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ОглавлениеEnterobius vermicularis, known as the pinworm or seatworm, is a roundworm parasite that has worldwide distribution and is commonly found in children. The adult female worm migrates out of the anus, usually at night, and deposits her eggs on the perianal area. The adult female (8 to 13 mm long) is occasionally found on the surface of a stool specimen or on the perianal skin. Since the eggs are usually deposited around the anus, they are not commonly found in feces and must be detected by other diagnostic techniques. Diagnosis of pinworm infection is usually based on the recovery of typical eggs, which are described as thick-shelled, football-shaped eggs with one slightly flattened side. Each egg often contains a fully developed embryo and will be infective within a few hours after being deposited.
The most striking symptom of this infection is pruritus, which is caused by the migration of the female worms from the anus onto the perianal skin before egg deposition. The sometimes intense itching results in scratching and occasional scarification. In most infected people, this may be the only symptom, and many individuals remain asymptomatic. Eosinophilia may or may not be present.
Infections tend to be more common in children and occur more often in females than in males. In heavily infected females, there may be a mucoid vaginal discharge, with subsequent migration of the worms into the vagina, uterus, fallopian tubes, appendix, or other body sites including the urinary tract, where they become encapsulated (1–8). Although tissue invasion has been attributed to the pinworm, these cases are not numerous. Symptoms that have been attributed to the pinworm infection, particularly in children, include nervousness, insomnia, nightmares, and even convulsions. In some cases, perianal granulomas may result (3).
In one report, a homosexual man presented with severe abdominal pain and hemorrhagic colitis, eosinophilic inflammation of the ileum and colon, and numerous unidentifiable larval nematodes in the stool. Using morphologic characteristics and molecular cloning of nematode rRNA genes, the parasites were identified as larvae of E. vermicularis; these larvae are rarely seen and are not thought to cause disease. The authors stated that occult enterobiasis is widely prevalent and may be a cause of unexplained eosinophilic enterocolitis (9).
The most widely used diagnostic procedure for pinworm infection is the cellulose tape (adhesive cellophane tape) method (5, 10–14) (Fig. 5.1 to 5.3). Several commercial collection procedures are also available. Specimens should be obtained in the morning before the patient bathes or goes to the bathroom. At least four to six consecutive negative slides should be observed before the patient is considered free of infection. Occasionally adult female pinworms are seen on the tapes or swabs.
Figure 5.1 Collection of Enterobius vermicularis eggs by the cellulose tape method. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch5.f1
Figure 5.2 Diagram of a commercial kit (Evergreen Scientific) for use in sampling the perianal area for the presence of pinworm (E. vermicularis) eggs. On the left is the vial containing the sampler, which has sticky tape around the end. Once this is applied to the perianal area and eggs are picked up on the tape, the label area is placed at one end of the slide. The sticky tape is rolled down the slide and attaches to the glass. This device is easy to use and provides an area sufficient for adequate sampling. A minimum of four to six consecutive negative tapes are required to rule out a pinworm infection; most laboratories are accepting four rather than requesting the full six. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch5.f2
Figure 5.3 (Top) Enterobius vermicularis (pinworm) eggs seen in a Scotch tape preparation; note the football-shaped eggs with one side a bit flatter than the other. In some preparations, eggs are seen that contain fully developed larvae; such eggs are infective. (Middle) Adult female pinworm found on a collection device. Note the large, round esophageal bulb. (Bottom) Adult female pinworm, enlarged anterior end. Note the cephalic expansions around the end of the head, as well as the large, round esophageal bulb. doi:10.1128/9781555819002.ch5.f3
Collection of the Specimen
1. Place a strip of cellulose tape on a microscope slide, starting 1/2 in. (1 in. = 2.54 cm) from one end and running toward the same end, continuing around this end lengthwise; tear off the strip flush with the other end of the slide. Place a strip of paper, 1/2 by 1 in., between the slide and the tape at the end where the tape is torn flush.
2. To obtain the sample from the perianal area, peel back the tape by gripping the label, and with the tape looped (adhesive side outward) over a wooden tongue depressor held against the slide and extended about 1 in. beyond it, press the tape firmly against the right and left perianal folds.
3. Spread the tape back on the slide, adhesive side down.
4. Write the name and date on the label.
Note Do not use Magic transparent tape; use regular clear cellulose tape. If Magic tape is submitted, a drop of immersion oil can be placed on top of the tape to facilitate clearing.
Examination
Lift one side of the tape, apply 1 small drop of toluene or xylene, and press the tape down on the glass slide. The preparation will then be cleared, and the eggs will be visible. Examine the slide with low power and low illumination.
The anal swab technique (15) is also available for the detection of pinworm infections; however, most laboratories use the cellulose tape method because it eliminates the necessity for preparing and storing swabs. At least four to six consecutive negative swabs should be obtained before the patient is considered free of infection.
Collection of the Specimen
Specimens should be obtained in the morning before the patient bathes or goes to the bathroom.
1. Prepare a mixture of 4 parts petrolatum to 1 part paraffin, and heat until liquid (just melted).
2. Dip the end of the cotton swab into the mixture, remove the swab, and allow it to cool. If the cotton is not thoroughly coated, dip it again.
3. Store the coated swab in a 100- by 13-mm tube, and plug the end of the tube with cotton. These tubes may be stored for long periods, preferably under refrigeration.
4. Rub the swab gently over the perianal surface and into the folds. Insert the swab into the anal opening about 1/4 in. and then replace it in the tube.
Examination
1. Fill the tube containing the swab half full of xylene or xylene substitute, and let it stand for 3 to 5 min.
2. Remove the swab, and centrifuge the tube at 500 × g for 1 min.
3. Remove the supernatant fluid with a pipette (do not pour it off).
4. Place the sediment on a slide, and examine the material for eggs. The fluid can be examined under a coverslip, in a depression slide, or in a wax pencil circle drawn on the slide (to prevent the fluid from spreading).
Material obtained from sigmoidoscopy can be helpful in the diagnosis of amebiasis that has not been detected by routine fecal examinations; however, a series of at least three routine stool examinations for parasites should be performed on each patient before sigmoidoscopy examination is done. Another option would be to use the immunoassay kits that are designed to detect either the Entamoeba histolytica/E. dispar group/complex or specifically pathogenic E. histolytica; fresh or frozen stools are required for these kits.
Material from the mucosal surface should be aspirated or scraped and should not be obtained with cotton-tipped swabs. If swabs are the only method available, a small amount of cotton should be used on the end of the stick and should be wound tightly to prevent absorption of the sigmoidoscopy material into the cotton. At least six representative areas of the mucosa should be sampled and examined (six samples, six slides) (8, 13).
The specimen should be processed immediately. Usually, the amount of material is limited and should be handled properly to ensure the best examination possible. Various methods of examination are available (direct mount, several options for permanent stains). All are recommended; however, depending on the availability of trained personnel, proper fixation fluids, or the amount of specimen obtained, one or two procedures may be used. If the amount of material limits the examination to one procedure, the use of fixative containing polyvinyl alcohol (PVA) is highly recommended.
If the material is to be examined by any of the new immunoassay detection kits (for Cryptosporidium spp. or Giardia lamblia [G. duodenalis, G. intestinalis]), 5 or 10% formalin or sodium acetate-acetic acid-formalin (SAF) or the Universal Fixative is recommended. However, as mentioned above, if immunoassay kits are to be used for detection of the E. histolytica/E. dispar group/complex or specifically pathogenic E. histolytica, fresh or frozen stools are required. It is always important to read the package insert to verify collection recommendations. Many physicians performing sigmoidoscopy procedures do not realize the importance of selecting the proper fixative for material to be examined for parasites. For this reason, it is recommended that a parasitology specimen tray (containing Schaudinn’s liquid fixative containing PVA, the Universal Fixative, and 5 or 10% formalin) be provided or that a trained technologist be available at the time of sigmoidoscopy to prepare the slides. Even the most thorough examination will be meaningless if the specimen has been improperly prepared.
If there is no lag time after collection and a microscope is available in the immediate vicinity, some of the material should be examined as a direct saline mount for the presence of motile trophozoites. A drop of material is mixed with a drop of 0.85% sodium chloride and examined under low light intensity for the characteristic movement of amebae. It may take time for the organisms to become acclimated to this type of preparation; therefore, motility may not be obvious for several minutes. There will be epithelial cells, macrophages, and possibly polymorphonuclear leukocytes and red blood cells (RBCs), which will require a careful examination to reveal amebae (Fig. 5.4).
Figure 5.4 Entamoeba histolytica trophozoites stained with trichrome stain. Note the clearly defined internal details (nuclear and cytoplasmic characteristics, plus the ingested red blood cells). doi:10.1128/9781555819002.ch5.f4
Note Since specific identification of protozoan organisms can be difficult when only the direct saline mount is used, this technique should be used only when sufficient material is left to prepare permanent stained smears.
Morphologic differentiation between the E. histolytica/E. dispar group/complex and Entamoeba coli can be difficult, in addition to the problem of differentiating human cells from protozoa. Also, unless trophozoites containing ingested RBCs are seen, it is impossible to differentiate organisms in the E. histolytica/E. dispar group/complex from the actual pathogen, E. histolytica.
Schaudinn’s Fixative
Most of the material obtained at sigmoidoscopy can be smeared (gently) onto a slide and immediately immersed in Schaudinn’s fixative. These slides can then be stained with trichrome stain and examined for specific cell morphology, either protozoan or otherwise. The procedure and staining times are identical to those for routine fecal smears.
PVA Fixative
If the material is bloody, contains a lot of mucus, or is a “wet” specimen, a few (no more than 2 or 3) drops of fixative containing PVA can be mixed with 1 or 2 drops of specimen directly on the slide, which is allowed to air dry (a 37°C incubator can be used) for at least 2 h before being stained. If time permits, the PVA smears should be allowed to dry overnight; they can be routinely stained with trichrome stain and examined as a permanent mount.
SAF Fixative
Material obtained at sigmoidoscopy can be placed in small amounts of SAF. After fixation for 30 min, the specimen can be centrifuged for 10 min at 500 × g, and smears from the small amount of sediment can be prepared for permanent staining with iron hematoxylin (trichrome stain would be the second choice). One of the organisms most strongly suspected when sigmoidoscopy is performed is E. histolytica, whose morphology is normally seen from the permanent stained smear; however, this identification assumes that RBCs are seen within the cytoplasm of the trophozoites (Fig. 5.4). If RBCs are not seen in the trophozoite cytoplasm, the report should indicate that organisms in the E. histolytica/E. dispar group/complex have been seen and identified. However, if SAF is used, both the permanent stained smear and a fecal immunoassay kit can be used. If enough material for only a single procedure is available, the permanent stained smear is recommended, particularly if the iron hematoxylin stain (incorporating the carbol fuchsin step) is used (34).
Universal Fixative
Material obtained at sigmoidoscopy can be placed in small amounts of the Universal Fixative (TOTAL-FIX). After fixation for 30 min, the specimen can be centrifuged for 10 min at 500 × g, and smears from the small amount of sediment can be prepared for permanent staining with trichrome or iron hematoxylin. The sediment should be smeared onto a slide and allowed to dry 30 min at 37°C prior to staining. If the material is thoroughly dry, no albumin or PVA is required. One of the organisms most strongly suspected when sigmoidoscopy is performed is E. histolytica, whose morphology is normally seen from the permanent stained smear; however, this identification assumes that RBCs are seen within the cytoplasm of the trophozoites (Fig. 5.4). If RBCs are not seen in the trophozoite cytoplasm, the report should indicate that organisms in the E. histolytica/E. dispar group/complex have been seen and identified. However, if the Universal Fixative is used, both the permanent stained smear and some molecular tests or panels can be used. If enough material for only a single procedure is available, the permanent stained smear is recommended.
In infections with Giardia, Strongyloides, or Cryptosporidium, routine stool examinations may not reveal the organisms. Duodenal drainage material can be submitted for examination as direct or concentration wet mounts or permanent stained smears, techniques that may reveal the parasites (Fig. 5.5). Use standard precautions and wear gloves when handling the specimen.
Figure 5.5 Duodenal aspirate specimens. (Top) Giardia lamblia in mucus from a duodenal aspirate stained with trichrome stain. Note the teardrop trophozoites (from the front view) and the darker-staining trophozoites that resemble the curved part of a spoon (side view). Also notice the number of organisms contained within the mucus. (Middle) A Strongyloides stercoralis rhabditiform larva in a wet mount. The genital primordial packet of cells is visible about halfway down the larva on the left side. (Bottom) Cryptosporidium spp. oocysts in duodenal aspirate material stained with a modified acid-fast stain. doi:10.1128/9781555819002.ch5.f5
The specimen should be submitted to the laboratory in a tube containing no preservative; the amount may vary from <0.5 ml to several milliliters of fluid. The specimen may be centrifuged (10 min at 500 × g) and should be examined immediately as a wet mount for motile organisms (iodine may be added later to facilitate the identification of any organisms present). If the specimen cannot be completely examined within 2 h after it is taken, any remaining material should be preserved in fixatives containing PVA, SAF, or the Universal Fixative. The “falling-leaf” motility often described for Giardia trophozoites is rarely seen in fresh, unpreserved preparations. The organisms may be caught in mucus strands, and the movement of the flagella on the Giardia trophozoites may be the only subtle motility seen for these flagellates. Strongyloides larvae are usually very motile. Remember to keep the light intensity low.
The duodenal fluid may contain mucus; this is where the organisms, particularly Giardia, tend to be found. Therefore, centrifugation of the specimen is important, and the sedimented mucus should be examined. Immunoassay detection kits (Cryptosporidium or Giardia) can also be used with fresh or formalinized material; however, the kits are specifically recommended for actual stool material.
If a presumptive diagnosis of giardiasis is made on the basis of the wet-preparation examination of the fresh specimen, the coverslip can be removed and the specimen can be fixed with one of the fecal fixatives for subsequent staining with either trichrome or iron hematoxylin. If the amount of duodenal material submitted is very small, permanent stains can be prepared rather than using any part of the specimen for a wet-smear examination. Some investigators think that this approach provides a more permanent record, and the potential visual problems with unstained organisms, very minimal motility, and a lower-power examination can be avoided by using oil immersion examination of the stained specimen at ×1,000 magnification.
Duodenal Capsule Technique (Entero-Test)
A simple and convenient method of sampling duodenal contents that eliminates the need for intestinal intubation has been devised (16). The device consists of a length of nylon yarn coiled inside a gelatin capsule (Fig. 5.6). The yarn protrudes through one end of the capsule; this end of the line is taped to the side of the patient’s face (Fig. 5.7). The capsule is then swallowed, the gelatin dissolves in the stomach, and the weighted string is carried by peristalsis into the duodenum. The yarn is attached to the weight by a slipping mechanism; the weight is released and passes out in the stool when the line is retrieved after a period of 4 h. Bile-stained mucus clinging to the yarn is then scraped off (mucus can also be removed by pulling the yarn between thumb and finger) and collected in a small petri dish; disposable gloves should be worn. Usually 4 or 5 drops of material are obtained.
The specimen should be examined immediately as a wet mount for motile organisms (iodine may be added later to facilitate the identification of any organisms present). If the specimen cannot be completely examined within an hour after the yarn has been removed, the material should be preserved in one of the fecal fixatives for subsequent staining. Organism motility is like that described above for duodenal drainage.
Note The pH of the terminal end of the yarn should be checked to ensure adequate passage into the duodenum (a very low pH means that it never left the stomach). The terminal end of the yarn should be a yellow-green color, indicating that it was in the duodenum (the bile duct drains into the intestine at this point).
Figure 5.6 Entero-Test capsule for sampling duodenal contents (HCD; Nutri-Link Ltd., Newton Abbot, United Kingdom; adult and pediatric capsules). The device consists of a length of nylon yarn coiled inside a gelatin capsule. The yarn protrudes through one end of the capsule; this end of the line is taped to the side of the patient’s face. The capsule is then swallowed, the gelatin dissolves in the stomach, and the weighted string is carried by peristalsis into the duodenum. (Illustration by Nobuko Kitamura.) doi:10.1128/9781555819002.ch5.f6
Figure 5.7 Entero-Test string test for sampling duodenal contents. (A) Capsule with partially extracted string; (B) capsule with string before being swallowed; (C) end of the string taped to the cheek. (From Leodolter A et al, World J Gastroenterol 11:584–586, 2005.) doi:10.1128/9781555819002.ch5.f7
The identification of Trichomonas vaginalis (Fig. 5.8) is usually based on the examination of a wet preparation of vaginal and urethral discharges and prostatic secretions or urine sediment. Multiple specimens may have to be examined before the organisms are detected. These specimens are diluted with a drop of saline and examined under low power and reduced illumination for the presence of actively motile organisms; as the jerky motility begins to diminish, it may be possible to observe the undulating membrane, particularly under high dry power. Recent data indicate that microscopic examination of a spun urine specimen performed in conjunction with microscopic examination of a vaginal fluid specimen improves the detection rate of T. vaginalis. While 73% of infections were detected by examination of vaginal fluid specimens and 64% were detected by examination of spun urine, the combined percentage using both methods was 85% (17). It is also important to remember that in older men with nongonococcal urethritis, diagnostic evaluation, empirical treatment, and partner management should include the possibility of infection with T. vaginalis (18).
Figure 5.8 (Top) Trichomonas vaginalis trophozoite. (Illustration by Sharon Belkin.) (Middle) T. vaginalis trophozoites seen in a wet mount preparation. (Bottom, left) Pentatrichomonas hominis (stool); (right) Trichomonas vaginalis (urinary-genital tract). doi:10.1128/9781555819002.ch5.f8
Stained smears are usually not necessary for the identification of this organism. The large number of false-positive and false-negative results reported on the basis of stained smears strongly suggests the value of confirmation by observation of motile organisms from the direct mount, from appropriate culture media (19–22), or from molecular testing (23–25). The ability of Amies gel agar transport medium to maintain the viability of T. vaginalis was examined by comparison with specimens immediately inoculated into culture medium. The immediate-inoculation method detected infections in 64 (94.1%) of 68 patients, while the transport method detected infections in 62 (91.2%) of 68 patients (26). However, the data depend on the type of medium used, the time taken for transport, and the temperature at which transport was carried out. Additional information on culture options can be found in chapter 7.
T. vaginalis infection is the most prevalent sexually transmitted disease in the world. To improve diagnostic results, molecular testing is now being used for the detection of this infection and reports excellent results (27–30). There are several FDA-approved molecular diagnostic tests for this organism. They include the APTIMA Trichomonas vaginalis Assay (GenProbe, San Diego, CA). This assay utilizes the same amplification technology that has been used for years to detect Neisseria gonorrheae and Chlamydia trachomatis. It also uses the same specimen types (i.e., clinician-collected vaginal and endocervical swabs, female urine, and PreservCyt solution). It has reported high sensitivity and specificity, and does not require organism viability or motility. It, as well as other antigen-based assays, represents advances in the detection of this parasite.
The Affrirm VPIII DNA probe technology (Becton, Dickinson and Co., Franklin Lakes, NJ) offers a dependable, rapid means for the early identification of three organisms causing vaginitis: Candida spp., Gardnerella vaginalis, and T. vaginalis. During the test protocol, the assay aligns complementary nucleic acid strands to form specific, double-stranded complexes called hybrids. For each organism, this hybrid is composed of capture and color development single-stranded nucleic acid probes, complementary to the released target nucleic acid analyte. Enzyme conjugate then binds to the captured analyte.
Examination of urinary sediment is indicated in certain filarial infections. The occurrence of microfilariae in urine has been reported with increasing frequency in Onchocerca volvulus infections in Africa. The triple-concentration technique is recommended for the recovery of microfilariae (13) (see chapter 7).
Urine is collected into a bottle, the volume is recorded, and thimerosal (1 ml/100 ml of urine) is added. The specimen is placed in a funnel fitted with tubing and a clamp; this preparation is allowed to settle overnight. The following day, 10 to 20 ml of urine is withdrawn and centrifuged. The supernatant fluid is discarded, and the sediment is resuspended in 0.85% NaCl. This preparation is again centrifuged, and 0.5 to 1.0 ml of the sediment is examined under the microscope for the presence of nonmotile microfilariae. The membrane filtration technique used with blood can also be used with urine for the recovery of microfilariae. Administration of the drug diethylcarbamazine (Hetrazan) has been reported to enhance the recovery of microfilariae from the urine (31).
A membrane filter technique for the recovery of Schistosoma haematobium eggs is also useful (32) (Fig. 5.9 and 5.10).
Figure 5.9 Membrane filtration system. (Upper) Membrane holder, which can be attached to a syringe for filtration of various types of specimens, particularly urine. (Lower) Package of membranes; different sizes with various mesh sizes are available, depending on the clinical specimen and suspected organism size. doi:10.1128/9781555819002.ch5.f9
Figure 5.10 Schistosoma haematobium eggs from a urine filtration. Note the terminal spines on the eggs. Since the specimens were not preserved prior to filtration, determination of the viability of the eggs is possible. The viability information should be conveyed to the physician as a part of the report. doi:10.1128/9781555819002.ch5.f10
Membrane Filter Technique
1. Collect a urine sample in a container (urine should be well mixed before step 2).
2. With a syringe, draw up 1 ml of urine into the syringe barrel.
3. Fill the rest of the barrel volume with air.
4. Attach the filter holder containing an 8-mm-pore-size Nuclepore membrane filter to the syringe.
5. Express the urine through the filter.
6. Remove the filter, and place it on a microscope slide face down.
7. Moisten the filter with 0.85% NaCl.
8. Examine the filter under ×100 power for the presence of eggs.
The efficiency of the polycarbonate membrane filtration technique for detecting S. haematobium eggs in urine is increased by using a pore size of 14 µm and the suction of a water jet pump. Egg concentrations of 1 egg in >1,000 ml of urine can be detected. Viability can be assessed after filtration by staining with trypan blue. This technique is highly recommended in light infections with small numbers of eggs in which previous diagnostic methods have not confirmed the infection (33).
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