 contains a typical trophozoite; note the odd shaped nuclear karyosome (nuclear variation very common in E. nana). Example 2 contains a single trophozoite with the typical large karyosomes. Note there also appears to be a bit of peripheral nuclear chromatin (nuclear variation very common). The other structure above the nucleus is merely debris. 5 AAB 3rd Quadrimester Parasitology, 2018
Example 3 contains a single trophozoite. Note the unusual appearance of the karyosomes (typical variation). Example 4 contains a single cyst with three of the four karyosomes being visible. Although many cysts are somewhat oval, there can be tremendous variation in shape. Example 5 contains a single trophozoite, with the large karyosome. Note there is no nuclear membrane visible around the karyosome (also typical). Example 6 contains a single trophozoites that is very typical; note the single large karyosomes and a faint outline of the nuclear membrane. Example 7 contains a single cyst with two of the four karyosomes visible. Also note what appears to be a vacuole; this can occasionally be confused with an Iodamoeba bütschlii (single nucleus only). See examples below. Endolimax nana cyst Iodamoeba bütschlii typical cysts Example 8 contains two trophozoites; the karyosomes are clearly visible. Example 9 contains one trophozoite that is a bit darker. Note the single karyosomes and faint outline of nuclear chromatin. Example 10 contains a cyst with two karyosomes visible (right side of the cyst). 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. 6 AAB 3rd Quadrimester Parasitology, 2018
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 nonpathogenic. 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. 7 AAB 3rd Quadrimester Parasitology, 2018