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Figure 10.4 Free-living freshwater ciliates. (Left) Tetrahymena. (Right) Paramecium. (Courtesy of David Nelson.)doi:10.1128/9781555819002.ch10.f4
Cryptosporidium spp. and Cyclospora cayetanensis
Cryptosporidium measures approximately 4 to 6 µm and overlaps in size with a number of objects, including yeasts and debris that are found in stool specimens. Cyclospora cayetanensis measures approximately 8 to 10 µm and can be easily confused with other coccidia or artifacts, especially if careful measurements are not taken. Without the use of modified acid-fast stains or immunoassay detection methods, a light infection with coccidia will probably be missed; the more normal the stool consistency, the fewer oocysts and more artifacts will be present. The oocysts can also be confused with artifact material when the staining results are of poor quality; they appear nonuniform and more like debris (Fig. 10.1).
When Giardia lamblia shrinks within the cyst wall, it can resemble Cystoisospora belli in the immature form (a single developing sporoblast; the two mature sporocysts within the oocyst wall have not yet formed). Although this error is occasionally made, the easiest way to differentiate the two is by size. Giardia cysts measure approximately 11 to 14 µm long by 7 to 10 µm wide, and C. belli measures 20 to 33 µm long by 10 to 19 µm wide. This represents another situation in which measurement of the organisms can help prevent diagnostic errors (Fig. 10.5).
Figure 10.5 Giardia lamblia and Cystoisospora belli. (Left) Giardia lamblia (G. duodenalis, G. intestinalis), trichrome stain. (Right) Cystoisospora belli, iodine wet mount. Note the size differences of the two organisms. doi:10.1128/9781555819002.ch10.f5
Microsporidian spores in humans measure approximately 1 to 4 µm, with the majority being 1 to 2 µm. These spores are round to oval and can mimic yeast cells and bacteria. Although they do stain in a modified trichrome procedure (see chapter 3), the color is usually pale and both the size and color overlap with those of many of the yeasts or bacteria present in the specimen (Fig. 10.1). The spores usually stain light to dark pink; they can be confused with bacteria, looking like large Gram-negative rods. Also, some of the bacilli contain terminal spores that mimic the large vacuole often seen at one end of the microsporidian spore. Size and staining color are rarely that helpful. It is important to prepare very thin smears prior to staining; the spores will be more visible, and the stain will penetrate more successfully. Without confirmation of the presence of the polar tubule in at least some of the spores (diagonal or horizontal line across the spore), it is almost impossible to confirm a microsporidial infection by using routine modified trichrome stains for fecal material. The microsporidia require molecular testing or electron microscopy to confirm the genus/species designations.
Malaria Parasites and Babesia spp.
One of the most common errors in examining blood smears is the incorrect identification of platelets as parasites. When mature schizonts rupture, the merozoites almost immediately penetrate another red blood cell (RBC) and are not seen outside of the RBCs. Extracellular “organisms” are almost always platelets. It is important to focus constantly when examining blood smears; the platelets are often on top of the RBCs. Another tip relates to color; parasites always have two colors, blue nuclei and red cytoplasm, and they are separate. Platelets tend to be uniform in color with no real internal structure; there are red and blue components, but the colors almost blend to form purple. Other internal structures within the RBCs such as Howell-Jolly bodies and Cabot’s rings may be confusing (Fig. 10.6 to 10.8). If the blood is held too long in EDTA anticoagulant or the ratio of blood to anticoagulant is incorrect, Plasmodium organisms can become distorted and may resemble other malaria stages or different species. Potential problems with using EDTA anticoagulant for the preparation of thin and thick blood films are discussed in chapter 31 (see Table 31.2).
Figure 10.6 Various structures within the RBC. (A) Malarial “ring” form (early trophozoite); (B) platelet on the RBC surface; (C) Howell-Jolly body; (D) Cabot’s ring. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch10.f6
FIGURE 10.7 (Top row and second row) Stain deposition on the surface of uninfected RBCs that could easily be confused with developing Plasmodium spp. stages. (Third row) Plasmodium falciparum gametocytes that have rounded up and no longer appear as the typical crescent-shaped gametocytes that are normally seen (could be due to low temperatures and/or storage for too many hours in EDTA blood). (Fourth row) Developing Plasmodium vivax trophozoites that appear to resemble P. falciparum gametocytes (found on blood smears prepared from EDTA blood that had been collected more than 8 h previously). (Bottom row) RBCs containing Howell-Jolly bodies that could be confused with very small, young ring forms of Plasmodium spp. doi:10.1128/9781555819002.ch10.f7
Figure 10.8 Exflagellation of Plasmodium vivax microgametocyte; these microgametes could easily be confused with some type of spirochete. These forms were seen in blood films prepared from blood stored for longer than 12 h in EDTA prior to additional smear preparation. (Left) Male Plasmodium gametocyte (microgametocyte) undergoing exflagellation. (Right) Single strand—very easily resembles a spirochete. doi:10.1128/9781555819002.ch10.f8
If a tube of blood containing EDTA cools to room temperature and the cap has been removed, the parasites can undergo several changes. The parasites within the RBCs will respond as if they were now in the mosquito after being taken in with a blood meal. The morphology of these changes in the life cycle and within the RBCs can cause confusion when examining blood films prepared from this blood (Fig. 10.8).
Although they are rarely seen, intracellular amastigotes called Leishman-Donovan bodies may be found in the monocytes in peripheral blood smears from patients with visceral leishmaniasis. When they are found in a bone marrow or splenic aspirate preparation, they are easier to find, probably because at that point there may be a high index of suspicion regarding the etiologic agent. In any suspected case of visceral leishmaniasis, peripheral blood buffy coat preparations are usually examined before more invasive procedures are undertaken. The amastigotes range from 3 to 5 µm, and a defined nucleus and kinetoplast may be difficult to see,
