American Association of Bioanalysts · 2020. 2. 13. · SPECIMEN 1: FORMALIN: The specimen was a fecal suspension in 10% formalin for direct wet mount examination; concentration was

AAB PARASITOLOGY – FIRST QUADRIMESTER, 2019

1 AAB 1st Quadrimester Parasitology, 2019

American Association of Bioanalysts

5615 Kirby Drive, Suite 870 Houston, TX 77005

800-234-5315 ♦ 281-436-5357 Q1 2019 Parasitology

Sample 1 Referees Extent 1 Extent 2 Total Code Organism Frequency % No. % No. % No. % No. 525 No parasites found 100.0% 11 94.1% 16 88.9% 24 90.9% 40 544 Endolimax nana 5.9% 1 7.4% 2 6.8% 3 533 Dientamoeba fragilis 0.0% 0 3.7% 1 2.3% 1 Total Population 44 Flagging for all extents appears for Reporting other than 525

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. 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, 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 1 with an overall 100% correct response. SPECIMEN 1: PERMANENT STAIN: The permanent stained smear contains no parasites. Artifact material is rarely consistent in either size or shape. This specimen contains the normal stool debris, most of which has no standard shape or size. In contrast to examination of the wet preparation, remember to use plenty of light when examining the permanent stained smears and to use the oil immersion objective (100X). You need to cover at least 300 oil immersion fields before you call the stained smear negative. Flagging appears for reporting other than No parasites found (Referees 11/11, 100%).



Specimen 2 Referees Extent 1 Extent 2 Total

Code Organism Frequency % No. % No. % No. % No. 571 Ascaris lumbricoides Few to Many 100.0% 11 27.8% 5 93.1% 27 72.7% 32

524 parasite(s) found referred for ID 66.7% 12 0.0% 0 27.3% 12

525 No parasites found 5.6% 1 3.4% 1 4.5% 2

578 Strongyloides stercoralis / sp. 0.0% 0 3.4% 1 2.3% 1

Total Population 47 Extent1 flagging appears for failure to report 571 or 524. Extent 2 flagging appears for failure to report 571. Flagging also appears in both extents for reporting other than 571 or 524.

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 contains Ascaris lumbricoides eggs; the referee laboratories (11/11, 100.0%) reported correctly with the overall participants reporting 94.5% to 100% correctly. Flagging appears in Extent 1 for failure to report Ascaris lumbricoides or parasite(s) found, referred for ID. Extent 2 flagging occurs for failure to report Ascaris lumbricoides. Fertilized and unfertilized eggs were seen. Some of the eggs (both types) are decorticate (loss of bumpy shell), and some of the fertilized eggs contain fully developed larvae. This is not unusual, since the thick shell often protects the developing embryo from fecal fixatives. In some clinical specimens, motile larvae within the egg shells can be seen (none seen in this specimen). Fertilized eggs exhibit the typical bumpy shell, while a few had lost the shell (decorticate). Although the bumpy shell of the unfertilized egg is more pronounced than that seen in the fertilized egg, it is not unusual to see both types (fertilized and unfertilized) of eggs and also not unusual to see some decorticate eggs of both types.

Fertile eggs, note larva, bumpy shell present Unfertilized eggs, note decorticate egg on the right Sample 3 Referees Extent 1 Extent 2 Total Code Organism Frequency % No. % No. % No. % No.

589 Schistosoma mansoni

Few to Many 100.0% 11 27.8% 5 96.4% 27 69.6% 32


525 No parasites found 5.6% 1 0.0% 0 2.2% 1 Total Population 46

Extent 1 flagging appears for failure to report 589 or 524. Extent 2 flagging appears for failure to report 589. Flagging also appears in both extents for reporting other than 589 or 524



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 Schistosoma mansoni (blood fluke) eggs. The eggs exhibit typical morphology and measure approximately 125 x 55 µm (114 to 180 µm by 45 to 73 µm). The eggs are passed fully embryonated and do not have an operculum. They tend to be a light yellowish-brown color; they are elongate and possess a large lateral spine projecting from the side near one end of the egg. These eggshells are acid fast when stained with a modified Ziehl-Neelsen staining method. On reaching water, the eggs hatch, liberating miracidia that must penetrate a suitable snail host. Cercariae liberated from the infected snail infect humans when the latter come in contact with cercaria-infested water. A period of 4 weeks or more from the time of first infection with a miracidium is required before the snail sheds cercariae. On infection, the cercariae lose their tails, and the body (now called a schistosomulum) migrates through the lungs to the liver, where it develops to a mature adult in the mesenteric veins of the large bowel. Some worms are also found in the superior mesenteric veins, vesical plexus, and intrahepatic portion of the portal veins. Disease syndromes associated with schistosomiasis are related to the stage of infection, previous host exposure, worm burden, and host response. Syndromes include cercarial dermatitis, acute schistosomiasis (Katayama syndrome), and related tissue changes resulting from egg deposition. The referee labs reported 11/11 S. mansoni. Extent 1 flagging appears for failure to report Schistosoma mansoni, or Parasites found referred for ID. Extent 2 flagging appears for failure to report Schistosome mansoni. Flagging also appears in both extents for reporting other than Schistosoma mansoni or parasites found referred for ID. Overall, participants performed very well with this challenge.

Schistosoma mansoni; note the large, lateral spine SPECIMEN 4: Permanent Stained Smear (Trichrome): Digital Image, Stool, Maximum Magnification is 1000X

Specimen 4 Referees Extent 1 Extent 2 Total Code Organism Frequency % No. % No. % No. % No.

549 Iodamoeba brutschii 100% 11 80.0% 12 96.0% 24 90.0% 36


Total Population 40 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 (11/11) 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 will with an overall percent of 90%. 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). Example 1 contains a single trophozoite, 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. Example 2 contains one typical cyst; note the large red/purple karyosome and the pale glycogen vacuole. Example 3 contains one typical cyst with the clear glycogen vacuole lying under the karyosome and large red/purple karyosome (granules on top of karyosome represent the “basket” nucleus). Example 4 contains one cyst; note that the pale glycogen vacuole and the somewhat oval karyosome. Also note the granules at the top of the karyosome; this represents the appearance of the typical “basket” nucleus. Example 5 contains a single cyst, which has a very large karyosome and typical glycogen vacuole that appears to be pushed in from the top. The nucleus is a typical “basket” nucleus with extra granules at the top of the karyosome. Example 6 contains one typical cyst with a distorted glycogen vacuole; note the basket nucleus (extra chromatin is at the bottom of the karyosome). Example 7 contains a single cyst. The glycogen vacuole is a bit shrunk, but the karyosome is very typical. Example 8 contains a single cyst. Both the karyosome and vacuole are a bit pale. Example 9 contains one cyst with a pale glycogen vacuole and typical large karyosome with granules at the top of the karyosome (“basket” nucleus). Example 10 contains a single cyst with an excellent nucleus and large glycogen vacuole.

Iodamoeba bütschlii cysts Iodamoeba bütschlii trophozoite



SPECIMEN 5: 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. _______________________________________________________________ Specimen 5 Referees Extent 1 Extent 2 Total

Code Organism Frequency % No. % No. % No. % No.

557 Plasmodium falciparum 100.0% 11 78.6% 11 83.3% 20 59.6% 31

524 parasite(s) found referred for ID 14.3 2 4.2% 1 16.7 3

556 Plasmodium sp.; undetermined 0.0% 0 8.3% 2 11.1% 2

558 Plasmodium malariae 0.0% 0 4.2% 1 5.6% 1

555

Plasmodium sp.; refer for identification to species level 7.1% 1 0.0% 0 5.6% 1

Total Population 38 Extent 1 flagging appears for failure to report 557, 556, 555 or 524. Extent 2 flagging appears for failure to report 557, 556, or 555. Flagging also appears in both extents for reporting other than 557, 556, 555 or 524.

SPECIMEN 5 (Digital Image): This stained thin blood film demonstrates excellent examples of Plasmodium falciparum ring forms and gametocytes. The nucleus appears as a dark dot, while the cytoplasm appears as a paler “ring” structure. Since P. falciparum tends to infect all ages of RBCs, the infected RBC sizes vary within the smear. Typically, with P. falciparum, only the ring forms and gametocytes are seen in the peripheral blood. However, in patients with early infections, they may present to the ER before gametocytes have formed. The majority of participants identified the organisms to the species level – good job NOTE: When Plasmodium (not identified to the species level). Is diagnosed, a report comment should always accompany the report:

Plasmodium species seen - UNABLE TO RULE OUT PLASMODIUM FALCIPARUM OR PLASMODIUM KNOWLESI.

Example 1 contains two ring forms, both within the RBC; the ring cytoplasm is visible in the bottom ring only. Example 2 contains a single ring; note it appears to be somewhat protruding from the surface of the RBC. This configuration is more commonly seen in P. falciparum. Example 3 contains a typical crescent-shaped gametocyte. Note the pale outline of the RBC can be seen at the right side of the gametocyte. The compact nuclear material suggests the female gametocyte. Example 4 contains a single infected RBC; the ring is in the “headphone” configuration with two chromatin dots. Example 5 contains several rings with multiple chromatin nuclear dots. Example 6 contains a typical gametocyte. Note the RBC pale outline can be seen at the left side of the gametocyte. The more compact nuclear material suggests the female gametocyte. Example 7 contains a large ring form; because the size is large, it may mean that the blood specimen was held in EDTA for some time prior to smear preparation (rings continue to grow) – not indicative of another malarial species. Example 8 contains a gametocyte that is seen from the top, rather than from the side. Therefore, the typical curved appearance is not visible. Note the diffuse nuclear material; this generally represents the male gametocyte or microgametocyte. More compact rather than diffuse chromatin would suggest the female gametocyte/macrogametocyte.



Example 9 contains two infected RBCs, both of which contains ring forms. Note some of the rings are in the “headphone” shape with two nuclear dots per ring. Also notice the two appliqué or accollé forms lying along the edge of the RBC (RBC at the left). Example 10 contains a single RBC infected with two rings (one appliqué form and a ring seen from the top). Example 11 contains a single RBC with an appliqué form that appears to be protruding from the cell. This is typical in P. falciparum. Example 12 contains a single RBC with both rings in the appliqué formation. Example 13 contains a single RBC with one ring (a bit enlarged). With no other forms on the smear, this morphology would make it difficult to identify to the species level. Example 14 contains two infected rings with some in the “headphone” configuration and one appliqué form. Example 15 contains a single RBC with several rings in the appliqué formation. Example 16 contains a single crescent-shaped gametocyte. The RBC outline is not visible.

Plasmodium falciparum rings and gametocyte 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 · 2020. 2. 13. · SPECIMEN 1: FORMALIN: The specimen was a fecal suspension in 10% formalin for direct wet mount examination; concentration was