Appendix 3 : APPENDIX 3 Common Problems in Parasite Identification

(a) Entamoeba histolytica/E. dispar trophozoite. Note the evenly arranged nuclear chromatin, central compact karyosome, and relatively clean cytoplasm. (b) Entamoeba coli trophozoite. Note the unevenly arranged nuclear chromatin, eccentric karyosome, and messy cytoplasm. These characteristics are very representative of the two organisms. However, in actual clinical specimens, there is great overlap between morphologic characteristics. When examining a patient specimen with a suspected mixed infection, one looks for two populations of organisms. When the majority of organisms seen appear to be from one genus/species, a rare one or two unusual organisms may just be outliers of the same genus/species that look a bit different. (Illustration by Sharon Belkin.)

(a) Entamoeba histolytica/E. dispar trophozoite. Note the evenly arranged nuclear chromatin, central compact karyosome, and clean cytoplasm. (b) Entamoeba coli trophozoite. Note that the nuclear chromatin appears to be evenly arranged, the karyosome is central (but more diffuse), and the cytoplasm is messy, with numerous vacuoles and ingested debris. The nuclei of these two organisms tend to resemble one another (a very common finding in routine clinical specimens). However, the karyosome in E. coli tends to be larger and more blot-like. (Illustration by Sharon Belkin.)

(a) Entamoeba histolytica/E. dispar trophozoite. Again, note the typical morphology (evenly arranged nuclear chromatin, central compact karyosome, and relatively clean cytoplasm). (b) Entamoeba coli trophozoite. Although the nuclear chromatin is eccentric, note that the karyosome seems to be compact and central. However, note the various vacuoles containing ingested debris. These organisms show some characteristics that are very similar (very common in clinical specimens). The nature of the karyosome tends to be very helpful; in E. histolytica/E. dispar the karyosome tends to be compact (may or may not be in the center). The karyosome of E. coli tends to be larger, more diffuse, and more irregular; it may or may not be eccentric. (Illustration by Sharon Belkin.)

(Top) Entamoeba histolytica/E. dispar trophozoites shown in a wet preparation (left) and a permanent stained smear (right). (Middle) E. histolytica/E. dispar trophozoite in permanent stained smear (left); Entamoeba coli trophozoite in permanent stained smear (right). (Bottom) E. histolytica/E. dispar trophozoite in permanent stained smear (left); E. coli trophozoite in permanent stained smear (right). Note the differences in the nucleus and cytoplasm: E. histolytica/E. dispar has even nuclear chromatin, a central, compact karyosome, and clean cytoplasm, whereas E. coli has uneven nuclear chromatin, eccentric karyosome, and messy/dirty cytoplasm. However, often there are nuclear and cytoplasmic characteristics that are not specific for one species or another; too many characteristics are seen that resemble either or both species. Again, the important differences can be seen as two populations of organisms. It may be difficult to assign a species designation to every organism seen, while the overall set of characteristics generally allow the organisms to be assigned to one or more species. All images stained with Wheatley's trichrome stain (routine trichrome for fecal specimens).

(a) Entamoeba histolytica trophozoite. Note the evenly arranged nuclear chromatin, central compact karyosome, and red blood cells (RBCs) in the cytoplasm. (b) Human macrophage. The key difference between the macrophage nucleus and that of E. histolytica is the size. Usually the ratio of nucleus to cytoplasm in a macrophage is approximately 1:6 or 1:8, while the true organism has a nucleus-to-cytoplasm ratio of approximately 1:10 or 1:12. The macrophage also contains ingested RBCs. In patients with diarrhea or dysentery, trophozoites of E. histolytica and macrophages are often confused, occasionally leading to a false-positive diagnosis of amebiasis when no parasites are present. Both the actual trophozoite and the macrophage may also be seen without ingested RBCs and can mimic one another. (Illustration by Sharon Belkin.)

(Upper) Entamoeba histolytica trophozoites on permanent stained smears; note the evenly arranged nuclear chromatin, central, compact karyosome, and ingested RBCs. The presence of the ingested RBCs indicates the presence of the true pathogen, E. histolytica. Without the presence of ingested RBCs, the organism would have been identified as E. histolytica/E. dispar (it is impossible to determine pathogenicity from the morphology on the permanent stained smear). (Lower) Two macrophages from human fecal specimen. The main difference between the macrophage nucleus and that of E. histolytica is the size. Usually the ratio of nucleus to cytoplasm in a macrophage is approximately 1:6 or 1:8, while the true organism has a nucleus-to-cytoplasm ratio of approximately 1:10 or 1:12. All images but one stained with Wheatley's trichrome stain (routine trichrome for fecal specimens); upper right stained with iron-hematoxylin.

(a) Entamoeba histolytica/E. dispar precyst. Note the enlarged nucleus (prior to division) with evenly arranged nuclear chromatin and central compact karyosome. Chromatoidal bars (rounded ends with smooth edges) are also present in the cytoplasm. (b) Polymorphonuclear leukocyte (PMN). The nucleus is somewhat lobed (normal morphology) and represents a PMN that has not been in the gut very long. Occasionally, the positioning of the chromatoidal bars and the lobed nucleus of the PMN will mimic one another. The chromatoidal bars stain more intensely, but the shapes can overlap, as seen here. (Illustration by Sharon Belkin.)

(a) Entamoeba histolytica/E. dispar mature cyst. Note that the four nuclei are very consistent in size and shape. (b) PMN. Note that the normal lobed nucleus has now broken into four fragments, which mimic four nuclei with peripheral chromatin and central karyosomes. When PMNs have been in the gut for some time and have begun to disintegrate, the nuclear morphology can mimic that seen in an E. histolytica/E. dispar cyst. However, human cells are often seen in the stool in patients with diarrhea; with rapid passage of the gastrointestinal tract contents, there will not be time for amebic cysts to form. Therefore, for patients with diarrhea and/or dysentery, if “organisms” that resemble the cell in panel b are seen, think first of PMNs, not E. histolytica/E. dispar cysts. (Illustration by Sharon Belkin.)

(Upper) PMNs. Note the appearance of “multiple nuclei” in the cell on the left, while the cell on the right contains the lobed nucleus, which has not yet fragmented to mimic an Entamoeba cyst. (Lower) PMNs in which the lobed nuclei have not yet fragmented; these cells should not be confused with Entamoeba cysts. When PMNs have been in the gut for some time and have begun to disintegrate, the nuclear morphology can mimic that seen in an Entamoeba cyst. However, remember that in patients with diarrhea, the gut contents move too rapidly for cysts to form; thus, cells with what appear to be multiple nuclei are actually PMNs.

(Upper) Entamoeba coli precyst with two enlarged nuclei, one on each side of the precyst) (left) and E. coli cyst (five or more nuclei) (right). These cells could be confused with PMNs. (Lower) Entamoeba histolytica/E. dispar precyst on the left with a single enlarged nucleus; E. histolytica/E. dispar mature cyst (containing four nuclei and one chromatoidal bar) on the right. All images stained with Wheatley's trichrome stain (routine trichrome for fecal specimens).

(a) Endolimax nana trophozoite. This organism is characterized by a large karyosome with no peripheral chromatin, although many nuclear variations are normally seen in any positive specimen. (b) Dientamoeba fragilis trophozoite. Normally, the nuclear chromatin is fragmented into several dots (often a “tetrad” arrangement). The cytoplasm is normally more “junky” than that seen in E. nana. If the morphology is typical, as in these two illustrations, differentiating between these two organisms is not very difficult. However, the morphologies of the two are often very similar. (Illustration by Sharon Belkin.)

(a) Endolimax nana trophozoite. Note that the karyosome is large and surrounded by a “halo,” with very little chromatin on the nuclear membrane. (b) Dientamoeba fragilis trophozoite. In this organism, the karyosome is beginning to fragment and there is a slight clearing in the center of the nuclear chromatin. If the nuclear chromatin has not become fragmented, D. fragilis trophozoites can very easily mimic E. nana trophozoites. This could lead to a report indicating that no pathogens were present when, in fact, D. fragilis is now considered a definite cause of symptoms. (Illustration by Sharon Belkin.)

Endolimax nana trophozoites. Note the tremendous nuclear variation; there is more nuclear variation in this organism than any of the other intestinal protozoa. The karyosome appears quite different from organism to organism; they can mimic Dientamoeba fragilis and/or Entamoeba hartmanni (particularly if the nucleus appears to have peripheral chromatin like the organisms in row 3 from the top).

Dientamoeba fragilis trophozoites. (Top three rows) Some have a single nucleus, while others have two nuclei; the nuclei tend to fragment into several chromatin dots. (Row 4) The image on the left shows the clearing within the karyosome prior to fragmentation into chromatin dots. Both images in this row are stained using iron-hematoxylin. All other images are stained using the Wheatley's routine trichrome stain. (Bottom) These two images show the newly discovered cyst stage, with the double cyst wall (Stark D et al, J Clin Microbiol 52:2680, 2014). However, the cystic forms tend to be rare in human specimens, a probable factor in the original failure to report and confirm these forms.

(a) Endolimax nana trophozoite. Note the large karyosome surrounded by a clear space. The cytoplasm is relatively clean. (b) Iodamoeba bütschlii trophozoite. Although the karyosome is similar to that of E. nana, note that the cytoplasm in I. bütschlii is much more heavily vacuolated and contains ingested debris. Often, these two trophozoites cannot be differentiated. However, the differences in the cytoplasm are often helpful. There is a definite size overlap between the two genera. (Illustration by Sharon Belkin.)

(Upper left) Endolimax nana trophozoite (note the large single karyosome with very little peripheral chromatin); (right) Iodamoeba bütschlii trophozoite (note the large karyosome, some light peripheral nuclear chromatin, and the messy/dirty cytoplasm containing many vacuoles). (Lower left ) E. nana cyst (note the four karyosomes with no peripheral chromatin; shape tends to be somewhat round to oval); (right) I. bütschlii cyst (note the large vacuole, single nucleus with large karyosome, and chromatin granules at one edge of the nucleus [basket nucleus]).

(Top) (a) RBCs on a stained fecal smear. Note that the cells are very pleomorphic but tend to be positioned in the direction in which the stool was spread onto the slide. (b) Yeast cells on a stained fecal smear. These cells tend to remain oval and are not aligned in any particular way on the smear. These differences are important when the differential identification is between Entamoeba histolytica containing RBCs and Entamoeba coli containing ingested yeast cells. If RBCs or yeast cells are identified in the cytoplasm of an organism, they must also be visible in the background of the stained fecal smear. (Illustration by Sharon Belkin.) (Middle) PMNs and RBCs (see arrows). The RBC cell shape can vary tremendously. (Bottom) Yeast cells in fecal material. Note the shape tends to be consistent and there appears to be no particular direction of placement such as is seen frequently with RBCs.

(Top) (a) Entamoeba histolytica/E. dispar cyst. Note the shrinkage due to dehydrating agents in the staining process. (b) E. histolytica/E. dispar cyst. In this case, the cyst exhibits no shrinkage. Only three of the four nuclei are in focus. Normally, this type of shrinkage is seen with protozoan cysts and is particularly important when a species is measured and identified as either E. histolytica/E. dispar or Entamoeba hartmanni. The whole area, including the halo, must be measured prior to species identification. If just the cyst is measured, the organism would be identified as E. hartmanni (nonpathogenic) rather than E. histolytica/E. dispar (possibly pathogenic). (Illustration by Sharon Belkin.) (Middle, left) E. histolytica/E. dispar cyst with four nuclei and one chromatoidal bar (note the shrinkage around the cyst); (right) Entamoeba hartmanni cyst with two of the four nuclei visible and two chromatoidal bars. (Note also the shrinkage around the cyst wall; this entire area would need to be measured for accurate species identification.) (Bottom) Entamoeba coli cysts. Note the shrinkage around the cyst wall; however, the identification to species could be made on the basis of five or more visible nuclei.

(a) Plasmodium falciparum rings. Note the two rings in the RBC. Multiple rings per cell are more typical of P. falciparum than of the other species causing human malaria. (b) Babesia rings. One of the RBCs contains four small Babesia rings. This particular arrangement is called the Maltese cross and is diagnostic for Babesia spp. (although this configuration is not always seen). Babesia infections can be confused with cases of P. falciparum malaria, primarily because multiple rings can be seen in the RBCs. Another difference involves ring morphology. Babesia rings are often of various sizes and tend to be very pleomorphic, while those of P. falciparum tend to be more consistent in size and shape. (Illustration by Sharon Belkin.)

(Top, left) Plasmodium falciparum rings. Note the “clean” morphology and headphone appearance of some of the rings; (right) Babesia spp. Note the “messy/pleomorphic” rings and the presence of the Maltese cross configuration of four rings within a single RBC; this does not occur in every species of Babesia. (Middle, left) P. falciparum ring forms in a heavy infection (note the clearly defined rings); (right) Babesia spp.; note smaller rings. Also note the rings outside of the RBCs, which does not occur in malarial infections. (Bottom, left) P. falciparum rings in a thick film (note the nuclei and cytoplasm portion of the ring forms); (right) Babesia spp. thick film; note the nuclei are seen and the nuclei appear smaller than those seen in the malaria thick film. However, differentiation of organisms based on thick film morphology can be quite difficult. Identification is best achieved from the thin blood film.

(a) Strongyloides stercoralis rhabditiform larva. Note the short buccal capsule (mouth opening) and the internal structure, including the genital primordial packet of cells. (b) Root hair (plant material). Note that there is no specific internal structure and the end is ragged where it was broken off from the main plant. Plant material often mimics some of the human parasites. This comparison is one of the best examples. These artifacts are occasionally submitted as proficiency-testing specimens. (Illustration by Sharon Belkin.)

(Upper) Root hair artifact that resembles actual nematode larva. (Lower) Strongyloides stercoralis rhabditiform larva. Note the internal structures, especially the large, genital primordium packet of cells (circle).

(a) Taenia egg. This egg has been described as having a thick, radially striated shell containing a six-hooked embryo (oncosphere). (b) Pollen grain. Note that this trilobed pollen grain has a similar type of “shell” and, if turned the right way, could resemble a Taenia egg. This similarity represents another source of confusion between a helminth egg and a plant material artifact. When examining fecal specimens in a wet preparation, you can tap on the coverslip to get objects to move around. As they move, you can see more morphologic detail. (Illustration by Sharon Belkin.)

(Left) Taenia egg, showing the striated shell and six-hooked embryo (oncosphere) within the egg shell. (Right) Pollen grain that can resemble an actual Taenia egg. Note that the shape may vary, especially depending on the way the pollen grain is lying in the wet preparation.

(a) Trichuris trichiura egg. This egg is typical and is characterized by the barrel shape with thick shell and two polar plugs. (b) Bee pollen. This artifact certainly mimics the T. trichiura egg. However, note that the shape is somewhat distorted. This is an excellent example of a parasite look-alike that could be confusing. (Illustration by Sharon Belkin.)

(Upper, left) Trichuris trichiura egg; (right) pollen that can mimic a T. trichiura egg. (Lower) Examples of pine pollen.

Relative sizes of helminth eggs.