Mycoplasma genitalium Co-infection In Women with Chlamydia trachomatis Infection

Abstract

We evaluated the prevalence of Mycoplasma genitalium co-infection in 302 chlamydia-infected women seen at a sexually transmitted disease clinic in Birmingham, Alabama. M. genitalium co-infection was detected in 22 (7.3%). No participant characteristics predicted co-infection. Among co-infected women, M. genitalium was detected again in 6 of 21 (28.6%) women returning for a 3-month follow-up visit after azithromycin treatment.

Short Summary

A study of Mycoplasma genitalium co-infection among 302 chlamydia-infected women seen at a sexually transmitted disease clinic found co-infection in 22 (7.3%). Repeat M. genitalium detection at a 3-month follow-up visit occurred in 6 of 21 (28.6%) women.

INTRODUCTION

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 (FDA)-cleared diagnostic test for MG in the U.S., a nucleic acid amplification test (NAAT) that was FDA-cleared in January 2019; however, there are also other research-based MG diagnostic NAATs available to some providers for MG testing. Due to 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 co-infections 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 co-infection 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 co-infection in CT-infected women and MG outcomes in women with CT and MG co-infection 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 1g 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 U.S. reported about half of MG-infected women had MG strains with MRMs.^3,14

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

MATERIALS & METHODS

Testing for MG and the MRM in the 23S rRNA gene was performed by a real-time PCR (MGMR PCR) on stored DNA samples isolated from cervical swab specimens that had been collected from a previously enrolled study cohort¹⁵ from March 2012 through 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 was comprised of CT-infected women ≥16 years of age seen at a sexually transmitted disease (STD) clinic in Birmingham, AL, USA 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 1g directly observed therapy for CT treatment. Analyses were performed with SAS software, version 9.4 (SAS Institute, Cary, NC). We used Fisher’s exact, Pearson’s chi-square, and Wilcoxon rank sum tests, as appropriate, to evaluate the association of participant characteristics (demographics, sexual history, prior STIs, hormonal contraceptive use, symptoms, clinical syndromes, and concomitant vaginal infections) with MG co-infection. 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 (PID) 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 (genome copy [GC]/uL) at baseline with repeat MG detection at follow-up. Missing values were excluded from the analysis. P <0.05 was considered statistically significant.

RESULTS

MG testing was performed on samples from 302 CT-infected women with the following characteristics (Table 1): 93% African American; median age of 22 (range 16–50); 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 PID; and 28.8% with bacterial vaginosis, 12.6% with vulvovaginal candidiasis, and 3.6% with Trichomonas vaginalis infection. MG was detected in 22 (7.3%) women. None of the above participant characteristics, including presence/absence of symptoms, were significantly associated with MG co-infection (Table 1).

Table 1.

Baseline Characteristics of Participants (N=302) by Mycoplasma genitalium (MG) Status

	Total (N = 302)	MG Positive (N = 22)	MG Negative (N = 280)	P-value
Age, median (range)	22 (16–50)	24 (16–34)	22 (16–50)	0.24*
African American, N (%)	281 (93.1%)	21 (95.5%)	260 (92.9%)	1.00†
Hispanic, N (%)	6 (2.0%)	0 (0%)	6 (2.1%)	1.00†
Asymptomatic, N (%)	158 (52.3%)	11 (50.0%)	147 (52.5%)	0.82‡
Genital bleeding, N (%)	61 (20.2%)	6 (27.3%)	55 (19.6%)	0.39‡
Prior chlamydia, N (%)	147 (48.7%)	11 (50.0%)	136 (48.6%)	0.91‡
Sexual partner number last 3 months, median (range)	1 (1–10)	1 (1–7)	1 (1–10)	0.47*
Hormonal contraceptive use, N (%)	127 (42.1%)	10 (45.5%)	117 (41.8%)	0.80‡
Concomitant infections
Candidiasis	38 (12.6%)	3 (13.6%)	35 (12.5%)	0.75†
Bacterial vaginosis	87 (28.8%)	6 (27.3%)	81 (28.9%)	0.87‡
Trichomoniasis	11 (3.6%)	1 (4.6%)	10 (3.6%)	0.57†
Pelvic inflammatory Disease	8 (2.7%)	1 (4.6%)	7 (2.5%)	0.46†
Cervicitis	57 (18.9%)	5 (22.7%)	52 (18.6%)	0.57†

^*= Wilcoxon rank sum test;

^†= Fisher’s exact test;

^‡= Chi-square test of independence

N=6 missing for hormonal contraceptives (all in MG negative group)

N=1 missing for genital bleeding (from MG negative group)

N=1 missing for prior CT (from MG negative group)

N=1 missing for candidiasis (from MG negative group)

N=2 missing for cervicitis (N=1 missing from MG positive group and N=1 missing from MG negative group)

We next evaluated frequency of MG detection at a 3-month follow-up visit in subjects with MG co-infection at baseline (at which time all received azithromycin 1g for CT treatment). Of the 22 participants with MG co-infection at baseline, 14 were determined have wild type MG strains. The remaining 8 had strains with MRM: 1 was A2071G (E. 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 (Figure 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 vs. 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 vs. 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 vs. 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 (GC/uL) was higher in the 6 participants with MG detected at the follow-up visit vs. 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)

Figure 1.

Outcome of Mycoplasma genitalium (MG) co-infection in a cohort of women with Chlamydia trachomatis (CT) infection who were treated with azithromycin (AZM) 1 gram, stratified by the presence of a macrolide-associated resistance mutation (MRM) vs. no mutation (Wild Type) in the MG strain.

DISCUSSION

Our study found that MG co-infection in CT-infected women seen in a STD clinic in Birmingham was uncommon, only occurring in 7.3%, which was lower than we anticipated. In two previous studies that evaluated MG co-infection in CT-infected women in STD clinics in U.S. cities, the MG-coinfection frequency was 25% (12 of 48 women) in one study¹¹ and 36.1% (13 of 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 co-infection (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 1g, a first-line urogenital CT infection treatment, in eradicating CT and MG in co-infected women. Among co-infected 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 due to azithromycin treatment failure rather than MG reinfection. Thus, azithromycin 1g, a current first line recommended CT treatment, was ineffective in eradicating MG infection in a significant number of CT-infected women with MG co-infection, 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 co-infection status and presence vs. 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 a 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 of 7 (28.6%) women with MRM strains became MG negative after treatment, suggesting 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 vs. without repeat MG detection to support this possibility.

While our study is unique in investigating whether participant characteristics predicted MG co-infection 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. While our study included MG MRM data, we did not investigate quinolone-associated resistance mutations.

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