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Category: Clinical Microbiology
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(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. (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 (very common finding in routine clinical specimens). (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 typical in clinical specimens). (Illustration by Sharon Belkin).
(a) Entamoeba histolytica trophozoite. Note the evenly arranged nuclear chromatin, central compact karyosome, and 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/cytoplasm ratio of approximately 1:10 or 1:12. The macrophage also contains ingested RBCs. In cases of diarrhea or dysentery, trophozoites of E. histolytica and macrophages can often be 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 they can mimic one another. (Illustration by Sharon Belkin.)
(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) 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 that of the lobed nucleus of the PMN can mimic one another. The chromatoidal bars will stain more intensely, but shapes can overlap, as seen here. (Illustration by Sharon Belkin.)
(a) Entamoeba histolytica/E. dispar 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 cases of diarrhea; with rapid passage of the gastrointestinal tract contents, there will not be time for amebic cysts to form. Therefore, in cases of diarrhea and/or dysentery, if “organisms” are seen that resemble the cell in panel b, think first of PMNs, not E. histolytica/E. dispar cysts. (Illustration by Sharon Belkin.)
(a) Endolimax nana trophozoite. This organism is characterized by a large karyosome with no peripheral chromatin, although there are normally many nuclear variations 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, then differentiating between these two organisms is not that difficult. However, the morphologies of the two will often be very similar. (Illustration by Sharon Belkin.)
(a) Endolimax nana trophozoite. Notice that the karyosome is large and surrounded by a “halo”, with very little, if any, 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 considered a definite cause of symptoms. (Illustration by Sharon Belkin.)
(a) Endolimax nana trophozoite. Note the large karyosome surrounded by a clear space. The cytoplasm is relatively “clean.” (b) Iodamoeba bütschlii. 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 will be a definite size overlap between the two genera. (Illustration by Sharon Belkin.)
(a) RBCs on a stained fecal smear. Note that the cells are very pleomorphic but tend to be positioned in the direction 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.)
(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.)
(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 of human malaria. (b) Babesia rings. In one of the RBCs are four small Babesia rings. This particular arrangement is called the Maltese cross and is diagnostic for Babesia spp. However, the Maltese cross configuration is not always present. 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.)
(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). Often plant material 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.)
(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 represents another confusion between a helminth egg and a plant material artifact. When examining fecal specimens in a wet preparation, tap on the coverslip to get objects to move around. As they move, you can see more morphological detail. (Illustration by Sharon Belkin.)
(a) Trichuris trichiura egg. This egg is typical and is characterized by the barrel shape with a thick shell and two polar plugs. (b) Bee pollen. This artifact certainly mimics the actual T. trichiura egg. However, note that the actual shape is somewhat distorted. This is an excellent example of a parasite “look-alike” and could be confusing. (Illustration by Sharon Belkin.)
Procedure for processing fresh stool for the ova and parasite examination. Special stains will be necessary for Cryptosporidium and Cyclospora (modified acid-fast) and the microsporidia (modified trichrome, calcofluor). Immunoassay kits are now available for some of these organisms. If the permanent staining method (iron hematoxylin) contains a carbol fuchsin step, the coccidia will stain pink. Symbols: †, some protozoa may not be identified using the wet examination only; ‡, protozoa (primarily trophozoites) can be identified and cysts can be confirmed.
Procedure for processing liquid specimens for the ova and parasite examination. Polyvinyl alcohol (PVA) and specimen will be mixed together on the slide, allowed to air dry, and then stained (fixation is sufficient for liquid specimen but not for formed stool). Symbols: †, some protozoa may not be identified from the concentration procedure; ‡, protozoa (trophozoites can be identified).
Procedure for processing preserved stool for the ova and parasite examination. Fixatives and effects: mercuric chloride, best polyvinyl alcohol (PVA) (trichrome, iron hematoxylin); zinc, current best substitute (trichrome, hematoxylin probably okay); copper sulfate, fair substitute (trichrome, iron hematoxylin: both fair to poor); sodium acetate-acetic acid-formalin, good substitute for PVA fixative (iron hematoxylin is best, and trichrome is okay).
Procedure for processing sodium acetate-acetic acid-formalin (SAF)-preserved stool for the ova and parasite examination. SAF can also be used with EIA, fluorescent-antibody, and cartridge immunoassay kits and the modified trichrome stain for microsporidia.
Procedure for processing stool specimens preserved in the Universal Fixative (TOTAL-FIX). Approaches are provided for immunoassays, stool sedimentation concentration, permanent stained smears (modified acid-fast, trichrome, modified trichrome, etc.), and molecular testing. This fixation contains no mercury, no formalin, and no PVA.
The traditional method for preparing a thin blood film; the blood can be either “pushed” or “pulled” by the spreader slide Note that “D” illustrates a thin film with a good feather edge. (Illustration by Sharon Belkin.)
Poorly prepared thin and thick blood films (dirty slides, oil on slides, too-thick preparations, poor spreading of the blood); organism morphology will be very poor on the stained films.
Method of thick-thin combination blood film preparation. (a) Position of drop of EDTA-containing blood. (b) Position of the applicator stick in contact with blood and glass slide. (c) Rotation of the applicator stick. (d) Completed thick-thin combination blood film prior to staining. (Illustration by Sharon Belkin.)
General diagram of a rapid malaria test; top, the negative test shows the control line only; middle, control line plus the Plasmodium vivax line indicates the presence of a panspecific antigen (common to all Plasmodium spp., but most sensitive for P. vivax rather than P. ovale and P. malariae); bottom, control line, panspecific antigen line, and line specific for Plasmodium falciparum antigen presence. Note that most cartridge rapid malaria tests have not been developed to detect Plasmodium knowlesi (under development). CI, confidence interval.
Body sites and possible parasites recovered (trophozoites, cysts, oocysts, spores, adults, larvae, eggs, amastigotes, and trypomastigotes) a
Body site, specimen, and recommended stain(s) a
Protozoa of the intestinal tract and urogenital system: key characteristics a
Helminths: key characteristics a
Parasites found in blood: characteristics a
Sources of commercial reagents and supplies a
Sources of available reagents for immunodetection of parasitic organisms or antigens a
Addresses of suppliers listed in Table 9.10.6–A3
Sources of parasitologic specimens (catalogs of available materials and price lists available from the -companies listed) a
Examination of blood specimens a