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Original Studies

High Prevalence of Vaginal and Rectal Mycoplasma genitalium Macrolide Resistance Among Female Sexually Transmitted Disease Clinic Patients in Seattle, Washington

Khosropour, Christine M. PhD, MPH; Jensen, Jørgen S. MD, PhD; Soge, Olusegun O. PhD‡,§; Leipertz, Gina BS; Unutzer, Anna MPH; Pascual, Rushlenne BS; Barbee, Lindley A. MD, MPH§,¶; Dombrowski, Julia C. MD, MPH∗,§,¶; Golden, Matthew R. MD, MPH∗,§,¶; Manhart, Lisa E. PhD, MPH∗,‡

Author Information
Sexually Transmitted Diseases: May 2020 - Volume 47 - Issue 5 - p 321-325
doi: 10.1097/OLQ.0000000000001148

Mycoplasma genitalium (MG) is a bacterial sexually transmitted infection associated with urethritis in men, and with cervicitis, pelvic inflammatory disease (PID), preterm birth, spontaneous abortion, and infertility in women.1,2 The prevalence of urogenital MG is approximately 1% to 9% in population-based samples of women3 and is approximately 15% to 20% in contemporary clinic-based samples of women in the United States.4,5

Like Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC), MG has been detected in female rectal specimens, but little is known about the epidemiology of rectal MG among women. Among clinic-attending women in the United States and South Africa, the prevalence of rectal MG is approximately 3% to 8%,6–9 which is in the range of that reported for rectal CT (6–9%) and rectal GC (2%) in comparable populations.10–12 Rectal MG does not appear to be associated with anal sex7,8 and, as has been suggested for rectal CT,13 may result from autoinoculation from the vagina. The health implications of rectal MG among women are largely unknown. Rectal MG is often asymptomatic, but could serve as a reservoir of transmission to men via anal or vaginal sex; the latter occurring if women autoinoculate MG from the rectum to the vagina. Although 2 commercially available MG diagnostic tests were approved by the U.S. Food and Drug Administration in 2019, there are no U.S. guidelines for routine vaginal or rectal MG screening.

The 2015 U.S. Centers for Disease Control and Prevention (CDC) sexually transmitted disease (STD) Treatment Guidelines recommends 1 g azithromycin (a macrolide antibiotic) for urethritis, cervicitis, and PID in cases where MG is the suspected causative agent.14 However, reports of MG azithromycin resistance have been increasing for well over a decade,15 and MG azithromycin cure rates have substantially declined since 2009.16 Azithromycin treatment failure is associated with genetic mutations in the 23S rRNA gene,17 and the prevalence of MG strains with these macrolide resistance-mediating mutations (MRM) varies widely, from 0% in South Africa18 to 63% in Australia19 to 100% in Greenland.20 Alarmingly, treatment failures with the CDC-recommended second-line antibiotic for suspected MG—moxifloxacin (a quinolone antibiotic)—have also been observed. These treatment failures are associated with mutations in the parC gene, mainly in codons coding for amino acids in position S83 or D87 of ParC (quinolone resistance-associated mutations [QRAMs]). The correlation with treatment failure is stronger in the presence of concurrent mutations in the gyrA gene.21 A prevalence of the S83I mutation in ParC as high as 84% has been reported in some settings.22 However, the prevalence of MRM and QRAM among rectal specimens from women has not been well described.

We have previously observed a high prevalence of rectal CT (22%) and rectal GC (8%) in a cohort of women at high risk of urogenital CT recruited from a municipal STD clinic in Seattle, WA.23 The goals of the current analysis were to examine the prevalence of vaginal and rectal MG and coinfection with CT and GC in this same population, and to describe the prevalence of macrolide and quinolone resistance mutations in women with vaginal and rectal MG.

METHODS

Study Design, Setting, and Population

This was a cross-sectional study, using data from the enrollment visit of a prospective study of women at high risk of urogenital CT. We have previously described the prospective study's methods and population.23 Briefly, patients attending the Public Health-Seattle & King County (PHSKC) STD clinic in Seattle, Washington between September 2017 and April 2018 were invited to participate in the study if they were female sex at birth, 16 years or older, not pregnant, reported sex with a man in the past 12 months, and self-reported one of the following: being a contact to a partner with CT or GC, urogenital symptoms, or a history of CT. At the enrollment visit, participants completed a detailed sexual behavior questionnaire and collected specimens for vaginal and rectal CT and GC testing, described below. Participants testing positive for vaginal or rectal CT were followed prospectively for up to 8 weeks. The results of the prospective study have been previously reported23 and are not discussed further here.

Participants were paid US $30 for completing the enrollment visit. This study was approved by the University of Washington Institutional Review Board. All participants provided written informed consent and consented to future testing of their specimens.

Clinical and Behavioral Data Collection and Measures

Per routine clinic practice, all patients presenting to the PHSKC STD Clinic for a new problem visit complete a computerized patient intake form, which queries patients on reason for visit, symptoms, and sexual behaviors. These data are included in the clinic's electronic medical record (EMR), which also contains information on clinical examination findings, results of laboratory tests, and treatment(s) received. We used these EMR data to obtain participants' clinical and treatment information (eg, diagnosis of cervicitis, bacterial vaginosis [BV], or proctitis, presence of vaginal discharge, and self-reported anal symptoms). Participants also completed a research questionnaire at enrollment that asked about demographics and sexual behaviors in the past 2 and 12 months. Questionnaire data were captured using Research Electronic Data Capture (REDCap).24,25

Specimen Collection and Testing

Women self-collected 1 vaginal and 1 rectal specimen for nucleic acid amplification testing (NAAT) testing at enrollment. Specimens were tested in real-time for CT and GC using the Aptima Combo-2 assay performed on the fully automatic Panther System (Hologic, Inc., San Diego, CA). Specimens were stored at −80°C for future testing and were subsequently tested for MG using the Aptima MG transcription-mediated amplification (TMA) assay (Hologic, Inc.). Women testing positive for CT or GC at enrollment received treatment per clinic protocol. Because MG testing occurred retrospectively (between 6 and 15 months after participants' enrollment visit) and the test was performed for research only, participants who tested positive for MG did not receive directed treatment as part of this study.

We retrospectively tested specimens that were MG-positive by Aptima for MRM17 and fluoroquinolone resistance-associated mutations in parC.26 We performed DNA extraction from specimens in Aptima transport medium using a MagNA Pure 96 Instrument (Roche, Pleasanton, CA) with large volume (1 mL) universal pathogen extraction protocol and elution in 50 μL. We used PCR and PyroMark Q96 sequencing to detect MRM.27,28 We amplified and sequenced the parC gene by conventional Sanger sequencing.29

Statistical Analysis

We present the prevalence of vaginal and rectal MG detection, coinfection with CT and GC, and the prevalence of MRM and quinolone resistance mutations. We describe characteristics associated with MG test positivity by anatomic site using Fisher exact test. Analyses were completed using Stata 13 (StataCorp, College Station, TX).

RESULTS

Of 50 enrolled women, 13 (26%) tested positive for MG, including 10 (20%) who tested positive for vaginal MG and 11 (22%) who tested positive for rectal MG. Of 13 women who tested positive for MG at either anatomic site, 8 (62%) tested positive for both vaginal and rectal MG, 2 (16%) tested positive for vaginal MG only, and 3 (23%) tested positive for isolated rectal MG only.

Characteristics of women with and without MG are presented in Table 1. Women with vaginal or rectal MG were younger than women without MG, though not statistically significantly so. Women with MG were more likely to be black, non-Hispanic compared with those without MG, but this difference was only statistically significant for rectal MG. There were no women with vaginal MG diagnosed with cervicitis and no women with rectal MG self-reported anal symptoms or were diagnosed with proctitis. Anal sex in the past 12 months was not associated with rectal MG (P = 0.47).

TABLE 1
TABLE 1:
Demographic and Clinical Characteristics and CT and GC Coinfection at Enrollment, by Anatomic Site and MG Test Positivity (N = 50)*

Among the 10 women with vaginal MG, 3 (30%) were coinfected with vaginal CT and 2 (20%) with rectal CT (Table 1). Forty-five percent (5 of 11) of women with rectal MG had concurrent rectal CT, and 36% (4 of 11) had concurrent vaginal CT. There were no women with MG who also tested positive for GC. Of the 11 women with vaginal CT, 3 (27%) had vaginal MG and 5 (45%) had rectal MG.

A line listing of ParC mutation results is provided in Table 2. We successfully tested 8 of 11 rectal specimens and 9 of 10 vaginal specimens for MRM, representing 13 unique women. Twelve (92%) of 13 women had MRM at either the rectal or vaginal site. All 8 rectal specimens and 8 (89%) of 9 vaginal specimens had MRM. We successfully sequenced the parC gene for 8 of 11 rectal specimens and 8 of 10 vaginal specimens. There were 6 vaginal specimens and 5 rectal specimens with parC mutations, though none were among those that have been associated with moxifloxacin treatment failure.

TABLE 2
TABLE 2:
MRM or ParC Mutations and Treatment Received Among Women With Vaginal or Rectal MG (n = 13)

Of 13 women with MG, 6 (46%) received standard therapy for diagnosed or presumptive vaginal CT within 1 week of enrollment, including 5 women who received azithromycin 1 g and 1 woman who received doxycycline (Table 2). Four of 5 women who received azithromycin for vaginal CT at enrollment had an MG MRM detected in their enrollment vaginal specimen (participants 7 and 12) and/or rectal specimens (participants 5, 7, 13).

DISCUSSION

In this cross-sectional study of women at high risk of urogenital CT, we observed a high prevalence of vaginal (20%) and rectal (22%) MG, and most women (62%) with MG were infected at both anatomic sites. Vaginal and rectal MG were not associated with vaginal discharge or cervicitis, or proctitis, respectively, and rectal MG was not associated with reporting anal sex. Approximately 30% of women with vaginal or rectal MG had a CT coinfection at the same anatomic site, but there were no women with MG and GC coinfection. Nearly 50% of women with vaginal CT were coinfected with either vaginal or rectal MG. All MG rectal specimens and nearly 90% of MG vaginal specimens had MRM, but no specimens had mutations in the parC gene associated with fluoroquinolone resistance. Our findings demonstrate that rectal MG has a similar epidemiologic pattern to rectal CT and highlight the high prevalence of potentially macrolide-resistant vaginal and rectal MG among a population of women routinely being treated with azithromycin for vaginal CT infection.

The prevalence of rectal MG in this study (22%) is identical to the prevalence of rectal CT in our study population,23 but is 2- to 3-fold higher than what has been previously reported among women. In 3 previous U.S. studies in New Orleans, Pittsburg, and Birmingham, the prevalence of rectal MG ranged from 4.3% to 8.1%,6,8,9 and in a study in South Africa, the prevalence was 2.7%.7 It should be noted, however, that only the study from Pittsburgh9 applied TMA detection of MG similar to the present study, suggesting that methodological aspects may play a role in the difference in prevalence.30 By definition, our study population was at high risk for vaginal CT, and it is likely that the women we enrolled were also at high risk for both vaginal and rectal MG. In fact, compared with 2 of the aforementioned studies,6,7 the prevalence of vaginal MG in our study population was also 2-fold higher. However, our findings are in line with 2 recent multisite studies in the United States,4,5 which found that the prevalence of vaginal MG was 15% to 20%, and the prevalence of vaginal CT and MG coinfection was 4%, which was similar to the 6% (3 of 50) that we observed. It is possible that having a prevalent CT infection provides a favorable environment for MG by some unknown mechanism, but to our knowledge, this has not been previously explored. Despite this high prevalence of vaginal and rectal MG, there were no women with signs or symptoms of cervicitis or proctitis, respectively.

Similar to previous studies, we did not find an association between reporting anal sex and rectal MG. Lillis and colleagues8 in New Orleans found that the prevalence of rectal MG was 6.8% among women who reported anal sex and 4.0% among women who did not report anal sex and Hay and colleagues7 in South Africa found that the prevalence of rectal MG was 7.7% among women who reported anal sex and 2.8% among those who did not report anal sex. Here, we identified rectal MG in 2 (12.5%) of 16 women who reported anal sex and 9 (26.5%) of 34 women who did not report anal sex (P = 0.47). It is not clear how women with rectal MG would have acquired their infection in the absence of anal sex, but it is possible that some women may have had persistent rectal MG for several months or years, beyond the recall period of 6 to 12 months used in our study and others. Alternatively, it is possible—as has been suggested for rectal CT—that women autoinoculate MG from the vagina to the rectum via sexual or hygiene practices (ie, wiping front to back), which is facilitated by the close proximity of the vagina and anus.13

The high prevalence of vaginal and rectal MG concurrent infection merits discussion. We found that 65% of women with MG were infected at both anatomic sites and that isolated rectal MG was more common (23% of MG-positive women) than isolated vaginal MG (15% of MG-positive women). This high prevalence of concurrent infection in the absence of anal sex is suggestive of autoinoculation between anatomic sites and is further supported by our resistance testing data, in which we observed identical mutations from different anatomic sites of the same woman. However, molecular typing is needed to confirm whether the MG strains in different anatomic sites were identical. Of particular concern is the potential for autoinoculation from the rectum to the vagina, which has the potential to result in adverse reproductive health outcomes among women and presents an opportunity for transmission to male partners via vaginal sex. It is important to note, however, that MG NAAT testing detects the presence of nucleic acid, not viable MG, so it is possible that the positive rectal MG tests identified in this study merely indicated the presence of nucleic acid from nonviable MG or the presence of viable MG in the perianal region but not causing infection.

We were alarmed that all rectal specimens and 90% of vaginal specimens had a MRM. A high prevalence of MRM has been identified in rectal specimens from men who have sex with men (range, 75%–88%),31s–36s but the prevalence of MRM we observed is considerably higher than that previously observed in female rectal specimens (range, 12.5%–57%).6,7 Further, the high prevalence of vaginal MG strains with MRM in our study is nearly double that reported in prior studies in Europe, Japan, and the United States (range, 0%–51%).4,18,37s–39s Although the MRMs we observed are known to cause treatment failure,17 we do not have information on MG positivity subsequent to treatment, so it is unclear if these women had persistent MG detection, subsequently experienced symptomatic infection, or spontaneously cleared their MG infection. In light of this high prevalence of MRM, it is particularly concerning that nearly 50% (5 of 11) of women with vaginal CT were coinfected with vaginal (n = 3) or rectal (n = 5) MG, of whom 4 received azithromycin for CT per routine clinical care. These findings highlight the fact that women are often treated for CT without regard to whether or not they have MG, which may lead to the further propagation and transmission of antimicrobial resistant MG.17 In particular, the high prevalence of vaginal CT and rectal MG coinfection underscores the potential for the rectum in women to serve as a reservoir or source of MG-resistant infection and transmission to male partners.

Despite the high prevalence of MRM in this population, it was reassuring that we did not observe any parC mutations that have been associated with quinolone treatment failure. Specifically, the S83N mutation in the quinolone resistance determining region has been shown to not result in elevated moxifloxacin minimum inhibitory concentrations in vitro.40s This is in line with other contemporary studies of women, which have found that between 0% and 14%6,18,37s,41s of vaginal specimens and 0% of rectal specimens6 from women have a quinolone resistance-associated mutation and is consistent with recent data from Seattle noting an absence of key quinolone resistance associated mutations among men who have sex with women.42s

There are several important limitations in this study. First, the number of women enrolled in this study was small (n = 50) and was purposefully selected to create a cohort of women at high risk for urogenital CT. As such, the prevalence of MG and genetic mutations may not be representative of other clinic-attending women and is not generalizable to nonclinic populations. Second, we do not have follow-up MG test results or data on signs/symptoms of infection from these women after enrollment. Only women with CT at enrollment were asked to self-collected vaginal and rectal specimens daily for 8 weeks, and, as described in this report, the majority of women with CT did not have MG. Third, we did not have data on organism load to be able to correlate to the presence of symptoms or to compare load between anatomic sites. Finally, we were unable to test all specimens for resistance mutations due to low levels of MG DNA in samples. Despite these limitations, the robust microbiologic and resistance testing data in this study provide a comprehensive picture of rectal sexually transmitted infections and the potential for transmission of antibiotic resistant infection in this clinic-based population of women.

In conclusion, we observed a high prevalence of rectal and vaginal MG—equivalent to the prevalence of CT in this same population—and found that nearly all MG strains had a macrolide resistance mutation. These findings are particularly concerning, and suggest high levels of previous exposure to azithromycin. Furthermore, nearly half of the women in this study with vaginal CT were coinfected with MG, received azithromycin as part of routine treatment, and likely remained infected with MG. This convergence highlights how the use of antimicrobials designed to treat an identified infection—in this case CT—has the potential to influence antimicrobial susceptibility patterns in other unidentified infections and reinforces the need for thoughtful testing and antibiotic prescribing practices. Our results also reinforce not only the longstanding concern that we are entering an era of potentially untreatable MG but also highlight our profound lack of understanding of the epidemiology of MG among women, in particular rectal MG. We do not know how long rectal MG persists in women, if women can autoinoculate MG to the vagina, or if the rectum serves as an incubator of potentially antimicrobial-resistant MG. Answers to these questions will improve our understanding of the health implications of rectal MG to inform screening and treatment guidelines.

REFERENCES

1. Lis R, Rowhani-Rahbar A, Manhart LE. Mycoplasma genitalium infection and female reproductive tract disease: A meta-analysis. Clin Infect Dis 2015; 61:418–426.
2. Wiesenfeld HC, Manhart LE. Mycoplasma genitalium in women: Current knowledge and research priorities for this recently emerged pathogen. J Infect Dis 2017; 216(suppl_2):S389–S395.
3. Baumann L, Cina M, Egli-Gany D, et al. Prevalence of Mycoplasma genitalium in different population groups: Systematic review and meta-analysis. Sex Transm Infect 2018; 94:255–262.
4. Getman D, Jiang A, O'Donnell M, et al. Mycoplasma genitalium prevalence, coinfection, and macrolide antibiotic resistance frequency in a multicenter clinical study cohort in the United States. J Clin Microbiol 2016; 54:2278–2283.
5. Sena AC, Lee JY, Schwebke J, et al. A silent epidemic: The prevalence, incidence and persistence of Mycoplasma genitalium among young, asymptomatic high-risk women in the United States. Clin Infect Dis 2018; 67:73–79.
6. Xiao L, Waites KB, Van Der Pol B, et al. Mycoplasma genitalium infections with macrolide and fluoroquinolone resistance-associated mutations in heterosexual African American couples in Alabama. Sex Transm Dis 2019; 46:18–24.
7. Hay B, Dubbink JH, Ouburg S, et al. Prevalence and macrolide resistance of Mycoplasma genitalium in South African women. Sex Transm Dis 2015; 42:140–142.
8. Lillis RA, Nsuami MJ, Myers L, et al. Utility of urine, vaginal, cervical, and rectal specimens for detection of Mycoplasma genitalium in women. J Clin Microbiol 2011; 49:1990–1992.
9. Cosentino LA, Campbell T, Jett A, et al. Use of nucleic acid amplification testing for diagnosis of anorectal sexually transmitted infections. J Clin Microbiol 2012; 50:2005–2008.
10. Chandra NL, Broad C, Folkard K, et al. Detection of Chlamydia trachomatis in rectal specimens in women and its association with anal intercourse: A systematic review and meta-analysis. Sex Transm Infect 2018; 94:320–326.
11. Dewart CM, Bernstein KT, DeGroote NP, et al. Prevalence of rectal chlamydial and gonococcal infections: A systematic review. Sex Transm Dis 2018; 45:287–293.
12. Lau A, Kong FYS, Huston W, et al. Factors associated with anorectal Chlamydia trachomatis or Neisseria gonorrhoeae test positivity in women: A systematic review and meta-analysis. Sex Transm Infect 2019; 95:361–367.
13. Rank RG, Yeruva L. Hidden in plain sight: Chlamydial gastrointestinal infection and its relevance to persistence in human genital infection. Infect Immun 2014; 82:1362–1371.
14. Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep 2015; 64(RR-3):1–140.
15. Manhart LE. Mycoplasma genitalium on the loose: Time to sound the alarm. Sex Transm Dis 2017; 44:463–465.
16. Lau A, Bradshaw CS, Lewis D, et al. The efficacy of azithromycin for the treatment of genital Mycoplasma genitalium: A systematic review and meta-analysis. Clin Infect Dis 2015; 61:1389–1399.
17. Jensen JS, Bradshaw CS, Tabrizi SN, et al. Azithromycin treatment failure in Mycoplasma genitalium-positive patients with nongonococcal urethritis is associated with induced macrolide resistance. Clin Infect Dis 2008; 47:1546–1553.
18. Muller EE, Mahlangu MP, Lewis DA, et al. Macrolide and fluoroquinolone resistance-associated mutations in Mycoplasma genitalium in Johannesburg, South Africa, 2007–2014. BMC Infect Dis 2019; 19:148.
19. Tabrizi SN, Su J, Bradshaw CS, et al. Prospective evaluation of ResistancePlus MG, a new multiplex quantitative PCR assay for detection of Mycoplasma genitalium and macrolide resistance. J Clin Microbiol 2017; 55:1915–1919.
20. Gesink DC, Mulvad G, Montgomery-Andersen R, et al. Mycoplasma genitalium presence, resistance and epidemiology in Greenland. Int J Circumpolar Health 2012; 71:1–8.
21. Murray GL, Bodiyabadu K, Danielewski J, et al. Moxifloxacin and sitafloxacin treatment failure in Mycoplasma genitalium infection: Association with parC mutation G248T (S83I) and concurrent gyrA mutations. J Infect Dis 2019. DOI: 10.1093/infdis/jiz550.
22. Li Y, Su X, Le W, et al. Mycoplasma genitalium in symptomatic male urethritis: macrolide use is associated with increased resistance. Clin Infect Dis 2019.
23. Khosropour CM, Soge OO, Suchland R, et al. Recurrent/intermittent vaginal and rectal chlamydial infection following treatment: A prospective cohort study among female sexually transmitted disease clinic patients. J Infect Dis 2019; 220:476–483.
24. Harris PA, Taylor R, Minor BL, et al. The REDCap consortium: Building an international community of software platform partners. J Biomed Inform 2019; 95:103208.
25. Harris PA, Taylor R, Thielke R, et al. Research electronic data capture (REDCap)—A metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inform 2009; 42:377–381.
26. Jensen JS, Bradshaw C. Management of Mycoplasma genitalium infections—Can we hit a moving target? BMC Infect Dis 2015; 15:343.
27. Jensen JS. Protocol for the detection of Mycoplasma genitalium by PCR from clinical specimens and subsequent detection of macrolide resistance-mediating mutations in region V of the 23S rRNA gene. Methods Mol Biol 2012; 903:129–139.
28. Salado-Rasmussen K, Jensen JS. Mycoplasma genitalium testing pattern and macrolide resistance: A Danish nationwide retrospective survey. Clin Infect Dis 2014; 59:24–30.
29. Deguchi T, Maeda S, Tamaki M, et al. Analysis of the gyrA and parC genes of Mycoplasma genitalium detected in first-pass urine of men with non-gonococcal urethritis before and after fluoroquinolone treatment. J Antimicrob Chemother 2001; 48:742–744.
30. Unemo M, Salado-Rasmussen K, Hansen M, et al. Clinical and analytical evaluation of the new Aptima Mycoplasma genitalium assay, with data on M. genitalium prevalence and antimicrobial resistance in M. genitalium in Denmark, Norway and Sweden in 2016. Clin Microbiol Infect 2018; 24:533–539.
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