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
