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Prevalence and Macrolide Resistance of Mycoplasma genitalium in South African Women

Hay, Bastien PharmD*†‡; Dubbink, Jan Henk Bsc§¶∥; Ouburg, Sander PhD; Le Roy, Chloé PhD*†; Pereyre, Sabine PharmD, PhD*†‡; van der Eem, Lisette Bsc§¶; Morré, Servaas A. PhD¶∥; Bébéar, Cécile MD, PhD*†‡; Peters, Remco P.H. PhD§**

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Sexually Transmitted Diseases: March 2015 - Volume 42 - Issue 3 - p 140-142
doi: 10.1097/OLQ.0000000000000246
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Mycoplasma genitalium is a sexually transmitted pathogen associated with several inflammatory reproductive tract syndromes in women such as vaginal discharge syndrome (VDS), cervicitis, pelvic inflammatory disease, and infertility.1,2 Recent improvements in diagnostic techniques for the detection of M. genitalium have enabled a broader understanding of the significance of this organism. Except for cohorts of Ugandan3 and Kenyan4,5 female sex workers (FSW), several studies about M. genitalium infections in women from Sub-Saharan Africa are available. In these studies, M. genitalium detection was only performed on genital samples, but not on rectal ones. Several studies recently showed a strong association between M. genitalium and HIV acquisition and transmission, especially in African populations.6,7 This study aimed to determine the prevalence and identify factors associated with vaginal and rectal M. genitalium infection in a cohort of women in rural South Africa.

We used remnant specimens collected from November 2011 to February 2012 from 601 women aged 18 to 49 years included in a cross-sectional study of vaginal, rectal, and oral chlamydial and gonococcal infections in women visiting primary health care clinics across Mopani District, Limpopo province, South Africa.8 Eligibility criteria were sexual activity (at least 1 sex act in previous 6 months), consent to have all 3 anatomical sites tested (vagina, anorectum, and oropharynx), and not having menses on day of recruitment. Demographic, clinical, and sexual behavioral data were collected by professional nurse–administered questionnaire. HIV status was self-reported and classified as positive, negative (tested <6 months ago), or unknown. Patient information was provided and written consent obtained. The study was approved by the Human Ethics Research Committee of the University of the Witwatersrand, South Africa (Reference No. M110726).

Collected swabs were processed for evaluation with the cobas CT/NG test (reported elsewhere).8Trichomonas vaginalis DNA was detected with the CE-IVD certified PrestoPlus test (Goffin Molecular Diagnostics, Houten, the Netherlands) using the LightCycler II (Roche Diagnostics, Indianapolis, IN) and with the T. vaginalis LightMix real-time polymerase chain reaction (PCR) kit (TIB MOLBIOL, Berlin, Germany) on the open channel of the cobas 4800 system (Roche Diagnostics). Residual purified DNA was tested for the presence of M. genitalium with the M. genitalium LightMix real-time PCR kit (TIB MOLBIOL), on 601 vaginal and rectal as well as 98 pharyngeal extracts, 80 obtained from all women reporting fellatio, and 18 randomly selected, using the open channel on the cobas 4800 system (Roche Diagnostics). The M. genitalium LightMix real-time PCR kit amplifies a 224-bp gap gene fragment with specific primers. It was previously evaluated in comparison to an in-house Taqman real-time PCR assay using the cobas z480 analyzer from the cobas 4800 system and shown to have comparable sensitivity, specificity, and positive and negative predictive values.9 For macrolide resistance, detection of mutations in the 23S rRNA gene was performed on M. genitalium–positive extracts by PCR and sequencing.10 Statistical analysis was performed using χ2 and Fisher exact tests and Mann-Whitney U test to compare dichotomous and continuous variables between groups, respectively. Multivariate analysis was conducted on variables with P value less than 0.1 in univariate analysis using a forward logistic regression analysis. A P value less than 0.05 was considered as statistically significant for all tests.

The median age of women was 30 years (range, 18–49 years); 31% of participants self-reported known HIV infection, where 46% of the study group reported a status of HIV uninfected, with 23% indicating unknown status. Prevalence of vaginal and rectal M. genitalium infection was 8.7% (52/601; 95% confidence interval [CI], 6.4%–10.9%) and 2.7% (16/601; 95% CI, 1.4%–3.9%), respectively. As already described for FSWs in Kenya5 and Uganda,3 prevalence of M. genitalium infection was higher in women aged 18 to 25 years than in older women, reporting 16.1% (vaginal 12.4%, rectal 4.3%) in younger women versus 8.2% (vaginal 6.8%, rectal 1.9%) in the older population (odds ratio [OR], 2.1; 95% CI, 1.3%–3.6%; P = 0.004). Concurrent infection with M. genitalium (i.e., present at both genital and rectal sites) was observed in only 3 women. Of women with rectal and vaginal infections, 81% (13/16) and 94% (49/52) had an isolated rectal or vaginal infection, respectively. There was no statistically significant association between genital and rectal infection (Fisher exact test: OR, 2.5; 95% CI, 0.7–9.1; P = 0.15). No oral infection was detected among the 98 selected pharyngeal DNA extracts tested. According to these negative results, we did not test other pharyngeal samples for M. genitalium detection. Overall, 10.8% (65/601; 95% CI, 8.3%–13%) of women were infected with M. genitalium at either anatomical site.

Assessment for other sexually transmitted infections showed that 16% of the women of the cohort were infected with Chlamydia trachomatis or Neisseria gonorrhoeae and 18% with T. vaginalis.8 Nineteen percent, 12%, and 27% of women M. genitalium–positive were coinfected with C. trachomatis, N. gonorrhoeae, and T. vaginalis, respectively.

Among the 52 vaginal and 16 rectal M. genitalium–positive samples, 2 vaginal and 2 rectal samples contained M. genitalium strains carrying mutations in the 23S rRNA, which are known to be associated with macrolide resistance. Thirty-one vaginal and 6 rectal specimens harbored wild-type M. genitalium sequences. Further characterization by sequencing was unsuccessful for 19 vaginal and 8 rectal samples, most likely due to a low concentration of M. genitalium DNA as indicated by cycle threshold (CT) values from the real-time PCR for these samples (mean CT value for all tested extracts 38.06, data not shown). Moreover, this low DNA load did not allow us to perform molecular typing on M. genitalium–positive extracts. All the mutated specimens harbored the A2058G mutation (Escherichia coli numbering), the most frequently observed mutation observed with macrolide resistance. In one vaginal specimen, a mixed population of wild-type bacteria and an A2058G mutant was observed as previously described.9

There was no significant association between individual vaginal symptoms as well as VDS or rectal symptoms and either M. genitalium genital or rectal infection, respectively. Only a trend for dysuria (P = 0.09) was observed. Adjustment for presence of genital C. trachomatis or N. gonorrhoeae coinfection did not change this association. Stratified analysis by HIV status did not reveal any effect of HIV infection on association of symptoms with infection.

Univariate analysis was performed to identify factors associated with genital M. genitalium infection (Table 1). Only age and coinfection with T. vaginalis were significantly associated with M. genitalium infection (P = 0.008 and P = 0.046, respectively), whereas there were tendencies observed for HIV status, current pregnancy, and reporting receptive anal intercourse (RAI; 0.06 < P < 0.1; Table 1). In multivariate analysis, a young age and HIV infection were significantly associated with M. genitalium infection (Table 1).

T1-7
TABLE 1:
Univariate and Multivariate Analyses of Factors Associated With Genital M. genitalium Infection*

This study shows that M. genitalium prevalence was relatively high in this cohort of rural South African women in comparison to those described in high-income countries. This prevalence is close to those described among FSWs (13%–16%), and comparable to symptomatic women, yet higher than in women tested in the general population in Sub-Saharan Africa (3%–4%).3–5,11–16

In our study, no significant association with individual vaginal or rectal symptoms and presence of M. genitalium was found. Indeed, women who are infected by M. genitalium are often asymptomatic, like those infected with C. trachomatis.17 The higher prevalence detected in younger women and association between HIV and M. genitalium have been previously reported in Sub-Saharan Africa.3,7

To our knowledge, this is the first report of M. genitalium rectal infection in Africa. Rectal infection (2.7%) was less common than vaginal infection (8.7%) in our study. In addition, RAI was not specifically associated with rectal M. genitalium infection; it should be noted that the reported frequency of RAI was low in our cohort (4.6%),8 with underreporting likely playing a role. Although not significant, rectal M. genitalium infection was more common among women reporting RAI (7.7%) than those not reporting RAI (2.8%). In comparison, 2 studies from the United Sates reported prevalence of M. genitalium rectal infection of 4.2% and 9.5% among women who were practicing RAI.18,19 In contrast to C. trachomatis and N. gonorrhoeae, M. genitalium does not seem to cause a syndrome of clinical proctitis considering the low frequency of localization of infections to the rectum, the lack of association between rectal symptoms and presence of M. genitalium, and infrequent concurrent genital and rectal infections. Furthermore, our data suggest that M. genitalium does not cause pharyngeal infection.

Macrolide resistance in M. genitalium through mutations in region V of the 23S rRNA has been described worldwide in specimens from Australia, Europe, and Japan1 and has been reported to be associated with up to 40% of characterized specimens.20 We describe for the first time macrolide resistance in M. genitalium–positive specimens from South Africa, with a prevalence of 9.8% (4/41). This prevalence should be confirmed by other studies according to the low number of samples evaluated. Furthermore, 36% of vaginal and 50% of rectal extracts could not be typed to look for macrolide resistance; thus, it is possible that the prevalence of macrolide resistance in extracts that could not be typed differed from the prevalence in those that could be. Syndromic treatment of VDS in South Africa does not include macrolides.21 Erythromycin belongs to the empirical treatment of genital ulcer disease; besides this indication, this macrolide is usually prescribed in South Africa as an alternative antibiotic treatment of skin and respiratory tract infections.22 Azithromycin and clarithromycin are currently available for specific indications only, such as for treatment of nontuberculous mycobacteria. As such, the macrolide pressure which could select a resistant mutant may be considered low in this population.

In summary, we confirm the presence of M. genitalium infection in women from rural South Africa and describe for the first time macrolide-resistant M. genitalium–positive specimens. To include an antimicrobial specifically active against M. genitalium in the syndromic treatment for women with VDS, in particular among those with HIV infection and not responding to initial syndromic treatment, requires further research. Indeed doxyxcycline, already included in the empirical VDS regimen, is not fully capable to eradicate M. genitalium infection.1

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