Mycoplasma genitalium: the need for testing and emerging diagnostic options

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LEARNING OBJECTIVES

1. Describe the characteristics that Mycoplasma genitalium possesses, including route of transmission, incidence, prevalence, and treatment.
2. Discuss the future need for accurate diagnosis of Mycoplasma genitalium through diagnostic testing.
3. List and describe the disease sequelae that Mycoplasma genitalium causes.
4. List and describe the testing methods that have been documented in the identification of Mycoplasma genitalium and the limitations that each method possesses.

Mycoplasma genitalium is a sexually transmitted pathogen which was first discovered in 1980 in the urinary tracts of two men with symptomatic nongonococcal urethritis (NGU).^1,2 The bacterium belongs to the class Mollicutes and is a flask-shaped pathogen.^1,3,4 M. genitalium is thought to be the smallest prokaryote currently known that is capable of self-replication.⁴ Surface lipid-associated membrane proteins (LAMPs) bind to the surface molecules of the host’s vascular endothelial cells using toll-like receptors (TLRs). This adhesion causes those cells to produce cytokines, resulting in an inflammatory response.³

Getting to know M. genitalium

The reduced genome is made up of 580 kb and has merely 482 protein-coding genes; as such, it was one of the first bacteria whose genome was fully sequenced and the first genome that was chemically synthesized.^1,4,5 It has an organelle on the tip of the terminal structure containing an adhesion molecule, also called the MgPa adhesion protein or P140, which is used to enter and infect a cell.³ The MgPa adhesion gene has a number of homologous repetitive DNA elements which help to target this gene.⁶ When polymerase chain reaction (PCR)-based detection assays targeting M. genitalium were introduced in the early 1990s, the impact of M. genitalium as a pathogen could begin to be investigated.⁴

Lisa Manhart, MPH, PhD, who co-authored a paper¹ on M. genitalium, says, “Among low risk young adults in the United States and the United Kingdom, the prevalence of M. genitalium ranges from one percent to three percent. This is quite a bit higher than the prevalence of gonorrhea (0.4 percent) and somewhat lower than the prevalence of chlamydia (four percent) in the U.S.”⁷ M. genitalium is very often asymptomatic and can go undetected. It colonizes the reproductive tracts of both men and women and has been shown to be particularly adept at eluding the host’s immune responses, possibly through intracellular localization and the recombinational variation found in genes responsible for encoding the surface-exposed antigens.^1,5 This results in long-term infections, many of which are resistant to single-dose azithromycin. Indeed, short-term macrolide antibiotic treatments such as this may even allow strains containing mutations in the 23s rRNA gene to predominate, resulting in drug resistance.⁵

Increasing concern over the implications of prolonged M. genitalium infection on reproductive and sexual health and the associated reproductive tract infections, inflammation, and disease sequelae has highlighted the need for diagnostic testing.¹ This is further emphasized by the specific treatment requirements for M. genitalium infections. However, M. genitalium has proven fastidious in its growth requirements, and cultures are time-consuming and labor-intensive to obtain.⁴

The need for diagnostic testing

An infection by M. genitalium has been found to result in a number of adverse sequelae, highlighting the need to test for this bacterium, either through targeted screening of high-risk individuals or through targeted diagnostic testing on presentation of symptoms.

Nongonococcal urethritis (NGU). M. genitalium has been associated in a number of studies with acute and persistent (or chronic) nongonococcal urethritis.^1,5 In addition to this, when treatment for acute urethritis fails to eradicate M. genitalium, the patient may present with persistent urethritis. Indeed, M. genitalium is thought to be responsible for 12 percent to 41 percent of cases of persistent or recurrent NGU.¹ Globally, the data suggest that M. genitalium may be present in six percent of asymptomatic men.⁴ Persistent NGU in men caused by M. genitalium is accompanied by inflammation in the urogenital system in response to cytokine secretion.^3,5

Cervicitis. The spectrum of clinical syndromes seen with M. genitalium infection is similar to those associated with Chlamydia trachomatis and Neisseria gonorrhoeae infections, which includes cervicitis in women.^2,3,4 Long-term infection with M. genitalium of the endocervical epithelial cells causes cytokine secretion, resulting in chronic inflammation and greater sensitivity to secondary TLR stimulation. A quantitative polymerase chain reaction (qPCR) assay has been used to demonstrate that low organism burdens are capable of causing chronic inflammation in endocervical epithelial cells in an animal model.⁵

Pelvic inflammatory disease (PID). M. genitalium has been associated with PID.^2,4,5 Samples taken from the vagina, endocervix, endometrium, and fallopian tubes have revealed M. genitalium DNA. This indicates that, after sexual transmission, the bacteria can disseminate from the primary point of contact through to the upper reproductive tract.⁵

Tubal factor infertility (TFI). PID is thought to be a precursor to TFI, and, by association, M. genitalium has been implicated in TFI.^1,2,4,5 This seems to be supported by a case of mild salpingitis which revealed an M. genitalium infection in the fallopian tube.¹ The organism can ascend from the vagina to the cervix, through the uterus and into the fallopian tubes and ovaries.⁵

Human immunodeficiency virus (HIV). Persistent and untreated M. genitalium infections could lead to the acquisition or pathogenesis of other sexually transmitted pathogens, including HIV. A high M. genitalium burden in HIV-infected individuals reportedly increases HIV shedding. M. genitalium infections, resulting in inflammatory cytokine secretion, have been found more often and are more persistent in HIV-positive patients.^3,5 Additionally, the increase in cytokine levels may play a part in increasing a patient’s susceptibility to HIV infections. M. genitalium is also implicated in the sexual transmission of HIV because it reduces the integrity of the epithelial barrier, thus increasing the potential of HIV to cross through the epithelium and to infect the target cells beneath.³ Thus, M. genitalium causes both increased susceptibility to HIV infection and heightened HIV shedding, resulting in potentially higher transmission rates.

Routine screening or targeted testing?

M. genitalium infections are often asymptomatic. Even so, there are potentially serious sequelae that can result from an untreated infection, regardless of whether the symptoms are immediately recognizable. In light of this, routine screening of high-risk individuals would be ideal for the detection and treatment of M. genitalium. However, culture techniques are extremely difficult and time-consuming, and the detection of M. genitalium is, at this time, dependent on nucleic acid amplification tests (NAATs), for which there are few commercial options available.² There is also a need for supporting data demonstrating that routine screening and treatment will indeed decrease the incidence of adverse sequelae, and such data is not currently available. As a result, clinicians seem unlikely to adopt routine screening to detect M. genitalium in the foreseeable future.

Patients who present with persistent NGU, cervicitis, or PID should be tested for M. genitalium through targeted testing. The more data that becomes available on M. genitalium and the adverse sequelae that can occur with infection, the better positioned clinicians will be to determine how urgent a cost- effective diagnostic test for routine screening is.¹ When such data is available, they will be able to consider conducting routine diagnostic testing on asymptomatic patients who may be at risk of further health complications.

The available testing alternatives

Culture. Culturing M. genitalium is time-consuming and costly; the fastidious nature of the bacteria makes it difficult to culture successfully. Specimen collection, handling, transport, and treatment require careful attention, and specialized nutritive transport media are required to keep the organisms viable for culture.⁸ Manhart confirms these difficulties: “The organism is very slow-growing and very difficult to culture; it requires tissue culture and takes approximately six months, and only a few laboratories in the world have successfully cultured clinical isolates of M. genitalium.”¹

Serological diagnosis. Serodiagnosis includes immunofluorescence, immunobinding, immunoblotting, and immunoperoxidase. In general, they have poor sensitivity and specificity and have not been widely used for diagnosis. Additionally, there are no standardized or commercially available serological tests, and molecular-based tests have proven more effective in detecting M. genitalium.⁶

Molecular-based tests. NAATs allow fastidious bacteria, such as M. genitalium, to be detected most effectively. Sample handling and collection is also easier for NAATs because the organism viability is not a criterion, as is the case in cultures.⁶

PCR methods are the most widely applied NAATs currently used as diagnostic tools for M. genitalium. Conventional PCRs require two to three days for results to be obtained, whereas the real-time PCR methods can yield results within one day and provide quantitative data, describing the bacterial load within the sample.⁶ The MgPa gene, which encodes the major surface protein MgPa, and the 16S rRNA encoding-genes are the more commonly used target genes when designing assays.⁴

One real-time quantitative PCR (qPCR) assay targeted the MG190 (MgPa) gene in a specific 92 bp region.⁵ Another real-time qPCR assay was found to have a detection limit of 300 genome copies/mL based on the amplification of the pdhD gene, the M. genitalium specific gene which encodes dihydrolipoamide dehydrogenase. The assay, which was both qualitative and quantitative, gave reproducible results and showed good specificity.⁴

Multiplex PCR (mPCR) tests allow for the detection of multiple pathogens. One such mPCR test searched for seven sexually transmitted pathogens, one of which was M. genitalium, targeting the MgPa adhesion gene. While there were only three samples that were positive for M. genitalium, the researchers claimed the test showed a specificity and sensitivity of 100 percent. If the mPCR assays have comparable clinical sensitivity to real-time PCR assay, they may be a low-cost alternative to consider for routine detection of STIs. However, simultaneously amplifying and detecting multiple gene targets can be technologically challenging and is not without drawbacks.⁸

A transcription-mediated amplification (TMA) assay has been developed^9,10 which uses analyte-specific reagents (ASRs) for M. genitalium. Damon Getman, PhD, a scientist affiliated with a major diagnostics manufacturer, says,“ The M. genitalium ASRs are oligonucleotides that can serve as the target capture oligo, primers, and probe for M. genitalium 16s rRNA. These oligo reagents are essentially the same as those used in a research use only (RUO) M. genitalium TMA assay that has been used for about 10 years for a number of research studies by academic investigators.”^9,10

These assays all use magnetic bead-based target capture to isolate the target 16s rRNA. The beads are magnetic particles that have been coated with 16s rRNA complementary oligonucleotides. The oligonucleotides bind to and capture the specific region of 16s rRNA that has been targeted. Getman explains that TMA is used to amplify the captured rRNA, and a hybridization-protection assay with chemiluminescent probes is used to detect the RNA amplicon produced in the TMA reaction. The M. genitalium TMA assays can be run manually on several different instrument platforms, or they can be run on automated instruments.

Commercially available options

At this time there is no FDA-cleared commercial standard detection method for M. genitalium. However, a number of NAAT-based systems have been developed, with a broad array of gene targets and methods used.² “Nucleic acid amplification testing is the best method to detect M. genitalium,” says Manhart. “Until recently, only research laboratories that had developed in-house PCR tests were able to perform M. genitalium testing. Now, several large commercial laboratories are offering NAAT testing for M. genitalium, but the sensitivity and specificity of those tests is not typically published.” This raises questions about the accuracy, sensitivity, and specificity of the assays, as many have not been compared and validated alongside other molecular-based or culture-based methods.⁶

A recent study was conducted to determine the performance of two commercial real-time PCR kits used to detect M. genitalium. The kits, which are used mainly in Europe, are not yet available in the United States. There are also a number of commercialized mPCR tests which include the detection of M. genitalium.² “Many medical laboratories have developed their own in-house PCR tests, and tests can be run on standard equipment,” says Manhart. With the recent availability of the analyte-specific reagents for the TMA assay, laboratories with an anticipated high testing volume can validate the test in their setting and run it on the same equipment that is used for the test for gonorrhea and chlamydia.

The future will no doubt include large-scale comparisons of NAATs using new and existing assay formats and gene targets. Commercially available and FDA-cleared standardized reagents and quality control will be possible after the accuracy and specificity of NAATs for the detection of M. genitalium have been determined.⁶

References

1. Manhart LE, Broad JM, Golden MR. Mycoplasma genitalium: should we treat and how? Clin Infect Dis. 2011; 53(suppl 3):129-142.doi:10.1093/cid/cir702.
2. Le Roy C, Pereyre S, Bébéar C. Evaluation of two commercial real-time PCR assays for detection of Mycoplasma genitalium in urogenital specimens. J Clin Microbiol. 2013;52(3):971-973. doi:10.1128/jcm.02567-13.
3. Das K, De la Garza G, Siwak EB, Scofield VL, Dhandayuthapani S. Mycoplasma genitalium promotes epithelial crossing and peripheral blood mononuclear cell infection by HIV-1. Int J Infect Dis. 2014;23(6):31-38.
4. Müller E, Venter J, Magooa M, Morrison C, Lewis D, Mavedzenge S. Development of a roto-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium. J Microbiol Methods. 2012;88(2):311-315. doi:10.1016/j.mimet.2011.12.017.
5. McGowin C, Annan R, Quayle A, et al. Persistent Mycoplasma genitalium infection of human endocervical epithelial cells elicits chronic inflammatory cytokine secretion. Infection and Immunity. 2012;80(11):3842-3849. doi:10.1128/iai.00819-12.
6. Waites KB, Xiao L, Paralanov V, Viscardi RM, Glass JI. Molecular methods for the detection of Mycoplasma and Ureaplasma infections in humans: a paper from the 2011 William Beaumont Hospital Symposium on Molecular Pathology. J Molec Diag. 2012;14(5):437-450. doi:10.1016/j.jmoldx.2012.06.001.
7. Oakeshott P, Aghaizu A, Hay P, et al. Is Mycoplasma genitalium in women the “new chlamydia?” A community‐based prospective cohort study. Clin Infect Dis. 2010;51(10):1160-1166. doi:10.1086/656739.
8. Muvunyi C, Dhont N, Verhelst R, et al. Evaluation of a new multiplex polymerase chain reaction assay STD Finder for the simultaneous detection of 7 sexually transmitted disease pathogens. DiagMicrobiol and Infect Dis. 2011;71(1):29-37. doi:10.1016/j.diagmicrobio.2011.06.005.
9. Wroblewski JK, Manhart LE, Dickey KA, Hudspeth MK, Totten PA. Comparison of transcription mediated amplification and PCR assay results for various genital specimen types for detection of Mycoplasma genitalium. J Clin Microbiol. 2006, 44(9):3306–3312.
10. Hardick J, Giles J, Hardick A, Hsieh YH, Quinn T, Gaydos C. Performance of the Gen-Probe transcription-mediated amplification research assay compared to that of a multi-target real-time PCR for Mycoplasma genitalium detection. J Clin Microbiol. 2006, 44(4):1236–1240.