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Original Studies

Mycoplasma genitalium Infections With Macrolide and Fluoroquinolone Resistance-Associated Mutations in Heterosexual African American Couples in Alabama

Xiao, Li PhD*; Waites, Ken B. MD; Van Der Pol, Barbara PhD, MPH; Aaron, Kristal J. PhD, MPH; Hook, Edward W. III MD; Geisler, William M. MD, MPH

Author Information
Sexually Transmitted Diseases: January 2019 - Volume 46 - Issue 1 - p 18-24
doi: 10.1097/OLQ.0000000000000891

Abstract

Among heterosexual African American couples, Mycoplasma genitalium was detected in 11.2% of men and 12.9% of women. Macrolide and fluoroquinolone resistance-associated mutations were detected in 60.7% and 11.1% of patients, respectively. By M. genitalium positivity status, 27.3% of couples were concordant.

Mycoplasma genitalium (MG), a sexually transmitted pathogen, is a common cause of urethritis and cervicitis1,2 and is associated with pelvic inflammatory disease, infertility, and pregnancy complications.3 Because culturing MG is slow and difficult, molecular MG tests are preferred. Despite several published MG nucleic acid amplification tests,4–7 there are no US Food & Drug Administration approved MG diagnostic tests available. Given the diagnostic challenges, MG treatment is usually empiric and mainly in the context of syndromic management.

The Centers for Disease Control and Prevention recommended first-line antimicrobial for urethritis, cervicitis, and pelvic inflammatory disease cases in which MG is suspected is azithromycin.2 However, MG infection cure rates with azithromycin 1 gram single dose are rapidly declining, averaging 67% since 2009.8 Azithromycin MG treatment failures have been primarily attributed to MG strains with macrolide resistance-associated mutations (MRMs) in the 23S rRNA gene, typically A2071 and A2072 (Escherichia coli numbering 2058 and 2059).9 The frequency of MG strains with MRMs detected in MG infections in the United States based on 2 studies ranges from 42% to 80%,10,11 which is similar to estimates reported outside the United States.12–14 The Centers for Disease Control and Prevention recommended second-line antimicrobial for suspected MG infections, in the case of azithromycin treatment failure, is moxifloxacin.2 However, MG quinolone treatment failures associated with MG quinolone resistance-associated mutations (qRMs) have also been reported.15–17 The MG qRMs are point mutations in the quinolone resistance-determining region of topoisomerase IV gene parC and DNA gyrase gene gyrA.15–17 Limited microbiological evidence has approved that S83I and G81C mutations in ParC are closely related to MG quinolone resistance, whereas the level of minimum inhibitory concentration may be modified by mutations in GyrA.18,19 The frequency of MG strains with qRMs detected in MG infections has been reported to be as low as 5% to 8% in Europe20–22 and as high as 47.1% in Japan.23 We published the only US study evaluating for MG qRMs, finding a prevalence of 29.6% in HIV-infected men who have sex with men with MG infection.11 There are no publications on frequency and mechanisms of MG qRMs in heterosexuals in the United States.

Because African Americans are at higher risk for sexually transmitted infections (STIs) compared with other races/ethnicities,24 and there is limited knowledge on the epidemiology and transmission of STIs within couples, we are conducting a longitudinal prospective study of STIs among African American couples in Birmingham, Alabama, in which the study’s primary objective is to assess STI concordance within couples and elucidating factors influencing sexual transmission of several pathogens. The objectives of the current study were to retrospectively evaluate MG infection prevalence, frequency of MG MRMs and qRMs, and MG infection concordance within couples enrolled in the parent study. Such knowledge could be very important in MG treatment considerations, considering the potential for MG infections with MRMs and qRMs to fail treatment and the limited availability of antibiotics for effectively treating MG infections in the United States.

METHODS

Participants

The study population was comprised of heterosexual couples with individuals aged 19 years or older. African American men presenting to the Jefferson County Department of Health STD Clinic in Birmingham, Alabama for STI evaluation were prospectively enrolled in a longitudinal study of STI concordance among couples in which males were asked to refer their primary female sexual partner for evaluation within 24 hours of enrollment. Primary partner was defined as a partner with whom the male had been having sex for more than 30 days, had sex more often than with other female partners, and planned to continue having sex with in the future. Exclusion criteria included inability to take azithromycin, antibiotic use in the prior 30 days, or immunosuppressive medical conditions (including HIV infection). Additional exclusion criteria for males included concomitant syphilis, chronic urethritis symptoms (>14 days), recurrent diagnoses of nongonococcal urethritis (>3 in the preceding 12 months), symptoms or signs of epididymitis or prostatitis, and having male sexual partners. Additional exclusion criteria for females included pregnancy, breast feeding, or being postmenopausal.

Clinical Procedures

At enrollment, participants were interviewed regarding demographics and medical and sexual history, underwent genital examination, and had the following specimens collected irrespective of whether the corresponding body site was reported to have been exposed in sexual activities: urine (first-catch), swabs from genital (males: clinician-collected urethral swab; females: self-collected vaginal swab), oral (clinician-collected), and anal (clinician-collected) sites. Gram stains were performed on urethral swab specimens (for counting polymorphonuclear leukocytes and looking for intracellular Gram-negative diplococci indicative of presumptive gonorrhea) and wet mounts on vaginal swab specimens (for detection of yeast, trichomonads, and bacterial vaginosis findings). Bacterial vaginosis was diagnosed by Amsel criteria.25 Swabs and urine specimens were tested for Chlamydia trachomatis and Neisseria gonorrhoeae by polymerase chain reaction (PCR) (Roche Diagnostics, Indianapolis, IN) and residual DNA was stored at −80°C. These same specimens were tested for Trichomonas vaginalis by transcription-mediated amplification (Hologic, San Diego, CA), and transcription-mediated amplification results were used for analyses. For this study, we retrospectively performed MG testing (for detection of MG, MRMs and qRMs, and MG genotypes) on DNA (from urine specimens from males and vaginal swab specimens from females, and oral and anal specimens from both males and females) stored frozen at −80°C for 4 to 6 weeks and analyzed clinical data for all couples enrolled between February 2015 and November 2017. The study was approved by the University of Alabama at Birmingham Institutional Review Board and Jefferson County Department of Health.

MG Testing

Mycoplasma genitalium detection was carried out on a Light Cycler 480 II (Roche Diagnostics) using a modified real-time PCR assay (MG macrolide resistance [MGMR] PCR) targeting the 23S rRNA gene with a limit of detection of 2.3 genome copy/μL in the PCR template.26 This quantitative assay also differentiates wild type versus MRMs by melting curve analysis. Briefly, 2 μL of template DNA was tested in a 20-μL PCR reaction mixture containing 0.375 μmol/L of MGMR-F primer, 0.5 μmol/L of MGMR-R primer, 0.1 μmol/L of each probe (MGMR-P1 and MGMR-P2–2), 0.4 U of LightCycler Uracil-DNA Glycosylase and 10 μL of LightCycler 480 Probes Master (both from Roche Diagnostics). quantified mutant and wild type positive controls and a negative control were included in each test. MG quantification was performed using an external standard curve that was established by serial dilutions of positive controls. Amplicons (wild type or mutant) were sequenced using the same PCR primers to verify mutations. Sequences were analyzed using CLC Main Workbench 7 (qiagen, CA).

quinolone resistance-associated mutations were detected by 4 traditional PCR assays amplifying the quinolone resistance-determining region of gyrA and parC, and the partial region of gyrB and parE (see Supplementary Table 1, https://links.lww.com/OLQ/A291).27–29 Nested PCR was performed if first amplicons were not detected by agarose gel. PCR was carried out with a 25 μL PCR reaction volume containing 0.4 μmol/L of each primer, 2.5 μL of 10× AccuPrime Pfx reaction mix and 0.5 U of AccuPrime Pfx DNA polymerase (Thermo Fisher, Fremont, CA). The PCR products were sequenced and analyzed as above.

The MG-positive samples were genotyped by a dual loci scheme, MG191 SNP and MG309 STR, which has been previously recommended for sexual network studies.30 MG191 and MG 309 loci were amplified using MgPa-1/MgPa-3 and MG309-STR-F/ MG309-STR-R primer sets, respectively. Polymerase chain reaction amplicons were sequenced. MG191 sequences were compared to published sequences in NCBI and a sequence type was assigned. The number of short tandem repeats of MG309 was counted. The types of the 2 loci were combined and renamed utilizing a numeric system for purposes of data analysis and presentation.

Statistical Analysis

We determined the prevalence of MG infection and frequency of MRMs and qRMs by gender and across specimen types. Comparisons across groups of categorical variables were based on Pearson χ2 or Fisher exact tests. Non-normally distributed continuous data were compared using a Kruskal-Wallis test. We calculated the crude prevalence ratio (PR) with 95% confidence intervals (CI) for MG infection in partners of enrolled participants who tested MG-positive. All statistical testings were performed using SAS 9.4 (Cary, NC).

RESULTS

Participant Clinical Characteristics

A total of 232 patients (116 heterosexual African American men and their primary female sexual partners) were enrolled in the study. All participants were African American, except for 1 white female. The median participant age was 25.0 years, and men were slightly older than women (median, 26.0 vs. 23.5 years; P = 0.02) (Table 1).

T1
TABLE 1:
Study Participant Characteristics and Association With MG Infection

MG Infection Prevalence and Organism Load

MG was detected in 28 (12.1%) participants: 23 were MG-positive at a single body site and 5 were positive at 2 body sites. MG positivity did not significantly differ by gender (11.2% for men and 12.9% for women) (Table 1). Urethral discharge was reported more often in men who had MG detected (46.2% vs. 19.6%, P = 0.03). MG positivity was also more common among men with gonorrhea (30.8% vs. 9.7%, P = 0.05). Among women, younger age was the only factor associated with a higher frequency of MG positivity (P = 0.001).

From 666 specimens (116 urine, 114 oral swabs, and 101 anal swabs from men; 116 vaginal swabs, 114 oral swabs, and 105 anal swabs from women), 33 (5.0%) were MG-positive (Table 2). The body site-specific MG infection prevalence did not significantly differ by gender (P = 0.14). MG infection prevalence was higher in participants with urogenital symptoms versus those without, though the difference did not reach statistical significance (males, 15.0% vs. 9.3% and females, 15.4% vs. 9.8%; P > 0.3 for both).

T2
TABLE 2:
MG Positivity by Gender and Body Site

The median MG load of all samples was 7.4 genome copy/μL (range, 0.5–5090) in the PCR template. Urogenital samples had a higher MG load (median, 10.7; range, 0.5–5090) than oral (median 7.2 [range 0.9–13.4]) and anal (median 2.0 [range 0.5–266]) specimens, but the difference was not significant (P = 0.31).

Frequency of MRMs and qRMs in MG-Infected Participants

Among 28 MG-positive participants, 17 (60.7%) carried strains with MRMs: 9 men and 8 women (Table 3A). The detected MRMs were A2071G (E. coli numbering 2058) in 12 participants (7 males and 5 females) and A2072G (E. coli numbering 2059) in 5 participants (2 males and 3 females) (see Supplementary Table 2, https://links.lww.com/OLQ/A292). Five couples had the same MRMs (see Supplementary Table 2, https://links.lww.com/OLQ/A292). The frequency of MRMs among MG-infected participants did not significantly differ by gender (69.2% vs. 53.3% in men vs. women) (Table 3A) or body site (Table 3B).

T3
TABLE 3:
Frequency of MG strains With MRMs, QRMs, and both MRMs and QRMs (MDR)

Among 28 MG-positive participants, 3 (11.1%, 1 failed testing) had strains with qRMs: 1 couple (urogenital) and 1 man (oral) (Tables 3A, 3B and see Supplementary Table 2, https://links.lww.com/OLQ/A292). All 3 participants harbored the same parC mutation, C248T (amino acid change S83I), a previously described MG qRM associated with quinolone resistance.19 We found 5 additional mutations in parC or parE that cause amino acid changes (see Supplementary Table 2, https://links.lww.com/OLQ/A292); however, the biological functions of these mutations are unknown. No mutations were found in gyrA or gyrB in successfully tested samples. All strains with qRMs also had MRMs, therefore the frequency of MG strains with both qRMs and MRMs (multidrug-resistant [MDR] MG) in the 28 MG-positive participants was 11.1%.

The MG organism load (genome copy/μL) was significantly higher in MG strains with resistance-associated mutations compared with wild-type strains (median (range): 14.8 [1.70–5090] vs. 1.69 [0.52–203]; P = 0.002).

Couple Concordance

The MG concordance in couples was analyzed initially by MG positivity status, then by MG strain genotype. Among 116 couples, there were 22 (19.0%) in which at least 1 partner was MG-positive: 6 couples (27.3%) were concordant vs. 16 (72.7%) discordant (Fig. 1). Of MG-positive males, 46.2% of their female partners were MG-positive (Fig. 2). Female partners had a 5.3-fold higher MG prevalence if their male partners were MG-positive (PR, 5.28; 95% CI, 2.24–12.44, P < 0.001). Of MG-positive females, 40.0% of their male partners were MG positive (Fig. 2). Male partners had a 5.8-fold higher MG prevalence if their female partners were MG-positive (PR, 5.77; 95% CI, 2.24–14.86, P < 0.001).

F1
Figure 1:
Concordance of MG infection in couples by MG positivity status and strain genotypes.
F2
Figure 2:
MG infection prevalence in participants and their partners, stratified by gender. A, MG infection prevalence in female partners based on the MG positivity status of the male participants. B, MG infection prevalence in male partners based on the MG positivity status of female participants.

Only 19 of 33 samples were successfully genotyped for both MG191 and MG309 loci (see Supplementary Table 2, https://links.lww.com/OLQ/A292). Among the 19 dual-typed samples there were 13 genotypes. Among the 6 MG concordant couples, 4 carried the same genotype and 2 were not successfully genotyped in one of the partners. If both MG positivity status and strain genotypes are considered in determining MG infection concordance, then only 4 (20.0%, 4/20) couples were concordant (Fig. 1).

DISCUSSION

The key findings from our study of MG infection within heterosexual African American couples in Birmingham, AL were that MG infection prevalence was high (12.1%), MG strains with MRMs were common (60.7%), MG strains with qRMs were not uncommon (11.1%), and the concordance of MG infection in couples was low (27.3% by MG positivity and only 20.0% when strain genotypes were also considered).

This study reported the frequency and mechanism of MRMs in MG-infected heterosexual African Americans in the United States. Compared with MG-infected African Americans in the Getman et al10 study, we found a similar frequency of MRMs in females (53.3% vs. 57.8%) but a higher frequency of MRMs in males (69.2% vs. 45.4%). These differences in frequency of MG strains with MRMs between the cohorts may reflect geographical differences in resistance rates and differences in prior azithromycin exposure in the study populations. Our smaller sample size could have also contributed to the difference. It has been reported that MRMs develop in about 10% of MG-infected patients after receiving azithromycin, most MG infection treatment failures are associated with MG strains containing MRMs, and longer azithromycin treatment courses do not improve the MG infection cure rate in strains with MRMs.31s Thus, azithromycin may no longer be an effective first-line treatment for MG infections. Since doxycycline is also ineffective against MG infections, this leaves the fluoroquinolone moxifloxacin as the best MG treatment option in the United States.

This study found that 11.1% of MG strains had qRMs in MG-infected heterosexuals in the United States, which is markedly lower than the frequency of MRMs. We recently reported that 29.6% of MG infections in HIV-infected men who have sex with men in Birmingham, Alabama had strains with qRMs.11 Two Canadian studies also showed similar frequencies (12.2%–20%) of qRMs in MG infections.12,32s All MG infections with strains carrying qRMs in our study harbored the ParC mutation S83I, a SNP associated with moxifloxacin resistance.19 We also detected other mutations in parC and parE that were nonsense or caused amino acid changes, but the impact of these mutations on MG treatment outcomes is unknown. All MG infections with strains carrying qRMs in our study also had MRMs, thus the frequency of potentially MDR strains among MG infections was 11.1%, higher than rates reported in Canada21 and European countries20,22 (<5%), similar to the frequency in Australia (8.6%),33s and lower than the frequency in Japan (30.8%).34s In the United States, there are limited other drugs available for treating the potentially MDR MG infections. An extended minocycline regimen has been shown to be effective in some cases in Japan,17 whereas doxycycline eradicates MG in only about 30% of the cases.35s

One of our study objectives was to examine MG infection concordance among couples, which could provide insight into MG transmission and the natural history of infection. Concordance was initially analyzed based on MG test positivity of each partner and 27.3% of couples were concordant. Taking into account MG strain genotypes, the MG concordance frequency was only 20.0%. It is interesting that 13 of the 33 MG-positive specimens had different MG genotypes, suggesting considerable MG strain diversity in our cohort. Our results also agree with previous reports that this dual typing system has the discriminatory power to be useful in sexual networks studies.31s,36s However, the concordance of this study was low compared to the previous studies using single locus or multi loci sequencing typing methods.37s,38s When considering risk of MG transmission from an infected participant to their partner, we found that the female partners were over 5 times more likely to be MG-positive if the male participant was MG-positive. From the perspective of female participants, male partners were also over 5 times more likely to be MG-positive if the female participant was MG-positive. Although these risk data may be overestimated, as individuals could have been infected from other casual partners, the trend of transmission risk is in agreement with 2 other studies that enrolled patients including African American females and their male partners showed that the male partners were 5- to 9-fold more likely to be MG-positive if the female participant was MG-positive.39s,40s These previous concordance studies only focused on MG positivity status and did not incorporate MG strain typing, which could have further discriminated concordance. It is interesting that the MG infection concordance rate was much lower than reported chlamydia concordance rates, which ranged from 68–76%.41s,42s Because only 58% of specimens could be successfully genotyped, our concordance analysis had limited power and it was challenging to interpret the significance of the proportion of couples concordantly infected with the same genotype; the low rate of successful genotyping was in part due to using archived frozen specimens in this study, the unknown sensitivity of genotyping PCRs, and our requiring both MG191 and MG309 loci be successfully genotyped to ensure sufficient discriminatory ability for concordance. It is possible that MG is biologically less transmissible than C. trachomatis per sexual encounter, an explanation for which there are no data to our knowledge. It is also possible that MG infections have a much shorter duration than chlamydia prior to detection or resolution. Findings from the 2 studies on the natural history of chlamydia suggest that about 50% of chlamydial infections may persist for 1 year after initial detection.43s,44s In contrast, 3 studies of the natural history of MG infection in females prior to 2018 showed that less than half of infections persisted longer than 3 months after initial detection39s,45s,46s and 2 more studies of MG infection in females published in 2018 showed about 80% of infections were cleared by 12 months after detection.47s,48s Thus, the natural history of MG infection, with respect to infection duration, is likely a major factor influencing infection concordance among couples. Studies on immune responses that mediate MG clearance are needed to better understand MG infection concordance and clearance. An improved understanding of the duration of MG infections will be useful in development of MG testing recommendations and in treatment decisions. For example, if MG screening becomes recommended in the future, the frequency of screening would likely be more often than annually (at least in higher prevalence populations) since most infections have cleared by 12 months. Also, if MG concordance rates are low and infection duration short, further consideration may be given as to whether contacts of MG-infected patients should be treated vs. whether there is a role for basing partner treatment on results of MG testing.

Our study evaluated MG infection prevalence, frequency of MG strains with MRMs and qRMs, and MG infection concordance only in heterosexual African American couples in Birmingham, AL. Findings could differ in populations of different races/ethnicities or geographic locations and in same sex partnerships. Because of the relatively small number of MG infections, the precision of our estimates on frequency of MG MRMs and qRMs and MG infection concordance may be limited. Because some individuals in our couples may have had casual partners, for which data were not collected in the study, this could have confounded the MG infection prevalence and concordance rate. It is also possible that presence of other STIs in our couples could confound concordance by affecting the natural history of MG infection; however, our study was not designed to sufficiently address this possibility. Because males with persistent or recurrent urethritis were already excluded from the longitudinal study that was the source of specimens and data for this retrospective study, our MG infection prevalence estimates may be lower than if we had included those males because studies suggest MG infection prevalence is higher in men with recurrent or persistent urethritis.2

In summary, we found a low frequency of MG infection concordance in heterosexual African American couples seen at a STD clinic in Birmingham, Alabama, which has implications in MG testing and treatment considerations. Among MG-positive participants, the majority had strains with MRMs and detection of qRMs was also not uncommon; all MG strains with qRMs also had MRMs. This finding of MG infections with potentially MDR strains (based on gene mutations) should sound the alarm that efficacious treatments may not be available for some MG-infected patients in the United States and therefore new MG treatments and/or treatment strategies (eg, dual therapy) are urgently needed. There is also a need for a MG surveillance program in the United States for monitoring frequency of MRMs and qRMs.

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