Читать книгу Diagnostic Medical Parasitology - Lynne Shore Garcia - Страница 19
ОглавлениеAlthough many body sites and specimens can be examined for the presence of parasites, the most difficult specimen in which to differentiate parasites from artifacts is usually fecal material. Feces consist of a number of components, including (i) undigested food residue; (ii) digestive by-products; (iii) epithelial and other human cells, mucus, and other secretions from the digestive tract; and (iv) many types of microorganisms such as bacteria and yeasts. Considering the ratio between fecal debris and parasites, it is not surprising that many artifacts are responsible for incorrect identifications of protozoan trophozoites and cysts and of helminth eggs and larvae. Often, many yeast cells and other artifacts are confused with coccidian oocysts or microsporidial spores. Appropriate training, adherence to protocols, use of quality control measures, and availability of reference materials and consultants should help minimize identification errors.
A number of cells and other organisms can easily be confused with intestinal protozoa. These are listed in Tables 10.1 and 10.2 and illustrated in Fig. 10.1.
Occasionally, free-living amebae are found in feces or as contaminants in water. Morphologically, they differ from parasitic amebae in having one or more large contractile vacuoles in the trophozoite form and having very thick cyst walls. They can also be differentiated on the basis of cultivation; i.e., they are much easier to culture than the pathogenic protozoa. Amebae which have been recovered from stool material include Entamoeba moshkovskii, Naegleria gruberi, Hartmanella hyalina, Sappinia diploidea, Vahlkampfia punctata, and Vahlkampfia lobospinosa. Contamination of specimens can be avoided by using dry collection containers, saline, or formalin for the concentration rinses and dilution of the specimen and by rapid fixation or examination of the specimen immediately after passage (1).
Some protozoa, such as Entamoeba coli, may contain fungi. Sphaerita spp. can be found in the cytoplasm, and Nucleophaga spp. can be found in the nucleus. Sphaerita spp. (sometimes called Polyphaga spp.) measure approximately 0.5 to 1.0 µm and are found in tightly packed clusters (Fig. 10.2) (2).
FIGURE 10.1 Various structures that may be seen in stool preparations. (Top row) Macrophage (left) and epithelial cells (right) that can be confused with Entamoeba histolytica/E. dispar trophozoites. (Second row) PMN with a fragmented nucleus (left) and artifact (right) that can be confused with Entamoeba spp. cysts. (Third row) Two artifacts that can resemble protozoan cysts. (Fourth row) Yeast cells (left) and an artifact (right) that can be confused with Cryptosporidium spp. and Cyclospora cayetanensis, respectively, on positive acid-fast stains; it is important to measure the structures and organisms carefully before confirming organism identification. (Bottom row) Yeast cells (left) that can be confused with microsporidial spores (however, notice the budding cell within the circle), and artifacts (right) that can also be confused with microsporidial spores; these were thought to be small yeast cells. doi:10.1128/9781555819002.ch10.f1
Figure 10.2 Fungi parasitizing Entamoeba coli. (Upper) Entamoeba coli containing ingested Sphaerita. (Lower) (A) Sphaerita (or Polyphaga) sp. within the cytoplasm; (B) Nucleophaga sp. within the nucleus. doi:10.1128/9781555819002.ch10.f2
Flagellates can be difficult to differentiate, and free-living organisms are occasionally seen in a stool specimen that has been contaminated with water or saline containing Bodo caudatus and Cercomonas longicauda. These two organisms are classified in the same family as Retortamonas intestinalis (Fig. 10.3).
Figure 10.3 Free-living freshwater flagellates. (Upper) Bodo caudatus. (Lower) Cercomonas longicauda. These organisms can be confused with parasitic protozoa, particularly if motility is seen in a wet mount of fresh stool contaminated with freshwater. doi:10.1128/9781555819002.ch10.f3
Apparently free-living ciliates are found in stagnant water, sewage, and soil and may be seen in fecal specimens contaminated with water or saline. Organisms that have been reported are Litonotus and Paramecium (1–3) (Fig. 10.4).
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, particularly if a number of organisms are packed in the cell (see chapters 7 and 19). The amastigotes may resemble Histoplasma capsulatum if the kinetoplast bar structure is not easily seen (Fig. 10.9).
Figure 10.9 (Left) Histoplasma capsulatum. (Right) Leishmania donovani. Note that the Leishmania amastigotes have the bar structure while the Histoplasma amastigotes do not; Histoplasma also has a “halo” around the organisms. doi:10.1128/9781555819002.ch10.f9
Any laboratory using staining reagents must use good quality control measures to ensure that the solutions do not become contaminated with artifacts or free-living organisms. Bits of cotton fiber, lint, and other components of dust can mimic microfilariae in wet mounts or when stained. However, the artifacts do not contain any internal nuclei. Rarely, nonhuman parasites can also be confused if they appear on the stained blood smears. One laboratory used to dry the blood films upright, leaning against a fish tank (many years ago, before more stringent safety measures were instituted). Examination of one of the stained blood films suggested that the patient had a filarial infection. Since the patient history did not support this diagnosis, further studies were performed. When samples of the fish tank water were centrifuged and examined, the “microfilariae” were found! This is just one example of the many unusual sources of artifact contamination (Fig. 10.10).
Figure 10.10 Fungal spores (Helicosporium type). Artifacts that can resemble microfilariae in stained blood films; these structures are not parasites but instead are some type of thread. Note that there is little to no internal structure visible. (Left) Courtesy of the CDC Public Health Image Library. (Middle and right) Photographed at a higher magnification than the image on the left. doi:10.1128/9781555819002.ch10.f10
Body Fluids: Ciliated Epithelial Cells
Detached ciliary tufts (ciliocytophthoria) have been seen in a variety of body fluids (especially peritoneal and amniotic fluids; also respiratory specimens). These tufts are the remnants of ciliated epithelium that are found as a part of normal cellular turnover in a number of sites: respiratory tract and sinuses, ventricles of the brain, central canal of the spinal cord, and epithelia of the male and female reproductive tracts. The tufts are motile, measure 10 to 15 µm in diameter, and can be confused with ciliated or flagellated protozoa. However, when they are carefully examined on a stained smear, there is no internal structure like that seen in protozoa (4–6). Ciliated epithelium cells can be seen in Fig. 10.11; ciliocytophthoria are anucleate remnants of these ciliated cells. However, in some cases, the cell nuclei are clearly visible. The ciliated tufts can be seen on the cells in this figure.
Figure 10.11 Bronchial epithelium cells. When these cells disintegrate, the ciliary tufts may be visible and may be confused with protozoan flagellates or ciliates (detached ciliary tufts = ciliocytophthoria). doi:10.1128/9781555819002.ch10.f11
These cells have been found in many clinical specimens, including peritoneal fluid (7). Recognition of these uncommon structures can be important in avoiding a misdiagnosis with a suspect protozoan infection. This has also been recognized as a potential problem in the virology laboratory (5). The difference between ciliocytophthoria and ciliated or flagellated parasites becomes very important during examination of specimens submitted for cytologic testing. Also, in performing immunofluorescence assays (direct fluorescent antibody [DFA] tests) used in the virology laboratory for the rapid detection of viruses, assessment of the cellularity of specimens is required for the most effective use of the DFA assay.
Plant or root hairs, such as the fuzz on peaches, may resemble nematode larvae. The root hairs tend to be clear and refractile, while the larvae pick up stain (iodine) which reveals internal structures (Fig. 10.12). It is important to recognize this potential error when examining formalin-fixed specimens submitted as proficiency testing specimens. In fecal specimens from patients with diarrhea, partially digested plant material, such as bean sprouts or other vegetable material, can mimic adult nematodes or tapeworm proglottids. Also, all stages of free-living nematodes can occur in feces or as contaminants of the water used in making fecal suspensions.
Figure 10.12 (Top) Root hair. (Middle) Root hair. Note that there is no internal structure visible within the root hairs. (Bottom) Strongyloides stercoralis rhabditiform larva. Note the short buccal cavity at the head end of the larva and the genital primordial packet of cells within the curved portion of the body (arrow). doi:10.1128/9781555819002.ch10.f12
Hairworms (often called horsehair worms) belonging to the phylum Nematomorpha can be confused with human parasites (8). These adult worms are slender, measuring 10 to 50 cm long, and have a blunt, rounded anterior end. “The caudal end of the male is bifurcate or has a dorsoventral groove; that of the female is entire or trilobate” (8) (Fig. 10.13). The adult worms are free living in water, while the larvae are parasites of insects. Human infection is quite rare and accidental, although in the past literature, serious health problems were attributed to “hair snakes” in the human body. Generally, human infection occurs through ingestion of free-living adult worms or adolescent worms within their insect hosts in drinking water or food. Worms have been reported as passing from the urethra in several cases. They have been identified as being in the genera Gordius, Chordodes, Parachordodes, Paragordius, Pseudogordius, and Neochordodes. Worms have been recovered in vomitus, urine, and feces; often, the stated origin of the worms in the body was not well documented. In spite of the reported cases, no evidence of pathogenicity has been demonstrated (8). Often symptoms were attributed to other causes or were psychological.
Figure 10.13 Gordius worms. (Upper) Adult worms (called hairworms or horsehair worms), which measure 20 to 50 cm long and are very slender. (Lower) Characteristic structure of Gordius worms. (A) Diagram of anterior end; (B) diagram of posterior end of a male worm in the genus Gordius; (C) whole worm. Bar, 1 cm. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch10.f13
In some cases, very large objects are recovered in patient stools. One structure resembled a trematode, but was actually a slug. These objects can be very confusing, particularly when it is unclear whether the object actually passed through the intestinal tract or entered the stool after passage (Fig. 10.14).
FIGURE 10.14 (Top) Slug recovered in stool; note the linear markings on the body. (Second) Underside of the slug. (Third) Fresh, living trematode; note the resemblance to the slug. (Bottom) Actual slug; note the markings on the body (this slug transmits Angiostrongylus). (Top and second, courtesy of Larry D. Gray, Clinical Microbiology Laboratory Consultants, LLC; bottom two images courtesy of the CDC Public Health Image Library.) doi:10.1128/9781555819002.ch10.f14
Plant cells tend to have thick, smooth walls and are not as symmetrical as helminth eggs (Fig. 10.15 and 10.16). Some of these plant cells range from 15 to 150 µm in diameter and may be confused with Ascaris eggs. Pollen grains are also thick-walled, symmetrical structures that stain very darkly with iodine, may be round or trilobed, and are 15 to 20 µm in diameter (Fig. 10.17). They may resemble Taenia eggs. Another example is the “Beaver body.” This structure is Psorospermium haeckelii, a stage of an alga that occurs in the tissues of crayfish. It is sometimes confused with helminth eggs when found in fecal specimens from individuals who have ingested crayfish (9) (Fig. 10.18). Accidental ingestion or contamination of food or water containing the eggs of plant nematodes, such as Heterodera species or mites or mite eggs, can also lead to confusing situations.
FIGURE 10.15 Various artifacts that may be seen in stool preparations (wet mounts or permanent stained smears). Many of these structures are pollen grains or egglike objects. Visually, they can be confused with some of the following helminth eggs: Hymenolepis nana, Ascaris lumbricoides, hookworm, and Enterobius vermicularis. doi:10.1128/9781555819002.ch10.f15
FIGURE 10.16 Various artifacts that may be seen in stool preparations (wet mounts). Note the egg-like structure in the fourth row (left). There is a small bubble (within the circle) that mimics the small knob found at the abopercular end of a Diphyllobothrium latum egg. doi:10.1128/9781555819002.ch10.f16
FIGURE 10.17 Various types of pollen grains and a root hair. These structures can mimic various helminth eggs (Ascaris lumbricoides, Trichuris trichiura), as well as nematode larvae. (Bottom row, left) Plant root hair (can mimic helminth larvae). (Row 4 from top: left, courtesy of Randy Oliver, photo by Gretchen D. Jones, United States Department of Agriculture; right, courtesy of http://cactiguide.com/forum/viewtopic.php?t=12480&view=next [accessed 7/23/13]). doi:10.1128/9781555819002.ch10.f17
Figure 10.18 (Upper) “Beaver bodies,” which are algae occasionally found in stool; (lower) leaf structure which resembles a trematode. (Courtesy of Joseph Dipersio.) doi:10.1128/9781555819002.ch10.f18
The human cells most likely to cause problems with identification are the polymorphonuclear leukocytes (PMNs) and the macrophages seen in stained fecal smears (Table 10.1; Fig. 10.19). These cells are frequently present in patients with nonspecific inflammatory bowel disease (ulcerative colitis). Therapy for this condition often includes immunosuppressive agents; this therapy would definitely be contraindicated in patients with amebiasis (2). Therefore, the correct identification between human cells and pathogenic protozoa is critical to good patient care. These cells should be reported and quantitated (rare, few, moderate, many). PMNs are frequently misidentified as a protozoan cysts, usually in the genus Entamoeba; as the PMNs begin to age, the lobed nucleus fragments into several parts, each resembling an amebic cyst nucleus. However, usually PMNs are seen in patients who tend to be symptomatic; patients with diarrhea do not tend to have protozoan cysts in the stool. The gut motility is so rapid that cyst formation does not occur; also, trophozoites do not encyst once they are outside of the body/gastrointestinal tract. The macrophage tends to have one large nucleus; the overall appearance can mimic the Entamoeba trophozoite.
Figure 10.19 (Left) PMNs in a fecal specimen stained with trichrome stain. Note the lobed nuclei; if these cells have been in the stool for some time (unpreserved), the nuclei may fragment into four or five pieces, thus resembling multiple nuclei seen in amebic cysts. Note the two RBCs in the upper left. (Middle) Macrophages. Although these cells often resemble amebic trophozoites, the ratio of nuclear material to cytoplasm is quite different from that seen in actual protozoa (more nuclear material per cytoplasm in human cells). (Right) PMNs and macrophages; also note the eosinophil (arrow). doi:10.1128/9781555819002.ch10.f19
Large numbers of PMNs are often found in patients with bacterial dysentery, and they may also be present in patients with intestinal amebiasis or ulcerative colitis (Fig. 10.19). These cells may be distinguished from Entamoeba histolytica as follows.
PMNs
1. Average size, 14 µm (10 to 12 µm on permanent stained smear)
2. Ratio of nuclear material to cytoplasm, 1:1
3. Nucleus: 2 to 4 segments connected by narrow, short chromatin bands. Segments may appear as separate nuclei like those of E. histolytica/E. dispar cysts—focus carefully to reveal connecting chromatin strands.
4. Granular cytoplasm
5. Trichrome staining characteristics similar to E. histolytica/E. dispar
E. histolytica/E. dispar (cysts)
1. Average size, 20 µm (less on permanent stained smear)
2. Ratio of nuclear material to cytoplasm, 1:10 to 1:12 (trophozoite), 1:2 to 1:3 (cyst)
3. Nucleus: round with central karyosome and peripheral chromatin
4. Uniform, agranular cytoplasm—may contain RBCs
5. Trichrome: green cytoplasm, dark red nuclear material
The identification of eosinophils in a fecal specimen usually indicates the presence of an immune response in the host. This allergic response may be caused by a parasitic infection or by other antigens such as pollen or food. Eosinophils are essentially the same size as PMNs and are characterized by the presence of large, purple-staining granules (trichrome) and usually a bilobed nucleus, which may be obscured by the granules (Fig. 10.19). Eosinophils can also be seen in other specimens such as blood specimens and sputum specimens from asthmatic patients (10, 11). When reviewing blood smears for parasites, it is helpful to recognize the typical white blood cells as seen in Fig. 10.20. Unfortunately, not all microbiologists have had training in hematology per blood cell recognition, although in fecal specimens the white blood cells do not tend to have the precise morphology as that seen in routine blood films.
Figure 10.20 White blood cells in a stained blood film. (1) Lymphocyte. (2) Basophil. (3) Eosinophil. (4) Monocyte. (5) PMN. doi:10.1128/9781555819002.ch10.f20
Macrophages (monocytes) are large, mononuclear, phagocytic cells that may resemble E. histolytica/E. dispar trophozoites (Fig. 10.19). These cells may be found in patients with intestinal amebiasis and ulcerative colitis and other inflammatory bowel diseases and can be differentiated from amebae as follows. One of the main differences is the ratio of nuclear material to cytoplasm, which is much larger in human cells.
Macrophages
1. Size: 30 to 60 µm, may be 5 to 10 µm less on permanent stained smear
2. Ratio of nuclear material to cytoplasm, 1:4–1:6
3. One large nucleus that may be irregular in shape (like monocyte nucleus)
4. Usually contains ingested debris, PMNs, and RBCs
5. May contain red-staining round bodies and nucleus may be absent
6. Trichrome staining characteristics similar to E. histolytica/E. dispar
E. histolytica/E. dispar (trophozoites)
1. Size: 12 to 60 µm; average, 20 µm (less on permanent stained smear)
2. Ratio of nuclear material to cytoplasm, 1:10 to 1:12
3. One nucleus, round, with central karyosome and peripheral chromatin
4. May contain RBCs and some debris; no PMNs
5. Nucleus always present
6. Trichrome: green cytoplasm, dark red nuclear material
Lymphocytes have a large, dense, dark-staining nucleus surrounded by very little cytoplasm. They are approximately two-thirds the size of PMNs (Fig. 10.20).
In a buffy coat preparation, some RBCs may be present. These cells measure ∼7.5 µm in a wet preparation and may be present in the stool as an indication of ulceration (parasitic or nonspecific) or other vascular or hemorrhagic problems. In the trichrome-stained slide, they appear as round or elongate (distortion may occur during smear preparation) red-purple bodies with no granules or inclusions and may be somewhat smaller than 7.5 µm (Fig. 10.19 and 10.20).
Charcot-Leyden (CL) crystals are formed from the breakdown products of eosinophils and basophils and may be present in the stool or sputum with eosinophils or alone. They are slender crystals with pointed ends, and they stain red-purple with trichrome stain. Many different crystal sizes can be seen in the same specimen (Fig. 10.21). They indicate that an immune response has taken place, but the cause may or may not be parasitic. The presence of eosinophils and/or CL crystals in the stool may not correlate with an increased eosinophilia on the peripheral blood smear.
Figure 10.21 Charcot-Leyden (CL) crystals. These crystals are formed from the breakdown products of eosinophils and basophils and may be present in the stool, sputum, or other specimens with or without eosinophils. They tend to stain red to red-purple on the permanent stained fecal smears, often darker than nuclear material, and although the shape is consistent, there is a large size range in a single fecal smear or sputum mount. (Upper, left and right) CL crystals in trichrome-stained fecal smear. (Lower, left) CL crystals in a sputum specimen. (Lower, right) Pineapple crystals in stool. These may be confused with CL crystals; however, the pineapple crystals are much more slender. doi:10.1128/9781555819002.ch10.f21
Identification of CL crystals in body fluids and secretions is considered an indicator of eosinophil-associated allergic inflammation. The overall structural fold of CL crystal protein is similar to that of galectins, and this is the first structure of an eosinophil protein to be determined (5). The protein exhibits weak carbohydrate binding activity for simple saccharides. There may be a potential intracellular and/or extracellular role(s) for the galectin-associated activities of CL crystal protein in eosinophil and basophil function in allergic diseases and inflammation (10).
Parasitic (primarily helminthic) infections push the immune system towards TH2 cytokine production and eosinophilia. Since eosinophil infiltration in infected organs and skin is a common finding, eosinophils are thought to have a specific role in parasite killing. Eosinophils have been considered as effectors of adaptive immune responses during parasitic infections and inflammatory processes. Their role in allergic and mucosal responses is mediated by membrane receptors that allow them to interact with IgE and IgA antibodies (10).
It has been reported that pineapple crystals sometimes mimic CL crystals in stool specimens. Apparently, these crystals can be found in fresh and canned pineapple and pineapple juice; also, they are not digested in the alimentary canal of humans. They range from 30 to 130 µm in length and 1 to 2 µm in width (Fig. 10.21). They appear to have parallel edges and are pointed at both ends. In wet preparations, they can resemble CL crystals. Those who routinely examine stool specimens for parasites should probably examine some pineapple juice under the microscope to become familiar with the appearance of these crystals.
Nonhuman Elements Seen in Feces (Yeast Cells)
There are many yeast cells that may be round to oval and measure ~4 to 8 µm which can be seen in fecal material. On a wet mount, they may resemble small protozoan cysts (Endolimax nana or Entamoeba hartmanni). After staining, they appear fairly uniform in color (red to green with trichrome stain) without many inclusions; if granules are seen, they are usually small but may resemble small protozoan karyosomes. Depending on the stain used, small yeast cells can be confused with coccidian oocysts or microsporidial spores. It is important to note the presence of budding yeast cells and/or pseudohyphae (clinically relevant only in freshly preserved specimens). The presence of branching pseudohyphae may be an indication of pathogenicity of the particular yeast present (usually Candida spp.) and should be reported. Large numbers of budding yeast cells in a fresh or freshly preserved specimen, indicating a potential source for a systemic infection, particularly in immunosuppressed patients, should also be reported (Fig. 10.22).
Figure 10.22 Yeast cells in clinical specimens. (Upper and middle) Trichrome-stained fecal smears. Depending on the size and permanent stain used (trichrome, modified acid-fast, modified trichrome), single yeast cells can often be confused with the coccidia or microsporidia. (Bottom left) Various yeasts in blood films; (right) Histoplasma within monocytes. doi:10.1128/9781555819002.ch10.f22
Note Because yeast can continue to grow if the stool is not immediately preserved, some laboratories do not report yeast, since the report can be misleading. They elect to call the physician and discuss the findings. Another option is to add a report comment indicating that reports of yeast (budding and/or pseudohyphae) might be misleading due to a lag time between stool passage and specimen fixation.
Finding insect larvae in stool is not common but may occur as a result of ingestion of whole larvae or adult insects with food. The presence of live larvae may suggest myiasis or, probably more common, contamination of the stool specimen. In these situations, it is always important to find out how and when the specimen was collected prior to submission, particularly if it was submitted as a fresh stool. Proper fixation of the suspected object is important for further identification (see chapter 9).
Spurious infections occur when individuals ingest liver from various animals. The various parasite eggs are digested free when the liver is eaten and will be passed in the stool for several days. Repeat ova and parasite examinations are recommended for several days to rule out a true infection. Examples are eggs of Fasciola hepatica, Dicrocoelium dendriticum, or Capillaria hepatica, which are present in the livers of cattle, sheep, and rodents, respectively (12). Occasionally, rarer eggs are found and may represent spurious infections acquired by eating the flesh of fish, birds, or other animals, both vertebrates and invertebrates (Fig. 10.23). In a true human infection with Capillaria hepatica, no eggs are found in the stool as they are in an infection with Capillaria philippinensis; diagnosis requires histologic examination. Eggs in liver biopsy specimens can be identified on the basis of their characteristic morphology.
Figure 10.23 (Top row, left) Fasciola hepatica egg (130 to 150 µm by 63 to 90 µm) (image is lower magnification than Dicrocoelium egg); (right) Dicrocoelium dendriticum egg (38 to 45 µm by 22 to 30 µm). (Middle row, left) Capillaria philippinensis egg (51 to 68 µm by 30 to 35 µm), passed in the stool (resembles egg of Trichuris trichiura); (right) Capillaria hepatica eggs in liver. (Bottom row, left) Capillaria philippinensis egg; (right) Trichuris trichiura egg. Note the striated shell of C. philippinensis compared with the nonstriated shell seen in the Trichuris egg. doi:10.1128/9781555819002.ch10.f23
Delusory Parasitosis (Delusional Infestation)
Occasionally, clinical specimens in which the patient has placed various objects or organisms to feign parasitism are submitted for examination. These patients are usually mentally disturbed and have often seen numerous physicians and submitted clinical specimens to many laboratories. The objects placed in the specimens range from pieces of thread to plant material to earthworms. Often, these patients call the laboratory with extensive histories of “parasitic infections” and seek referrals to other experts or consultants. They may bring photographs or samples of the “parasites” that they have found, all of which will be submitted for identification. These “infections” are not limited to specimens such as stool but may include skin, urine, and other samples for analysis. Often, these patients are women older than 50 years, with possible other medical problems, who present with a wide variety of symptoms. It is likely that dopaminergic and serotonergic dysfunction may play a role in delusional parasitosis; dopamine and serotonin antagonists may be relevant for the treatment of this disorder (13–16).
This disease can become a tremendous burden both for the patient and for the family. Patients can appear to be very rational about everything else, with the exception of their beliefs concerning infection or infestation with parasites. However, actual infections have been misdiagnosed as delusory parasitosis when appropriate diagnostic procedures have not been used and the true infections have been missed. A thorough investigation for parasites on the patient, on pets, and in the work and home environment should be completed before assigning a diagnosis of delusory parasitosis.
Patients with delusory parasitosis pose a difficult interdisciplinary problem for the medical system. Such patients avoid psychiatrists and consult dermatologists, microbiologists, or general practitioners but often lose faith in professional medicine. Epidemiology and history suggest that the imaginary pathogens may continually change, while the delusional aspect remains constant. Patients with self-diagnosed “Morgellons disease” can be seen as a variation of this delusional theme (15). Reference 15 provides very comprehensive information on all aspects of this disorder, including recommendations for physician/patient interactions and guidance algorithms.
The minimal criteria for delusional infestation are as follows:
• A strong conviction of being infested by pathogens (small, vivid, inanimate [rare], often “new to science”) without any medical or microbiological evidence for this, ranging from overvalued ideas to a fixed, unshakable belief.
• Report of abnormal sensations in the skin explained by the first criterion.
Additional symptoms may include visual illusions or hallucinations. Although the “infestation” is generally associated with the skin, all parts of the body may be involved. Often this condition has gone on for months or years (15).
References
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12. Fuehrer HP, Igel P, Auer H. 2011. Capillaria hepatica in man—an overview of hepatic capillariosis and spurious infections. Parasitol Res 109:969–979. PMID 21717279
13. Narumoto J, Ueda H, Tsuchida H, Yamashita T, Kitabayashi Y, Fukui K. 2006. Regional cerebral blood flow changes in a patient with delusional parasitosis before and after successful treatment with risperidone: a case report. Prog Neuropsychopharmacol Biol Psychiatry 30:737–740. PMID 16431007
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15. Aw DC, Thong JY, Chan HL. 2004. Delusional parasitosis: case series of 8 patients and review of the literature. Ann Acad Med Singapore 33:89–94. PMID 15008571
16. Wenning MT, Davy LE, Catalano G, Catalano MC. 2003. Atypical antipsychotics in the treatment of delusional parasitosis. Ann Clin Psychiatry 15:233–239. PMID 14971869
