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Mycoplasma genitalium Coinfection in Women With Chlamydia trachomatis Infection

Harrison, Sally A. BS; Olson, Kristin M. MPH∗,†,‡; Ratliff, Amy E. BS§; Xiao, Li PhD; Van Der Pol, Barbara PhD, MPH; Waites, Ken B. MD§; Geisler, William M. MD, MPH∗,†

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doi: 10.1097/OLQ.0000000000001028
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Mycoplasma genitalium (MG) infection is a sexually transmitted infection associated with cervicitis, pelvic inflammatory disease, and infertility in women.1 There is currently only one Food and Drug Administration–cleared diagnostic test for MG in the United States, a nucleic acid amplification test (NAAT) that was Food and Drug Administration cleared in January 2019; however, there are also other research-based MG diagnostic NAATs available to some providers for MG testing. Because of the challenges with MG culture, most studies on MG infection have used NAATs for MG detection. Studies on the prevalence of MG infection in females have demonstrated that MG infections are sometimes accompanied by coinfections with other sexually transmitted pathogens, including Chlamydia trachomatis (CT).2–12 However, MG screening, either in conjunction with CT screening or on its own, is not recommended and therefore not routinely performed.

There are a limited number of studies on the prevalence of MG coinfection in CT-infected women, and the reported frequency of MG detection in CT-infected women ranges from 4.8% to 42.9%.4–12 These studies have not evaluated predictors of MG coinfection in CT-infected women and MG outcomes in women with CT and MG coinfection who are treated with azithromycin; the latter is especially important because the first-line antibiotics used to treat CT infection, azithromycin and doxycycline, have low cure rates against MG,13 which could lead to persisting MG infection and potentially reproductive morbidity. Treatment of MG with azithromycin 1 g single dose is strongly associated with MG treatment failure in persons infected with MG strains with macrolide resistance–associated mutations (MRMs) in the 23S rRNA gene.13 Two recent studies evaluating MRM frequency in female populations in the United States reported that approximately half of MG-infected women had MG strains with MRMs.3,14

Our study had 2 major objectives that were aimed at further understanding the epidemiology and clinical significance of MG coinfection in CT-infected women: (1) investigate MG coinfection frequency and participant characteristics that predicted coinfection in a cohort of CT-infected women and (2) assess the frequency of MG strains with MRMs among coinfected participants and their association with repeat MG detection at a 3-month follow-up visit after CT treatment with azithromycin.

MATERIALS AND METHODS

Testing for MG and the MRM in the 23S rRNA gene was performed by a real-time polymerase chain reaction (MGMR PCR) on stored DNA samples isolated from cervical swab specimens that had been collected from a previously enrolled study cohort15 from March 2012 to September 2017. The methods for the real-time PCR and its validation have been previously reported.16 Briefly, 2 μL of DNA template was tested by the MGMR PCR (which has a limit of detection of 2.3 genome copy [GC]/μL) on the Roche LightCycler 480. The bacterial load was determined according to a previously generated external standard curve. The mutant amplicons were sequenced to identify the specific gene mutations. The cohort comprised CT-infected women ≥16 years of age seen at a sexually transmitted disease (STD) clinic in Birmingham, AL, for treatment of a positive screening CT NAAT result.15 Women with gonorrhea, HIV infection, syphilis, current pregnancy, prior hysterectomy, a current immunosuppressed state due to illness or medical treatment, or treatment with antibiotics with anti-CT activity in the previous 30 days were excluded from the study. All women received azithromycin 1 g directly observed therapy for CT treatment. Analyses were performed with SAS software, version 9.4 (SAS Institute, Cary, NC). We used the Fisher exact, Pearson χ2, and Wilcoxon rank sum tests, as appropriate, to evaluate the association of participant characteristics (demographics, sexual history, prior sexually transmitted infections, hormonal contraceptive use, symptoms, clinical syndromes, and concomitant vaginal infections) with MG coinfection. Regarding clinical syndromes, cervicitis was diagnosed by visualization of mucopurulent or purulent endocervical discharge or easily induced endocervical bleeding after insertion of a swab through the cervical os, and pelvic inflammatory disease was diagnosed by pelvic examination findings of cervical motion tenderness, uterine tenderness, or adnexal tenderness. The Wilcoxon rank sum test was used to assess the association of MG load (in GC per microliter) at baseline with repeat MG detection at follow-up. Missing values were excluded from the analysis. P < 0.05 was considered statistically significant.

RESULTS

The MG testing was performed on samples from 302 CT-infected women with the following characteristics (Table 1): 93% African American; median age of 22 years (range, 16–50 years); median number of sex partners in the last 3 months of 1 (range, 0–10); 42.1% on hormonal contraception; 48.7% with prior CT infection (by self-report or medical record review); 52.3% without urogenital symptoms; 18.9% with cervicitis; 2.7% with pelvic inflammatory disease; and 28.8% with bacterial vaginosis, 12.6% with vulvovaginal candidiasis, and 3.6% with Trichomonas vaginalis infection. The MG was detected in 22 (7.3%) women. None of the above participant characteristics, including presence/absence of symptoms, were significantly associated with MG coinfection (Table 1).

T1
TABLE 1:
Baseline Characteristics of Participants (n = 302) by MG Status

We next evaluated frequency of MG detection at a 3-month follow-up visit in subjects with MG coinfection at baseline (at which time all received azithromycin 1 g for CT treatment). Of the 22 participants with MG coinfection at baseline, 14 were determined have wild-type MG strains. The remaining 8 had strains with MRM: 1 was A2071G (Escherichia coli numbering 2058), 6 were A2072G (E coli numbering 2059), and 1 was mixture of wild type and mutant (we were unable to determine the specific gene mutation). There were 21 patients who returned for a 3-month follow-up visit and had a cervical specimen for MG testing, of whom 6 (28.6%) had MG detected again (Fig. 1). All 6 participants with MG strains detected at the 3-month follow-up visit had strains with an MRM; 5 (83.3%) of these participants also had a strain with an MRM detected at the baseline versus 1 (16.7%) having a wild-type strain at baseline. Thus, azithromycin treatment may have contributed to development of an MRM in 1 of the 6 subjects with repeat MG detection. There was no difference in frequency of participants with unprotected sexual activities or new sexual partners in those with versus without MG detected at follow-up. Of the 15 participants who did not have MG detected at follow-up, 13 (86.7%) had wild-type strains at baseline versus 2 (13.3%) having MRM strains at baseline (thus, 2 women with MRM strains became MG negative after azithromycin treatment). We also found that the baseline MG load (in GC per microliter) was higher in the 6 participants with MG detected at the follow-up visit than in the 15 without MG detected at follow-up (median [range], 178.6 [21.7–2670.0] vs. 46 [1.7–18,100.0]), but the difference did not reach statistical significance (P = 0.23).

F1
Figure 1:
Outcome of Mycoplasma genitalium (MG) coinfection in a cohort of women with Chlamydia trachomatis (CT) infection who were treated with azithromycin (AZM) 1 g, stratified by the presence of a macrolide-associated resistance mutation (MRM) versus no mutation (wild type) in the MG strain.

DISCUSSION

Our study found that MG coinfection in CT-infected women seen in an STD clinic in Birmingham was uncommon, only occurring in 7.3%, which was lower than we anticipated. In 2 previous studies that evaluated MG coinfection in CT-infected women in STD clinics in US cities, the MG coinfection frequency was 25% (12/48 women) in one study11 and 36.1% (13/36 women) in the other.12 Our patient cohort differed from these studies in that all women were CT infected (in contrast to having CT-negative women in the cohort), and our study evaluated a larger number of CT-infected women for MG coinfection (n = 302). Other notable differences in our cohort included a lower frequency of symptomatic patients and patients with a cervicitis diagnosis.

Because our study had data from a 3-month follow-up visit, we could observe the effectiveness of azithromycin 1 g, a first-line urogenital CT infection treatment, in eradicating CT and MG in coinfected women. Among coinfected women, MG was detected again in 29% at the follow-up visit, and all of these MG strains had an MRM detected. Given the reported low cure rates of MG infection with MRM strains treated with azithromycin,17 most of these repeat MG detections in our study likely reflected persisting MG infection because of azithromycin treatment failure rather than MG reinfection. Thus, azithromycin 1 g, a current first-line recommended CT treatment, was ineffective in eradicating MG infection in a significant number of CT-infected women with MG coinfection, especially those with an MRM strain. Importantly, 5 of the 6 women with repeat MG detection had an MRM strain at baseline, and therefore, resistance to azithromycin likely contributed to its ineffectiveness in these women. Knowledge of their MG coinfection status and presence versus absence of an MRM strain at the time of CT treatment could have affected treatment choice (use of moxifloxacin instead of azithromycin), which could have helped to prevent MG treatment failure. Also important to note was that 1 of the 6 women with repeat MG detection (with an MRM strain) had a wild-type MG strain at the time of treatment, raising the possibility that azithromycin exposure selected for an MRM that contributed to treatment failure; azithromycin exposure could lead to an MRM in a wild-type strain, and then the resulting MRM strain could become the dominant strain in a mixed infection with a wild-type strain or the only strain that persists. Interestingly, 2 (28.6%) of 7 women with MRM strains became MG negative after treatment, suggesting that either azithromycin treatment was still effective or there could have been spontaneous clearance of MG infection (i.e., immune mediated).

It is possible that a higher MG load at the time of azithromycin treatment could also contribute to treatment failure, which has been reported previously,18 but the sample size of MG-infected subjects in our study was likely too low to be sufficiently powered to demonstrate this association. It is also possible that some of the baseline MG infections were cleared with treatment and the repeat MG detection at follow-up indicates MG reinfection; we did not find significant differences in sexual behaviors in those with versus without repeat MG detection to support this possibility.

Although our study is unique in investigating whether participant characteristics predicted MG coinfection in CT-infected women, there were no significant associations found. This may have in part been due to the small sample size of MG-infected women in our cohort of CT-infected women. Although our study included MG MRM data, we did not investigate quinolone-associated resistance mutations.

In conclusion, our study demonstrated that MG coinfection occurred infrequently in our cohort of CT-infected women; however, when considering findings from other published studies, it seems that prevalence of MG coinfection in CT-infected women may depend on the population that is being investigated. We did not identify participant characteristics associated with MG coinfection in our cohort. However, 29% of CT-infected women with MG coinfection who were treated with azithromycin 1 g for their CT infection had repeat MG detection at follow-up, which likely reflected treatment failure due to their MG strains having MRMs.

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