Abstract
Background: Mycoplasmoides genitalium remains a difficult sexually-transmitted infection (STI) to manage due to its potential for antimicrobial resistance and post-infection sequelae. University students are especially vulnerable, as this demographic has the highest rate of STI in the United States. As a result, investigating prevalence rates and therapeutic outcomes in this population is essential to minimize future impact of M. genitalium. The purpose of this study was to investigate a university student population for M. genitalium distribution and treatment outcome.
Design: Retrospective chart-review of university health clinic attendees, augmented by laboratory detection of M. genitalium following therapeutic intervention.
Methods: A total of 1617 student encounters at a midwestern United States university health clinic over a 28-month interval from November 2017 through February 2020 were analyzed for M. genitalium and Chlamydia trachomatis positivity rates and prevalence. Detection of these sexually-transmitted pathogens occurred by commercial RNA amplification testing. Chart review was focused on participant outcomes following initial M. genitalium detection and therapeutic intervention.
Results: C. trachomatis positivity and prevalence rates were 7.05% and 9.00%, respectively, while analogous rates for M. genitalium were 7.05% and 6.51%, respectively. An average of 1.83 positive results was generated from participants infected with M. genitalium at any time, with an average of 1.17 positive results for C. trachomatis (P < 0.0002). For students treated with azithromycin, 30.3% generated a negative M. genitalium result upon follow-up, with 1g daily and 2-day 500mg dosing regimens demonstrating less efficacy than a 4-day 250mg regimen or moxifloxacin.
Conclusion: Data indicate a need for molecular M. genitalium macrolide resistance determination from primary specimens in the university setting.
Individuals aged 15-24 years account for approximately half of the 26 million newly-diagnosed sexually-transmitted infections (STI) in the United States on an annual basis.1,2 Data from the United States Centers for Disease Control and Prevention (CDC) have revealed that the Milwaukee-Waukesha-West Allis (Wisconsin) Metropolitan Statistical Area produces Chlamydia trachomatis and Neisseria gonorrhoeae infection rates per 100,000 population that each rank within the top five nationally.3 Furthermore, surveys of both male4 and female5 demographics within the Milwaukee metropolitan area have documented detection rates of the STI agent Mycoplasmoides genitalium which either rival or exceed those of C. trachomatis.
M. genitalium remains a difficult STI to manage due to its potential for antimicrobial resistance6 and post-infection sequelae.7,8 Complications of M. genitalium infection in females (namely cervicitis, pelvic inflammatory disease, spontaneous abortion, preterm delivery, and infertility) have demonstrated stronger statistical associations8 than sequelae in males. University students potentially become vulnerable to these outcomes, as this demographic exhibits the highest rate of STI in the United States. As a result, investigating prevalence rates and therapeutic outcomes in this population is essential to minimize future impact of M. genitalium infection.
A previous assessment of STI detection within Milwaukee area university students, performed by nucleic acid amplification testing on primary first-void urine or genital swab specimens, revealed significant incidence rates of both C. trachomatis and M. genitalium.9 In light of potential limitations relative to efficacious treatment of M. genitalium infection, we investigated a surrogate of microbiologic outcome (i.e., organism-specific RNA detection) in a subset of university students following therapeutic intervention for M. genitalium infection.
Methods
Participants and Enrollment
Under the auspices of a Marquette University Institutional Review Board-approved protocol, university students voluntarily consented to enrollment in and authorization to use or disclose protected health information in research for a cost-free STI screening program at a university health clinic. University enrollment approximated 10,000 undergraduates (~55% female; ~30% non-Caucasian; ~2% international). Signed forms were maintained in the participant health record. Participants were also advised that no financial compensation would be available.
Specimens and Testing
Single collections of first-void urine (both genders), self- or provider-collected vaginal swabs, or endocervical swabs were procured within the clinic setting. Swabs were placed into Aptima transport media (Hologic, Incorporated; San Diego, CA) at time of collection; urine was aliquoted to Aptima urine transport tubes (Hologic) at the clinic. Specimens were maintained at 2-30°C and tested within 30 days of collection, per package insert guidelines.
Molecular Detection of STI Agents
Transcription-mediated amplification-based detection of a C. trachomatis-specific 23S ribosomal (r)RNA genetic sequence was performed using the Aptima Combo 2 Assay; detection of a M. genitalium-specific 16S rRNA genetic determinant was performed using the Aptima Mycoplasma genitalium Assay (both from Hologic). All testing was performed via direct tube sampling on the Panther System (Hologic). The analyzer generated qualitative interpretive data for both STI analytes.
Data Analysis
Over a 28-month interval from November 2017 through February 2020, C. trachomatis- and M. genitalium-specific positive results were translated into positivity rates (on the basis of all testing performed) and prevalence rates (on the basis of each enrolled participant). Only a single positive test result (and single test performance) contributed to C. trachomatis and M. genitalium prevalence data. Participants yielding detectable M. genitalium rRNA were subject to retrospective chart review to ascertain treatment outcome on the basis of follow-up Aptima Mycoplasma genitalium Assay testing. The significance test of proportions was used to determine if comparative proportion or frequency value differences were significant. Differences in testing interval data were analyzed by the t-test for independent samples. An a priori decision was made to examine statistical significance using a two-tailed test with an alpha level of 0.05.
Results
Differing C. trachomatis and M. genitalium Prevalence Rates Within University Students When Compared to Detection Rates
A total of 1617 specimens were submitted for commercial transcription-mediated amplification testing during the investigation. Encounters involved 63% female participants and an overall provider-assessed symptomatic rate of 36.1% (41.7% in females; 26.6% in males). Approximately 13% of participants reported same-gender sexual practices. The overall positivity rate for either C. trachomatis- and M. genitalium-specific rRNA was 7.05% and did not differ when stratified by gender (P ≥ 0.33; Table 1). A total of 1167 unique participants were identified in the investigation. The overall C. trachomatis prevalence rate was 9.00%. The M. genitalium prevalence rate (6.51%) was lower than its positivity rate, with significantly lower prevalence in males (5.42%) compared to that for C. trachomatis (P = 0.03; Table 1).
Transcription-mediated amplification-based Chlamydia trachomatis and Mycoplasmoides genitalium positivity and prevalence rates in a university student population, stratified by gender.
Within 54 multi-encounter study participants with detectable C. trachomatis-specific rRNA during the study period, 85.2% yielded a single positive test result (Figure 1). In contrast, 63.0% of the 46 multi-encounter participants with detectable M. genitalium-specific rRNA were positive on more than one encounter (P < 0.0002 versus study participants yielding more than one positive C. trachomatis test result). Individual participants known to harbor C. trachomatis rRNA had an average of 1.17 positive test results throughout the course of the study, while individual participants known to have detectable M. genitalium rRNA yielded an average of 1.83 positive test results (P < 0.0002).
Percentages of Chlamydia trachomatis (blue) or Mycoplasmoides genitalium (red) rRNA-positive participants yielding one or more positive test results throughout the course of a university sexually-transmitted infection screening program.
Treatment Outcome and Post-Treatment Testing of Participants With Detectable M. genitalium rRNA
Participants that were prescribed therapeutic regimens following a positive M. genitalium rRNA test result, then re-tested for M. genitalium rRNA upon follow-up, were subject to chart review. These criteria were met by 30 participants, receiving a total of 33 regimens of azithromycin therapy. Follow-up testing of participants occurred at a mean of 129.9 days after initial encounter (median 100 days; range 7 to 424).
Four-day courses of 250mg azithromycin therapy resulted in M. genitalium rRNA clearance in 42.9% of participants (Figure 2); 2-day courses of 500mg azithromycin resulted in rRNA clearance in 29.4% of participants; no follow-up rRNA clearance was observed in participants receiving either a single 1g or 2g azithromycin regimen. In contrast, administration of an initial 500mg azithromycin dose, followed by 250mg doses for 4 additional days, demonstrated M. genitalium rRNA clearance at follow-up. No significant difference in mean follow-up testing intervals was noted between participants with M. genitalium rRNA clearance (144.0 days) versus detectable M. genitalium rRNA (123.8 days; P = 0.63).
Mycoplasmoides genitalium clearance (denoted by transcription-mediated amplification-based testing) following therapeutic regimens initiated by a detectable M. genitalium rRNA test result, stratified by azithromycin dosing regimen.
Participants (n = 16) receiving moxifloxacin therapy (400mg qd for 7 days or 10 days) had a M. genitalium rRNA clearance rate of 75.0%. This compared favorably to a 33.3% rRNA clearance rate for six participants receiving doxycycline (100mg either qd for 7 days or bid for 10 days) and a 30.3% clearance rate following the cumulative 33 regimens of azithromycin (Figure 3).
Mycoplasmoides genitalium clearance (denoted by transcription-mediated amplification-based testing) following therapeutic regimens initiated by a detectable M. genitalium rRNA test result, stratified by class of antimicrobial agent.
Discussion
The prospect of laboratory detection of M. genitalium infection has improved with the advent of molecular diagnostics,10,11 from which three assays have received Food and Drug Administration clearance in the United States.12-14 Performance of one of these assays within a university student population has been described.9 In our current investigation, we demonstrate M. genitalium prevalence rates to be lower than positivity rates (and also lower than C. trachomatis prevalence data). These findings appeared to reflect an increased number of participants testing positive for M. genitalium upon multiple encounters (Figure 1). Odds ratio data from a longitudinal men-who-have-sex-with-men (MSM) cohort15 revealed that an initial detectable M. genitalium rectal swab or first-void urine result was more predictive of a subsequent positive result from the same specimen source when compared to similar analyses relative to C. trachomatis. Within the MSM cohort, differences reported at 6-month follow-up reflected three- to four-fold increases in odds ratios (i.e., repeated M. genitalium detection versus repeated C. trachomatis detection), while a greater than two-fold increase was demonstrated at 12-month follow-up.15
Both the increased odds ratios described in the MSM cohort15 and the propensity of participants in our study to repeatedly test positive for M. genitalium rRNA may suggest non-efficacious therapeutic intervention. Prior to 2021, therapeutic management of M. genitalium infection largely defaulted to guidelines for syndromic management of cervicitis and urethritis16 due to diagnostic challenges that were antecedent to molecular diagnostics. Antimicrobial regimens largely centered around a recommended 1g single dose of azithromycin,17,18 though published data have reported only an approximate 40% microbiologic cure rate.19 Extended dosing has been investigated, involving a 500mg initial azithromycin dose followed by 250mg daily for 4 days, and shown to be marginally superior to the 1g single dose azithromycin.20-22 Wood et al.23 reported that treatment failures with the 1g azithromycin dosing regimen were associated with macrolide-resistant strains of M. genitalium.
In our study, permutations of azithromycin treatment demonstrated variable efficacy in producing M. genitalium rRNA clearance. Single-dose regimens resulted in no rRNA clearance whereas multi-dose regimens showed increasing rates of rRNA clearance. However, overall limited efficacy of azithromycin (30.3%; Figure 3) suggests the increased potential of macrolide-resistant M. genitalium causing urogenital infection in this university setting. Indeed, data have chronicled increased macrolide resistance rates within M. genitalium on a worldwide basis. Earlier reports documented 40-60% macrolide resistance rates in Western Europe.24-26 In North America, contemporary data have reported 45% macrolide resistance in the context of female urogenital infection27 and 70% resistance in one study of heterosexual couple urogenital, pharyngeal, and rectal specimens.28
As a result, the latest CDC STI treatment guidelines for M. genitalium29 are algorithm-based and largely incumbent on access to macrolide resistance data derived from primary clinical specimens. Scenarios in which macrolide resistance determination is unavailable or in which M. genitalium macrolide resistance is detected within a primary clinical specimen are to be managed with 100mg doxycycline PO bid for 7 days, followed by 400mg moxifloxacin PO qd for 7 days. In our study, fluoroquinolone therapy yielded rRNA clearance rates that exceed those of doxycycline (in a small study set) and azithromycin (Figure 3). M. genitalium-infected patients without detectable macrolide resistance are to be managed with 100mg doxycycline PO bid for 7 days, followed by 2.5g azithromycin (1g initial dose, followed by 500mg PO qd for 3 additional days).29
Our findings should be viewed in the context of potential study limitations. From a pharmacokinetic perspective, perceived treatment failures could simply be the result of ineffective macrolide dosing, irrespective of macrolide resistance detection. Secondly, a total of 30 M. genitalium-infected participants (65.2%) presented for follow-up encounter. It is possible that some participants elected to seek follow-up healthcare from an alternative local healthcare system or a healthcare provider at their permanent residence. Thirdly, follow-up encounters that did commence solely entailed M. genitalium molecular testing and did not document patient symptomatic status. It is possible that post-treatment M. genitalium rRNA detection could represent residual nucleic acid from treated organisms. However, this possibility is less likely as the median and mean intervals between therapeutic intervention and post-treatment testing approximated 3 to 4 months. At the same time, the possibility that post-treatment M. genitalium rRNA detection indicated infection from a new sexual partner cannot be eliminated. Previous data involving this cohort9 revealed that female participants self-reported an average of approximately 3.2 sexual partners over a previous 12-month interval, with males self-reporting 3.8.
Conclusion
M. genitalium remains a difficult bacterium to treat due to potentially high levels of macrolide resistance, including in the university setting where STI is prevalent. While certain therapeutic regimens were effective to varying degrees in treating infection by this bacterium, this report, chronicling therapeutic intervention failure at a high frequency, is illustrative of the incorporation of drug-resistant M. genitalium onto the CDC “Watch List” for antibiotic resistance threats in the United States30 and can be extrapolated to other areas of public health and clinical patient management. On-site M. genitalium macrolide resistance determination, with one example being molecular analyte-specific reagent assays under development for commercial use,31 could prevent failed therapeutic interventions and limit post-infection complications.
Acknowledgments
The authors gratefully acknowledge student participants and medical staff from the participating university health clinic. S.C.L. was supported by the Carleton College Career Center’s Internship Funding program. E.M. has received honoraria and travel grants from Hologic, Incorporated.
- Received August 28, 2023.
- Revision received January 9, 2024.
- Accepted February 7, 2024.
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