CHAPTER 12 Ken B. Waites1 and CécileBébéar2 1 University of Alabama at Birmingham, Birmingham, AL, USA 2 Université de Bordeaux, Bordeaux, France Mycoplasmas and ureaplasmas are the smallest free-living microorganisms. Their simplicity is reflected by their fastidious nature and complex cultivation requirements. They demand special techniques for their laboratory detection and characterization that are often not available in hospital microbiology laboratories. Their fastidious nature and lack of appreciation for their importance as human pathogens has led to them being ignored by many diagnostic laboratories. This situation has changed somewhat in recent years because of greater appreciation for their clinical importance, improved methods for detection, including nonculture-based molecular assays such as polymerase chain reaction (PCR), availability of commercially manufactured growth media, serological assays, and products sold as complete diagnostic kits. However, the number and types of products available for detection and characterization of mycoplasmas is still very limited in comparison to products sold commercially for other types of bacteria. A number of products available for use in mycoplasma detection and characterization have never been subjected to rigorous external evaluation so that recommendations for their use cannot be made based on objective evidence of performance. Readers interested in an in-depth discussion of the taxonomy, disease associations, and biological characteristics of mycoplasmas and ureaplasmas of humans are referred to other reference texts [50] for more detailed information on these aspects of human mycoplasmas. Only a brief synopsis of this information is included here. Class Mollicutes (“soft skin”), includes the bacteria commonly referred to as mycoplasmas (“fungus-form”). The class is comprised of four orders, five families, eight genera, and about 200 known species. Sixteen species have been isolated from humans, excluding occasional animal mycoplasmas that have been detected in humans from time to time, but are generally considered transient colonizers. Mollicutes are the smallest life-forms capable of an independent existence. They permanently lack cell walls and peptidoglycan precursors and require a complex enriched medium containing serum, amino acid precursors, nucleotides, yeast extract, and other supplements. Mycoplasma pneumoniae is the best known and most intensely studied mycoplasma that occurs in humans. This organism causes pharyngitis, tracheobronchitis, and pneumonia in persons of all age groups. Some people may experience extrapulmonary complications including skin rashes, hemolytic anemia, arthritis, meningoencephalitis, peripheral neuropathy, and pericarditis. Mycoplasma pneumoniae has been detected by culture or PCR from extrapulmonary sites such as synovial fluid and cerebrospinal fluid, pericardial fluid, and skin lesions. Clinical manifestations are not sufficiently unique to allow differentiation from infections caused by other common microorganisms. Ureaplasma urealyticum, U. parvum, and M. hominis are commensal organisms in the lower genital tract in many sexually active adults. Their common occurrence in healthy persons has lead to difficulty in accepting these organisms as independent causes of disease. Nevertheless, there is evidence that these organisms play etiologic roles in some conditions. Ureaplasma spp., particularly U. urealyticum, and M. genitalium are causes of nongonococcal urethritis in men. Mycoplasma hominis causes a small portion of cases of pyelonephritis and has been isolated from endometrium and fallopian tubes of women with salpingitis. Mycoplasma genitalium also plays a significant role in female cervicitis and salpingitis. Ureaplasmas can cause placental inflammation and may invade the amniotic sac early in pregnancy in the presence of intact fetal membranes, causing persistent infection and adverse pregnancy outcome, including premature birth. M. hominis and Ureaplasma spp. have been isolated from the blood of women with postpartum or postabortal fever. Congenital pneumonia, bacteremia, progression to chronic lung disease of prematurity, and even death have occurred in very low birthweight infants and have been attributed to ureaplasmal infection of the lower respiratory tract. Both M. hominis and Ureaplasma spp. have been isolated from maternal and umbilical-cord blood, as well as the blood of neonates. Both organisms can also invade the cerebrospinal fluid of neonates. Mycoplasmas and ureaplasmas can be important pathogens in individuals with immunodeficiencies and in other immunosuppressed persons such as organ transplant recipients. They can cause invasive disease of the joints and respiratory tract with bacteremic dissemination, especially individuals with antibody deficiencies. M. hominis has been isolated in pure culture from the bloodstream after renal transplantation, trauma, and genitourinary manipulations, and it has also been found in women with postpartum endometritis and in wound infections. Mycoplasma fermentans, Ureaplasma spp., and M. hominis have been detected in synovial fluid of persons with rheumatoid arthritis and other inflammatory arthritides, although their precise contribution to these diseases is still uncertain. There is also some evidence that Ureaplasma spp. are involved in reactive arthritis. Mycoplasma fermentans has been detected in adults with an acute influenza-like illness, and in bronchoalveolar lavages, peripheral blood lymphocytes, and bone marrow from patients with AIDS. Due to the relatively mild clinical course of most respiratory tract infections caused by M. pneumoniae, lack of widespread availability, and considerable expense of rapid diagnostic tests, many clinicians choose to treat empirically when infection with this organism is strongly suspected. However, laboratory testing would almost certainly become more common in the United States if rapid molecular-based assays become more readily available as they have in Europe. It is reasonable to obtain laboratory testing for M. pneumoniae by molecular-based procedures when respiratory illness in an adult or child is of sufficient severity to warrant hospitalization and no other causative microorganism is readily apparent. Testing should also be considered if there is an unsatisfactory clinical response to macrolide treatment because this may indicate the presence of a macrolide-resistant strain; in patients with an underlying comorbid condition or immunodeficiency that would make severe and disseminated disease more likely; and when there are significant extrapulmonary symptoms present. Measurement of serum antibodies, though widely used, has limited value for laboratory diagnosis of M. pneumoniae infection if direct detection methods such as PCR are available, especially in the first few days of illness. Some infections that are well documented by seroconversion are negative by the PCR assay, so a single test may not always yield the microbiological diagnosis. Lack of commercial molecular-based tests for detection of M. genitalium in the United States has greatly limited interest among physicians in seeking this mycoplasma in clinical specimens since it cannot be satisfactorily isolated by culture and no serological tests are available. Given the growing evidence for the importance of M. genitalium in a variety of urogenital conditions and the likelihood of venereal transmission to other individuals, use of molecular-based assays would undoubtedly increase if they become available. If M. genitalium is detected in lower urogenital tract specimens from cervix, urethra, endometrium, or fallopian tube from a symptomatic patient, it should be considered clinically significant. Unlike M. hominis and Ureaplasma spp., M. genitalium is less likely to be carried by asymptomatic persons. Since M. hominis and Ureaplasma spp. are commonly found in the lower urogenital tracts of healthy adults, and healthy infants may be transiently colonized on the skin or in the upper respiratory tract with organisms of maternal origin, a positive PCR or culture result from specimens from these sites can usually be expected and has little clinical value. Positive PCR or culture results for Ureaplasma spp. from the urethra in men with urethritis, from tracheal aspirates of neonates with respiratory distress, from the bloodstream or cerebrospinal fluid in neonates with pleocytosis, or from sterile extragenital sites should be considered diagnostic of clinically significant infection. Likewise, detection of M. hominis in any quantity from sterile body fluids or tissues of neonates or adults is significantly associated with disease. Ureaplasma spp. and M. hominis can be relatively common opportunistic pathogens causing systemic infections involving various body sites in immunosuppressed people. There are many reports of systemic infections that were caused by M. hominis or Ureaplasma spp. for which diagnosis was delayed simply because no one attempted to perform any diagnostic laboratory tests. Even though PCR may be more sensitive than culture for detection of ureaplasmas and M. hominis, it is less widely available since there are no commercial PCR assays available in the United States and it requires submission to a reference laboratory with molecular diagnostic facilities. Culture for these organisms is available in reference laboratories and many larger hospital laboratories and usually requires about 2–5 days for detection of growth. No specific recommendations can be made for laboratory diagnosis of M. fermentans since its role in human disease is much less established than the other species previously discussed. PCR is the best diagnostic approach since optimum cultivation conditions are not well described and a PCR assay would be necessary anyway to identify the organism to species level. Some reference laboratories offer their own PCR assay to detect M. fermentans. Successful cultivation of mycoplasmas and ureaplasmas from clinical specimens involves careful consideration of the type of specimen to be obtained, the type of patient suspected of having infection, and organisms sought. For respiratory specimens, if M. pneumoniae is the only organism of interest, it is sufficient to inoculate a single general-purpose medium type such as SP4 broth and agar, using a broth-to-agar technique [45]. Culture is less likely to be used than PCR because of its lower sensitivity and slow turnaround time, up to 6 weeks. However, culture is sometimes useful to obtain clinical isolates of M. pneumoniae for epidemiological purposes or to measure in vitro antimicrobial susceptibilities. Likewise, if only M. hominis or Ureaplasma spp. are of interest, it is sufficient to set up cultures using a single broth and agar formulation such as 10B broth and A8 agar. All of these media are available from various commercial sources. Mycoplasmas of human origin grow best in an atmosphere supplemented with 5–10% CO2 at 37°C. If the specimen to be cultured is from an extragenital or extrapulmonary site, is a sterile body fluid or tissue, and/or the patient is immunosuppressed, any mycoplasma of human origin or accidental infection with a mycoplasma of animal origin should be considered. Many animal mycoplasmas are not reliably detected in culture using protocols described for the more common species, and alternative nonculture-based tests such as PCR assays should also be performed, if suitable reagents are available. However, isolation may occasionally be successful, usually after prolonged incubation in culture. SP4 broth and agar, containing both glucose and arginine, should also be inoculated and incubated under anaerobic conditions. Gram stains can assist in excluding the presence of other bacteria from a clinical specimen, even though mycoplasmas cannot be visualized since they lack cell walls. In order to stain mycoplasmas in clinical specimens or cultures, a DNA fluorochrome stain such as Hoechst 33258 can be used. These types of stains are commercially available from several vendors. Procedures for performance and interpretation of mycoplasma cultures and identification of species are available in other reference texts [45,50]. Therefore, only brief comments will be presented here. Ureaplasma spp. can be easily identified to the genus level by the characteristic brown granular colonial morphology that occurs due to urease activity on an agar such as A8 that contains a CaCl2 indicator. A number of different techniques are available to identify an unknown large colony mycoplasma definitively to species level. Using a combination of biochemical reactions, colony morphologies, and growth rates, a preliminary characterization of the most frequently encountered isolates in clinical settings often can be achieved [50]. From a practical standpoint, PCR assays available through specialty or reference laboratories have now replaced older phenotypic methods for definitive mycoplasmal species identification from culture-positive clinical specimens and differentiation of U. urealyticum from U. parvum, as well as providing an alternative to primary culture-based detection of the organisms from clinical specimens. Culture of Ureaplasma spp. and M. hominis is still widely used because of its relative simplicity, rapid turnaround time, low cost, and because it does not require laboratory facilities and staff experienced with molecular-based techniques. Respiratory tract specimens, including nasopharyngeal and throat swabs, sputum, pleural fluid, bronchoalveolar lavage fluid, endotracheal aspirates, and lung tissue obtained by biopsy are all acceptable specimens for cultural detection of M. pneumoniae. Cultures of cerebrospinal fluid, other sterile body fluids, or tissues can also be performed for detection of M. pneumoniae if extrapulmonary manifestations are present. However, PCR will provide a more sensitive and rapid means for detection that is particularly important for extrapulmonary sites. Urethral swabs in men are preferred over urine for detection of M. hominis and Ureaplasma spp. Prostatic secretions, semen, and urinary calculi can be cultured. For females, urine and cervical or vaginal swabs are acceptable. Avoid specimens that are contaminated by lubricants or antiseptics. 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 pelvic inflammatory disease or post-partum fever. For women with clinical amnionitis, amniotic fluid, blood, and placenta should be cultured. Culture of nasopharyngeal, throat, and endotracheal or gastric secretions of neonates is appropriate, especially if birthweight is < 1000 g., and there is clinical, radiographic, laboratory, or other evidence of pneumonia. Extragenital or extrapulmonary specimens submitted for culture should reflect the site of infection and disease process. Ureaplasmas and mycoplasmas should always be sought from synovial fluid in the setting of acute arthritis in hypogammaglobulinemia. Other sterile fluids, including peritoneal fluid, pericardial fluid, cerebrospinal fluid, and blood are suitable for culture if clinical conditions warrant. Bone chips from patients with chronic osteomyelitis without a proven bacterial etiology are also appropriate for culture as are wound aspirates and tissue collected by biopsy or autopsy. Mollicutes are inhibited by sodium polyanethol sulfonate (SPS) present in most commercial blood culture media, but the effect can be overcome by adding 1% gelatin. 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. Successful isolation of M. hominis and Ureaplasma spp. from blood can be achieved by inoculating > 10 mL directly into liquid mycoplasmal growth media in at least a 1:10 ratio. Smaller volumes can be used for neonates or children. There is sometimes difficulty in detecting a color change in liquid media in the presence of large amounts of blood due to hemolysis, and there may be a slight color change immediately after introducing blood into liquid media. Serial dilution of the original specimen and subcultures to agar will help distinguish such nonspecific color changes. Due to their lack of a cell wall, mycoplasmas are extremely sensitive to adverse environmental conditions, particularly to drying and heat. Therefore, a suitable transport system to preserve specimen viability until cultures can be inoculated is essential under most conditions. If shipping is to occur, it is imperative to have a transport system containing some type of cryoprotectant in order to prevent lysis of the organisms during freezing. Specimens should be inoculated at bedside whenever possible, using appropriate transport, or mycoplasma culture medium. Liquid media including SP4 glucose, arginine or urea broths, or 10B arginine/urea broths, manufactured and sold in the United States by various commercial vendors can serve as transport as well as growth media. 2 SP (10% v/v heat inactivated fetal calf serum with 0.2 M sucrose in 0.02 M phosphate buffer, pH 7.2), and trypticase soy broth with 0.5% v/v bovine albumin are also acceptable transport media. Specialized commercial liquid transport media such as A3B have been designed by deletion of some of the growth supplements present in other growth media so that increase in pH caused by urea hydrolysis by ureaplasmas will be delayed and result in less toxicity to the organisms during transport. Some transport media also contain buffers to maintain a neutral pH. Laboratories may choose to stock a single universal transport medium that can be used for mycoplasmas, ureaplasmas, chlamydiae, and viruses. Most universal transport media contain inhibitors to prevent bacterial or fungal overgrowth from specimens obtained from nonsterile sites that have their own indigenous microbial flora. Avoid using any transport system that contains gentamicin or another aminoglycoside since these antibiotics can be inhibitory to mycoplasmas and ureaplasmas. The M4, M5, and M6, UTM, and BD UVT media are examples of universal transport systems available from various suppliers. Additional specialized transport media for genital mycoplasmas are also available in Europe from various suppliers. Fluids or tissues do not require special transport media if cultures can be inoculated within 1 h, provided the specimens are protected from drying. Tissues can be placed in a sterile container, which can be delivered to the laboratory immediately. Otherwise, all specimens should be placed in transport media if delay in culture inoculation is anticipated. When swabs are obtained, care must be taken to sample the desired site vigorously to obtain as many cells as possible since mycoplasmas are cell-associated. Flocked swabs can be used to obtain more cells from the specimen. Dacron, or polyester swabs with aluminum or plastic shafts are preferred. Wooden shaft cotton swabs should be avoided because of potential inhibitory effects. Specimens should be refrigerated if immediate transportation to the laboratory is not possible. If specimens must be shipped and/or if the storage time is likely to exceed 24–48 h prior to processing, the specimen in transport medium with cryoprotectant should ideally be frozen and shipped on dry ice to prevent loss of viability. ThermoFisher transport media are reported by their manufacturer to maintain mycoplasma viability for up to 48 h when stored at 2–8°C. Becton Dickinson Diagnostics provides data on their internet web site to support storage at 2–25°C for up to 48 h using their UVT transport media while maintaining viability. Mollicutes 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 can result in loss of viability. Frozen specimens may be shipped with dry ice to a reference laboratory if necessary. When specimens are to be examined, they should be thawed rapidly in a 37°C water bath. In order to maximize yield of mycoplasmas from clinical specimens, serial tenfold dilution in liquid media to 10–3 to remove inhibitors and inoculation of broth dilutions to appropriate agar has been suggested [45,50]. Tissues should be minced prior to dilution. No single medium formulation is ideal for all pertinent species due to their different properties, optimum pH, and substrate requirements. Mycoplasmas of importance for human infections utilize glucose and/or arginine as metabolic substrates, whereas ureaplasmas utilize urea. The various medium formulations designed for their cultivation must provide these substances along with other essential ingredients, including serum as a cholesterol source, yeast extract, and a pH indicator for detection of growth when the substrate is metabolized. SP4 broth and agar can be used for both M. pneumoniae and M. hominis [45]. These media can also be used to cultivate other fastidious and slow-growing species. Shepard’s formulations of U9 or 10B broth have been used successfully for cultivation of Ureaplasma spp., with A7 or A8 agar as the corresponding solid media [45]. Other media such as bromothymol Blue (B broth), Hayflick’s medium, Boston broth, pleuropneumonia-like organisms (PPLO) medium, and several others have also been used for detection of mycoplasmas and/or ureaplasmas in clinical specimens. Detailed description of essential components of growth medium, formulations, instructions for preparation of various nonproprietary types, and incubation conditions for different mycoplasmas can be found in other reference texts [45]. Multiple vendors including ThermoFisher and Hardy Diagnostics in the United States market various formulations of growth media, including 10B broth, A7 and A8 agars for cultivation of M. hominis and Ureaplasma spp.,10B broth with arginine and arginine broth for M. hominis; and SP4 broth and agar for M. pneumoniae, M. hominis, and other Mycoplasma spp. The PPLO broth and agar can be used for general purpose cultivation of Mycoplasma spp. Since the broth does not contain a pH indicator, it is not possible to detect growth by direct observation of the liquid medium. Some manufacturers will produce customized orders of various sizes for most of their media, with or without inhibitors, or special additives, as desired by the purchaser. Most broths and transport media can be obtained in lyophilized form to prolong shelf-life. For laboratories that prefer to prepare their own media, individual media components described in other reference texts [45] can be obtained from various commercial sources. Mycoplasma Experience manufactures and sells a variety of diagnostic products for use in detecting mycoplasmas of veterinary importance, primarily marketing their products in the United Kingdom and Europe. However, they also produce their own line of liquid and solid media suitable for use in detecting human mycoplasmas and ureaplasmas. Mycoplasma Experience manufactures their own broth formulations containing specific biochemical substrates, e.g., for glucose or arginine metabolism, to aid in preliminary characterization of species. Media can be purchased as ready to use broths and agar plates. Liquid media is also available frozen in various volumes. Frozen agar supplements that can be added to agar are also available for purchase. Quality control strains of M. pneumoniae and U. urealyticum are available in 96-well microtiter plates. Several French companies produce a variety of liquid and solid media for urogenital mycoplasma and ureaplasma detection, with many of the same names and formulations as those sold in the United States. Additionally, there are some kits that can be used for detection as well as presumptive species identification. Some of the products are the companies’ versions of the traditional media initially described by mycoplasma researchers, while others are proprietary products of their own design. The diagnostic kits have been marketed primarily in Europe, but some have now been introduced into the United States. The ELITech products MYCOSCREEN PLUS and MYCOFAST US are now distributed in the United States by Wescor, Inc. (Figures 12.1 and 12.2). Figure 12.1 MYCOSCREEN PLUS (ELITech). MYCOSCREEN PLUS is a commercial kit for specimen transport, enumeration, and identification of Ureaplasma spp. and M. hominis in the urogenital tract. The kit consists of UMMt liquid transport medium and UMMlyo lyophilized growth medium in microplate wells. Detection of growth of the organisms is based on color change in liquid medium containing urea and arginine. Species identification is based on differential inhibition by erythromycin and lincomycin. This product is available in the United States. (Photograph courtesy of ELITechGroup, Inc.) Figure 12.2 MYCOFAST US (ELITech). MYCOFAST US is a commercial kit for the detection, identification, and enumeration of Ureaplasma spp. and M. hominis in clinical specimens. The kit consists of microplates containing lyophilized growth substrates and antimicrobials. Detection of growth of the organisms is based on color change in liquid medium containing urea and arginine. Species identification is based on differential inhibition by erythromycin, trimethoprim/sulfamethoxazole, and lincomycin. This product is available in the United States. (Photograph courtesy of ELITechGroup, Inc.) Diagnostic kits typically consist of strips with wells containing specific dried or lyophilized substrates and inhibitors. Specimens are placed in a suspension-transport medium that is used to inoculate wells. The detection, identification, and quantitation of organisms are based on the color change of specific wells containing substrates and inhibitors. Some products also contain an antimicrobial susceptibility testing component. Descriptions of some of the commercial diagnostic kits along with their manufacturers are provided in Table 12.1. Some commercially sold diagnostic products have undergone external validation in comparison to one another and with other non-proprietary products in clinical trials that have been published in peer-reviewed journals. Citations, when available, are also indicated in Table 12.1. Some products included in earlier published evaluations are now sold by different companies than when the original evaluations were performed. Other commercial products from various manufacturers and countries exist, but to our knowledge, have not been subjected to rigorous comparison with standard methods of testing. None of these diagnostic kits has undergone complete evaluation and received approval by the United States Food and Drug Administration (FDA) as of this writing. Table 12.1 Commercial detection and susceptibility test kits for Ureaplasma spp. (Us) and M. hominis (Mh). The information included in this table was current as of the time of writing CFU, colony-forming unit; CCU, color-change unit; NA, not applicable/available; CLSI, Clinical and Laboratory Standards Institute. * S, I, R: Susceptible, intermediate, or resistant. Abele-Horn et al. [1] evaluated the MYCOSCREEN broth and agar (ELItech), arginine broth and A7 agar (bioMérieux), U9 broth, arginine broth, and A7 agar (Bio-Rad), and mycoplasma broth and A7 agar (Biotest AG) in 298 clinical specimens and concluded that the commercial media were generally satisfactory for organism detection and identification when compared to unspecified noncommercial media. These authors also evaluated diagnostic kits for quantitative organism detection and identification, which included the Mycoplasma IST (bioMérieux), Mycoplasma Duo (Bio-Rad), and the Mycoplasma “All-In” (no longer available). Performance of these kits was generally satisfactory, but when quantitation is desired, standard methods were better. Cheah et al. [8] compared the Mycoplasma Duo kit with PCR for detection of Ureaplasma spp. in endotracheal aspirates from 60 premature neonates. The overall agreement for 13 total Ureaplasma-positive infants between the two tests was 96%. Evans et al. [16] reported that the Mycoplasma Duo kit actually detected more positive urogenital and placental specimens than traditional culture using A7 agar. Clegg et al. [10] compared the Mycoplasma IST kit with arginine broth, 10C broth, and A7 agar to determine the prevalence of M. hominis and Ureaplasma spp. in 100 vaginal specimens. The Mycoplasma IST kit gave a sensitivity and specificity of 92.9% and 86.7% for M. hominis versus 97.4% and 72.7% for Ureaplasma spp., respectively. They noted that false-positive reactions may occur in the Mycoplasma IST kit if contaminating bacteria are present. Therefore, organism identification and purity should be verified by colony morphology on agar plates. Vasquez and colleagues [44] evaluated Mycoplasma IST and MYCOFAST All-In for detection of M. hominis and Ureaplasma spp. against isolation on A7 agar (bioMérieux), testing 123 vaginal and cervical swabs in women and urethral swabs in men. The sensitivity was 100% for M. hominis and Ureaplasma
Mycoplasma
12.1 Introduction and clinical considerations
12.1.1 Mycoplasmas and ureaplasmas as human pathogens
12.1.2 Indications for diagnostic testing
12.2 Culture-based diagnosis
12.2.1 Specimens appropriate for culture
12.2.2 Specimen collection and transport
12.2.3 Commercial growth media and diagnostic kits
Product name
(Company, number of tests)
Parameters
Samples
Format
Detection, identification and enumeration
Susceptibility testing*
Antibiotics tested
References
Mycoplasma IST2 (bioMérieux, 25)
Cervico-vaginal, urethral swabs, semen
Microplates of 22 wells
Yes
Us ≥ 104 CFU/mL
Mh ≥ 104 CFU/mL
Yes (categorization S, I, R)
Tetracycline, doxycycline, azithromycin, clarithromycin, josamycin, clindamycin, pristinamycin, ofloxacin, ciprofloxacin
1, 10, 16, 44
Renaudin and Bébéar, Abstract, 15th Congress of the International Organization for Mycoplasmology, 2004
Mycoplasma Duo (Bio-Rad, 20)
Vaginal, endocervical, urethral swabs, semen, urine
Microplates of 6 wells
Yes
Us ≥ 104 CCU/mL
Mh ≥ 104 CCU/mL
No
NA
1, 10, 16, 44
SIR Mycoplasma (Bio-Rad, 10)
Broth culture of Us or Mh from well X of Mycoplasma Duo or from A3 broth inoculated with the sample and enriched with mycoplasmas
Microplates of 16 wells
No
Yes (categorization S, I, R)
Tetracycline, doxycycline, erythromycin, azithromycin, josamycin, clindamycin, pristinamycin, ofloxacin
1, 10, 16, 44
Mycokit-NUM
(Eurobio, 24)
Vaginal, endocervical, urethral swabs
Microplates of 16 wells
Yes
Uu ≥ 104 CCU/mL
Mh ≥ 104 CCU/mL
No
NA
NA
Mycokit-ATB
(Eurobio, 6)
Broth culture of Us or Mh A3 broth inoculated with the sample and enriched with mycoplasmas
Microplates of 16 wells
No
Yes (categorization S, I, R)
Fusidic acid, doxycycline, erythromycin, lincomycin, minocycline, pristinamycin, josamycin
NA
MYCOSCREEN Plus (ELITech, 16 or 32)
Cervico-vaginal, urethral swabs, semen, urine
Microplates of 6 wells
Liquid medium UMMt
Yes
Us ≥ 104 CCU/mL
Mh ≥104 CCU/mL (to be read on the microplate for urethral and cervicovaginal swabs)
Us or Mh ≥ 102 CCU/mL if the UMMt medium is positive
No
NA
NA
MYCOFAST Evolution 2 (ELITech, 12 or 30)
Cervico-vaginal, urethral, endotracheal swabs, semen, urine
Microplates of 20 wells
Yes
Us ≥ 104 CCU/mL for urethral swabs, ≥ 103 CCU/mL for urines, semen, and endotracheal swabs
Mh ≥ 104 CCU/mL
Yes (categorization S, I, R)
Doxycycline, roxythromycin, ofloxacin
Poulin and Kundsin, Abstract, 97th American Society for Microbiology General Meeting, 1997
MYCOFAST Evolution 3 (ELITech, 12 or 30)
Cervico-vaginal, urethral, endotracheal, semen, urine
Microplates of 20 wells
Yes
Us ≥ 104 CCU/mL for urethral swabs, ≥ 103 CCU/mL for urines, semen, and endotracheal swabs
Mh ≥ 104 CCU/mL
Yes (categorization S, I, R)
Doxycycline, azithromycin, roxythromycin, josamycin, pristinamycin, ofloxacin, ciprofloxacin
NA
MYCOFAST US
(ELITech, 30)
Cervico-vaginal, urethral, endotracheal, semen, urine, gastric secretions
Microplates of 20 wells
Yes
Us 103 to > 105 CCU/mL
Mh > 104 CCU/mL
No
NA
NA
MYCOFAST RevolutioN (ELITech, 50 microplates for screening, 25 microplates for numbering and susceptibility testing)
Cervico-vaginal, urethral, endotracheal, gastric, semen, urine
Microplates of 6 wells for screening
Microplates of 20 wells for numbering and susceptibility testing
Yes
Us ≥ 104 CCU/mL for urethral swabs, ≥ 103 CCU/mL for urines, semen, and endotracheal swabs
Mh ≥ 104 CCU/mL
Yes (categorization S, R)
Tetracycline, erythromycin, clindamycin, levofloxacin, moxifloxacin: at the breakpoint concentrations recommended by the CLSI guidelines
Renaudin and Bébéar, Abstract, 19th Congress of the International Organization for Mycoplasmology, 2012
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