Mycoplasma genitalium is the second most common cause of nongonococcal urethritis (NGU) after Chlamydia trachomatis accounting for up to 30% of NGU in men.1–5 M . genitalium -positive NGU (M. genitalium NGU) is more common in men with only female sexual partners (MSW) in Australia.3,4,6–8
Antimicrobial resistance is now a major problem in the treatment of M . genitalium infections.5,9–13 M. genitalium strains harbored macrolide resistance-associated mutations (MRM), and MSM were twice as likely to be infected with resistant strains compared with MSW.14 M. genitalium has been reported to be 79.4% among MSM in Sydney, including 87.5% in urethral infections, and 63.6% in banked specimens from men and women in Queensland.11,13 Mycoplasma genitalium MRM prevalence has been increasingly reported internationally, including in Spain (35%), the United States (48%), and Canada (57%).5,15,16 9 5,9,12,17
Australian and European guidelines have recently been updated to replace azithromycin with doxycycline for first-line therapy for NGU due to concern that the widespread use of azithromycin for syndromic management can induce macrolide resistance.18–22
The last published study of M. genitalium NGU in Sydney, Australia, reported an overall M. genitalium prevalence of 5.2% and found that infection was much more common in MSW compared with MSM.4 M. genitalium and M. genitalium MRM in first-void urine (FVU) specimens from men with NGU and to investigate associations with infection or macrolide resistance, including differences between MSM and MSW.
MATERIALS AND METHODS
Study Population
Sydney Sexual Health Centre and Western Sydney Sexual Health Centre provide comprehensive testing and management of STIs, human immunodeficiency virus (HIV) and viral hepatitis to at-risk priority populations in metropolitan Sydney. Between April 2017 and May 2018, consecutive men 16 years or older presenting with symptoms of acute NGU were prospectively enrolled by the attending clinician. Acute NGU was defined as having symptoms of urethral discharge or dysuria for less than 1 month. Urethral Gram stain microscopy was conducted in eligible men with signs of urethral discharge but microscopic confirmation (ie, ≥5 polymorphonuclear leukocytes/high-powered field on urethral Gram stain) was not a requirement in this study because local guidelines support the diagnosis of NGU in symptomatic men with dysuria in the absence of signs of urethral discharge.
Men with presumptive urethral gonorrhea based on Gram-negative intracellular diplococci in urethral smear samples at the point of testing or a positive Neisseria gonorrhoeae PCR were excluded, as were those with a previous diagnosis of M. genitalium at any site and those with persisting or recurrent NGU.
Participants were offered comprehensive STI and HIV testing, and management of positive M. genitalium results was guided by the resistance assay result and local clinical guidelines for treatment, test of cure and partner notification.
Clinical Methods
The following data were collected at enrolment: age, condom use for anal or vaginal sex in the past 3 months, sex and number of partners in the past 3 months, treatment of C. trachomatis or N. gonorrheae infection with a regimen including azithromycin 1 g in the past 12 months, HIV serostatus, current use of HIV preexposure prophylaxis (PrEP) and the presence of urethral signs and symptoms. Participants with symptoms of urethral discharge were examined and urethral discharge specimens were collected for Gram stain microscopy by the attending clinician during the consultation. Nongonococcal urethritis was diagnosed on the basis of having symptoms of urethral discharge or dysuria and the absence of N. gonorrhoeae . FVU specimens were transported to the microbiology laboratory, stored at 2°C to 8°C and processed within 7 days of collection.
Laboratory Methods
The ResistancePlus MG assay (SpeeDx) is a multiplex quantitative (quantitative polymerase chain reaction) assay that uses novel PlexZyme and PlexPrime technology to simultaneously detect M. genitalium and five 23S rRNA mutations associated with macrolide resistance: A2058C, A2058G, A2058T, A2095C, and A2059G.20 M. genitalium that harbors no resistance mutations. Assay sensitivity and specificity for the detection of M. genitalium and the detection of macrolide resistance-associated mutations have been previously reported.23 11
Data analysis to identify the presence or absence of M. genitalium , 23S rRNA mutation and internal control was performed using the FastFinder ResistancePlus MG LC480 (UgenTec) analysis software.
Chlamydia trachomatis and N. gonorrhoeae nucleic acid amplification test testing of first FVU was performed by the clinics' local laboratories using the Cobas 6800 system (Roche, Mannheim, Germany) for patients from Sydney Sexual Health Centre and the Viper system with XTR technology (BD Diagnostics, Franklin Lakes, NJ) for patients from Western Sydney Sexual Health Centre.
Statistical Methods and Analysis
A sample size of 616 was selected to detect a 5% difference in the proportion of M. genitalium detected in MSM compared with MSW, assuming 80% power at a significance value of 0.05. Data were entered into Microsoft Excel and analyzed to investigate any associations with M. genitalium infection or the presence of macrolide resistance mutations using Stata Statistical Software: Release 12 (StataCorp, College Station, TX). Relationships between variables were first examined using the χ2 test for independence or Fisher exact tests. The associations between study factors and presence of M. genitalium and of M. genitalium MRM were evaluated using log-binomial regression with a significance level of 5%. We report unadjusted and adjusted risk ratios (RRs) with the corresponding 95% confidence intervals (95% CIs). Variables significantly associated with the outcome of interest in unadjusted regression models were selected for multivariate model building, which was conducted using a forward step-wise technique.
RESULTS
Study Population
A total of 616 men aged 19 to 70 years were consecutively enrolled in the study. Of these, 28 were excluded: 27 because their N. gonorrhoeae PCR test results were positive and one because the urine M. genitalium PCR failed to give a result due to inhibition. Thus, 588 participants were included. All men who met study criteria were included in the study. Informed patient consent was not required as we used standardly collected data and routine tests.
Median age was 30 years (interquartile range, 26–37 years). Overall, the majority of participants (n = 480, 81.6%) reported having condomless anal or vaginal sex in the preceding 3 months, approximately half (n = 306, 52.0%) reported having male sexual partners and 102 (17.5%) were taking HIV PrEP. Twenty-three percent (n = 135) of participants tested positive for C. trachomatis and 24% (n = 141) had received azithromycin treatment in the preceding 12 months. The HIV antibody test results were available for 501 of 588 participants of which 10 (2.0%) were HIV seropositive. Participant characteristics were similar between clinics in terms of age, number of partners and sex of partners.
Prevalence of Infection and Macrolide Resistance-Associated Mutations
The prevalence of M. genitalium was 12.8% (75 of 588) overall and 12.8% in both MSW and MSM (Table 1 ). Macrolide resistance-associated mutations were present in 70.7% of participants overall, including 50.0% of MSW and 89.7% of MSM (Table 2 ). Prevalence of M. genitalium and of M. genitalium MRM was similar for the two clinics (P = 0.141 and 0.195, respectively). Chlamydial coinfection was present in 5.9% of men with M. genitalium infection .
TABLE 1: Associations With M. genitalium Infection
TABLE 2: Associations With M. Genitalium Macrolide-Resistant Mutations
Associations With M. genitalium and C. trachomatis Infection
Associations between M. genitalium infection and demographic, behavioral and clinical variables are presented in Table 1 . Men with M. genitalium infection were significantly less likely to be infected with C. trachomatis but there were no significant associations between M. genitalium infection and age, number of sexual partners, condom use, azithromycin treatment in the last 12 months, HIV serostatus, or PrEP use.
Infection with C. trachomatis was significantly less likely among MSM compared with MSW (RR, 0.68; 95% CI, 0.50–0.91; P = 0.011). Overall, there was no significant association between age (≥30 years compared with ≤29 years and chlamydial infection (RR, 0.93; 95% CI, 0.69–1.25; P = 0.639), including for MSM (RR, 1.47; 95% CI, 0.88–2.47; P = 0.144) and MSW (RR, 0.77; 95% CI, 0.52–1.15; P = 0.205) when the groups were examined separately, although the numbers in separate groups may not have been large enough to detect any difference.
Associations With M. genitalium Macrolide Resistance
Factors associated with presence of MRM among men with M. genitalium infection on unadjusted analysis are presented in Table 2 . The MSM were significantly more likely to have macrolide-resistant M. genitalium infections than MSW. Men 30 years and older were also more likely to have M. genitalium MRM, as were those who did not always use condoms for anal or vaginal sex. Prevalence of MRM was not associated with treatment with azithromycin in the last 12 months. On multivariate analysis, having male sexual partners compared with only female sexual partners was the only independent predictor of MRM (RR, 1.55; 95% CI, 1.02–2.38; P = 0.042).
DISCUSSION
The present study confirms that M. genitalium infections are common in men with NGU. Key findings are that nearly 90% of M. genitalium infections among MSM with NGU are macrolide-resistant and that macrolide resistance is significantly more prevalent among MSM compared with MSW. In contrast to other studies, we did not find an association between macrolide-resistant M. genitalium and recent use of azithromycin, most likely because macrolide-resistant strains are now very commonly transmitted and acquired.17
We also found that M. genitalium infection among men with NGU is now equally likely among MSM and MSW in Sydney, whereas most local infections were previously among MSW.4 M. genitalium to cases of NGU has also increased locally. In cases of NGU in 2006 to 2007, M. genitalium was detected in 4.5%, and C. trachomatis in 18.7%, whereas in 2017 to 2018, M. genitalium was detected in 12.8% and C. trachomatis in 23.0%.4
This study is valuable for estimating the current proportion of NGU associated with M. genitalium in our population and potentially in regard to other populations, for demonstrating change in the pattern of infection over the last 10 years (particularly in MSM), and the high and disparate rates of macrolide-resistant infections among MSM and MSW.
The findings of this study have several implications for M. genitalium testing, treatment, and surveillance. The results provide further evidence to test all men with NGU for M. genitalium , preferably using an assay which identifies resistance, to optimize initial antibiotic treatment and contact management. Read et al24 M. genitalium, resulted in cure of ≥92% infections, and reduced the expected rate of macrolide resistance selection during treatment.
There is little likely benefit and potential harm in continuing to use azithromycin in the syndromic management of NGU given that most M. genitalium infections in MSM, and half of those in MSW, harbor macrolide resistance mutations. There is an urgent need to reduce antibiotic resistance selective pressure by minimizing the use of azithromycin in general, and in particular among MSM, who are more likely to be exposed to azithromycin because they test more frequently and have higher rates of sexually transmissible infections compared to the general population. Real-time testing for resistance is the only reliable way to guide appropriate treatment choices, and monitor changing patterns of resistance.24
In light of these findings and to prevent the onward transmission of resistant strains, men with M. genitalium NGU should be advised to abstain from sex until they and their current partner/s have completed treatment and had a negative tests of cure.25 M. genitalium may be more common in people with HIV however there was no association between HIV seropositivity and M. genitalium in our study.25
Strengths of this study are that it was large, prospective and multicenter but there are several limitations. First, we did not use a control group to measure the presence of M. genitalium or macrolide resistance in asymptomatic men. M. genitalium is an accepted cause of NGU, and the main focus of our study was to investigate differences between MSM and MSW, including differences in M. genitalium macrolide resistance. A recent study of M. genitalium prevalence among MSM in western Sydney found that most urethral M. genitalium infections were asymptomatic, and that most infections were macrolide-resistant.11 22
Second, the increased prevalence of M. genitalium among men with NGU in this study compared to the previous study at the same clinics may partly be attributable to the different inclusion criteria.4 M. genitalium infection compared to studies that included men with urethral irritation only. However, the large increase in overall prevalence, from 4.5% 10 years ago to 12.8% today, is unlikely to be solely due to differences in inclusion criteria because M. genitalium infection among MSM with NGU was rare in the previous study and is now equivalent to MSW suggesting an increase in M. genitalium prevalence in our local MSM population and overall. It is also unlikely that the increase in prevalence is due to differences in the detection method because in-house evaluations found the sensitivities of the previous and current M. genitalium to be highly comparable.
In this study, microscopy was not done or there was insufficient sample to be tested in 41% of cases and only 65% of those with urethral smear Gram stain results had microscopic urethritis (≥5 polymorphonuclear leukocytes/high-powered field). Microscopic confirmation of NGU was not an inclusion requirement for this study and this should be considered when interpreting our results in relation to other studies.
HIV testing was not a protocol requirement for the study and 81 of 588 participants were not tested at study enrolment, either because a test was not indicated, was declined, or there was a history of recent testing elsewhere.
Lastly, although multidrug-resistant M. genitalium is on the rise, we did not measure resistance to second-line fluoroquinolone therapy in this study.9,12,26
In conclusion, M. genitalium is an increasingly common cause of NGU. Almost 90% of MSM and half of MSW with M. genitalium NGU have macrolide-resistant infections. Our findings support recommendations to test all men with NGU for M. genitalium , to incorporate real-time resistance assays into clinical care to guide treatment decisions and to limit use of azithromycin when treating NGU and other STIs.6,26
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