Stains and Culture-Based Tests
Culture is still the most widely used means for detection and identification of genital mycoplasmas in clinical specimens, and it remains the accepted reference standard (14,62). The relative rapidity of their growth will allow cultural detection and presumptive identification of these organisms within 2-5 days with an analytical sensitivity comparable to that of the PCR assay. The main challenges that may be encountered by clinicians are finding an experienced laboratory capable of performing and interpreting the results of mycoplasmal cultures and the careful attention required for proper specimen collection and transport to ensure that viable organisms are obtained and preserved until received in the diagnostic laboratory.
Mycoplasmal and ureaplasmal cells are too small to be visualized in Gram-stained preparations of clinical specimens or cultures, and the lack of a cell wall precludes uptake of crystal violet or safranin. However, the Gram stain may prove useful to exclude contaminating bacteria. Giemsa stains may be used, but the results can be difficult to interpret because of debris and artifacts in clinical specimens that can be confused with mycoplasmas because of their small size. Deoxyribonucleic acid (DNA) fluorochrome stains may be useful to determine whether micro-organisms are present in a clinical specimen or culture, but they do not distinguish mycoplasmas from other bacteria (62).
Cultures of nasopharyngeal, throat, and endotracheal secretions of neonates are appropriate to evaluate respiratory infection. Gastric aspirates and throat and nasal swabs are less desirable because they may not always accurately reflect the microbiology of the lower respiratory tract because some infants may be colonized in other locations without ill effects. If urogenital specimens from adults are of interest, urethral swabs, urine, and cervical or vaginal swabs are acceptable. Urine samples from females are most meaningful when obtained by catheter or suprapubic aspiration and if numbers of organisms are quantitated. Endometrial tissue, tubal samples, or pouch of Douglas fluid can be obtained to confirm mycoplasmal etiology of postpartum fever. For women with clinical amnionitis, amniotic fluid, blood, and placenta should be cultured. Other sterile fluids from neonates, such as CSF and blood, are suitable for culture, as are wound aspirates, abscess fluid, and tissue collected by biopsy or autopsy.
Mollicutes are extremely sensitive to adverse environmental conditions, particularly to drying and heat, so great care must be taken to ensure proper specimen collection and transport. Clinical specimens from neonates will usually be of very small volumes or quantities. Therefore, they should always be collected and placed in appropriate transport medium immediately to prevent desiccation and loss of organism viability. If larger samples, such as lung tissue collected at autopsy or placenta, can be placed in a sterile screw-capped container and sent to the laboratory immediately, no additional transport medium is necessary. However, if there is any delay anticipated or if specimens have to be shipped to a laboratory off site, addition of transport medium is essential.
Transport medium such as Shepard's 10B broth is acceptable for transport of M. hominis as well as Ureaplasma spp. This medium is available commercially (Remel
Laboratories, Lenexa, KS) or can be prepared locally (62). 2 SP (10% v/v heat-inactivated fetal calf serum with 0.2M sucrose in 0.02M phosphate buffer, pH 7.2), which is also used for transport of specimens for chlamydial cultures, is also acceptable. Other media available commercially for transport and storage of specimens include Stuart's medium, A3B, and arginine broth. From a practical standpoint, transport media can be kept frozen in small volumes in a freezer located in a clinical unit so that it can be rapidly thawed and used to inoculate specimens at bedside.
When swabs are obtained, care must be taken to sample the desired site vigorously to obtain as many cells as possible because mycoplasmas are cell associated. Calcium alginate, Dacron, or polyester swabs with aluminum or plastic shafts are preferred. Wooden shaft cotton swabs should be avoided because of potential inhibitory effects. Swabs should be vigorously swirled in the appropriate transport broth, pressed against the side of the tube to express as much fluid as possible, and discarded.
Endotracheal secretions from neonates who are intubated can be collected using a small-bore suction catheter connected to a vacuum outlet. The catheter is passed through the endotracheal tube, and suction is applied. The tip of the suction catheter is cut with a sterile scalpel blade. Then, 1 mL of 10B broth is drawn into a 3-mL syringe to which a 21-gage needle is attached. The tip of the suction catheter is placed into the tube from which the broth was drawn, and the needle is used to flush the catheter with broth, forcing the respiratory secretions into the tube, which is then transported to the laboratory.
Fluids such as CSF or urine should be obtained according to standard clinical procedures and inoculated immediately into transport media in an approx 1:10 ratio. Mycoplasmas and ureaplasmas are inhibited by sodium polyanethol sulfonate present in most commercial blood culture media, so this is not an acceptable means for detection. Commercial blood culture media designed for use in automated instruments may support growth of M. hominis, but the instruments usually do not flag the bottles containing this organism as positive (63). Successful isolation of M. hominis and Ureaplasma spp from blood can be achieved by inoculating blood directly into liquid mycoplasmal growth media such as 10 B broth in at least a 1:10 ratio.
Specimens should be refrigerated if immediate transportation to the laboratory is not possible. If specimens must be shipped or if the storage time is likely to exceed 24 hours prior to processing, the specimen in transport medium should be frozen to prevent loss of viability. Specimens can be stored for long periods in appropriate growth or transport media at -70°C or in liquid nitrogen. Storage at -20°C for even short periods will result in loss of viability. Frozen specimens may be shipped with dry ice to a reference laboratory if necessary.
There are a variety of commercial and nonproprietary culture medium formulations that have been used to detect M. hominis and Ureaplasma spp. Their merits and relative disadvantages have been reviewed (62). Shepard's 10B broth is an ideal choice for general use because it can be used for cultivation of both M. hominis and Ureaplasma spp with A8 agar (62) as the corresponding solid medium. In the event that other mycoplasmal species are to be sought, alternative methods such as the PCR assay should be utilized because culture techniques are not well established for other species. Even though 10B broth and A8 agar are available commercially, the comparability of the commercially prepared products to nonproprietary formulations in their ability to support the growth of these fastidious organisms has not been documented, so internal quality control should be practiced (62). There are a number of complete diagnostic kits for detection and identification of M. hominis and Ureaplasma spp; these are sold commercially in several European countries, but none is available in the United States. A comprehensive discussion and description of these products is available elsewhere (64).
It is beyond the scope of this chapter to describe in depth the procedures that must be undertaken in a diagnostic laboratory to recover mycoplasmas and ureaplasmas from clinical specimens. Refer to reference microbiology texts (14,62,64) that deal with this topic for specific information, and only general comments are provided here.
Specimens should be mixed, and fluids should be centrifuged and the pellet serially diluted to at least 10-3 and inoculated into liquid and solid medium. Tissues should be minced in broth prior to diluting. Subculture of each dilution onto agar is an extremely important step in the cultivation process because it will help overcome possible interference by antibiotics, antibodies, and other inhibitors, including bacteria that may be present in clinical specimens. Omission of this critical dilution step can be one reason why some laboratories have difficulty in recovering the organisms. Dilution also helps to overcome the problem of rapid decline in culture viability, which is particularly common with ureaplasmas, and it also provides information about the number of organisms present in the specimen. Colonies develop best when agar plates are incubated in an atmosphere of 95% N2 plus 5% CO2 (14), but successful isolation is also possible using an atmosphere of room air plus 5% CO2 or in a candle jar.
Growth in 10B broth is suggested by an alkaline shift and change in color of the medium from yellow to pink because of urea hydrolysis by Ureaplasma spp or argin-ine hydrolysis by M. hominis. Such changes may be evident in 24 hours or less in the case of Ureaplasma spp and in 24-28 hours for M. hominis. Turbidity in broth cultures indicates bacterial contamination. Positive broths should always be subcultured to agar immediately because the primary inoculum does not always grow. Subcultures must be performed soon after the color change occurs, particularly if the organism is Ureaplasma, because the culture can lose viability within hours. Occurrence of pinpoint colonies on bacteriological media such as Columbia agar that do not produce a recognizable Gram reaction warrants subculture to mycoplasma media because of the possibility they may be M. hominis.
Colonies of Ureaplasma spp and M. hominis growing on A8 agar are shown in Figs. 1 and 2. Except for hydrolysis of urea and development of characteristic colonies on A8 agar that are unique for ureaplasmas, biochemical and colonial features are insufficient for definitive species distinction. However, colony morphology in conjunction with the biochemical profile, body site of origin, and rate of growth will often allow presumptive identification of the most common clinically significant species of large colony mycoplasmas. An arginine-hydrolyzing, urease-negative organism that produces fried egg colonies within 3-4 days of incubation may be presumptively identified as M. hominis, and in most instances no further work-up is required. Mycoplasmas that require more definitive identification can be submitted to a reference laboratory for characterization by PCR or other immunologic procedures in the event that this is o
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