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Clinical
Original article
Efficacy of standard therapies against Ureaplasma species and persistence among men with non-gonococcal urethritis enrolled in a randomised controlled trial
  1. Christine M Khosropour1,
  2. Lisa E Manhart1,2,
  3. Catherine W Gillespie1,3,
  4. M Sylvan Lowens4,
  5. Matthew R Golden1,4,5,
  6. Nicole L Jensen5,
  7. George E Kenny5,
  8. Patricia A Totten5
  1. 1Department of Epidemiology, University of Washington, Seattle, WA, USA
  2. 2Department of Global Health, University of Washington, Seattle, WA, USA
  3. 3Children's National Health System, Washington, DC, USA
  4. 4Public Health - Seattle and King County HIV/STD Program, Seattle, WA, USA
  5. 5Department of Medicine, University of Washington, Seattle, WA, USA
  1. Correspondence to Dr Lisa E Manhart, UW Center for AIDS and STD, 325 9th Avenue, Seattle, WA 98104, USA; lmanhart{at}u.washington.edu

Abstract

Objective Ureaplasma urealyticum biovar 2 (UU-2), but not Ureaplasma parvum (UP), has been associated with non-gonococcal urethritis (NGU), but little is known about species-specific responses to standard therapies. We examined species-specific treatment outcomes and followed men with treatment failure for 9 weeks.

Methods From May 2007 to July 2011, men aged ≥16 attending a sexually transmitted disease (STD) clinic in Seattle, Washington, with NGU (urethral discharge or urethral symptoms plus ≥5 polymorphonuclear leucocytes /high-powered field) enrolled in a double-blind, randomised trial. Participants received active azithromycin (1 g) + placebo doxycycline or active doxycycline (100 mg twice a day ×7 days) + placebo azithromycin. Ureaplasma were detected in culture followed by species-specific PCR. Outcomes were assessed at 3, 6 and 9 weeks. At 3 weeks, men with persistent Ureaplasma detection received ‘reverse therapy’ (eg, active doxycycline if they first received active azithromycin). At 6 weeks, persistently positive men received moxifloxacin (400 mg×7 days).

Results Of 490 men, 107 (22%) and 60 (12%) were infected with UU-2 and UP, respectively, and returned at 3 weeks. Persistent detection was similar for UU-2-infected men initially treated with azithromycin or doxycycline (25% vs 31%; p=0.53), but differed somewhat for men with UP (45% vs 24%; p=0.11). At 6 weeks, 57% of UU-2-infected and 63% of UP-infected men who received both drugs had persistent detection. Failure after moxifloxacin occurred in 30% and 36%, respectively. Persistent detection of UU-2 or UP was not associated with signs/symptoms of NGU.

Conclusions Persistent detection after treatment with doxycycline, azithromycin and moxifloxacin was common for UU and UP, but not associated with persistent urethritis.

Trial registration number NCT00358462.

https://doi.org/10.1136/sextrans-2014-051859

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    Introduction

    Non-gonococcal urethritis (NGU) is a common syndrome among male patients attending sexually transmitted disease (STD) clinics. Several organisms have been implicated in the aetiology of NGU; Chlamydia trachomatis and Mycoplasma genitalium account for 15–40% and 15–25% of all NGU cases, respectively; Trichomonas vaginalis, herpes simplex virus and adenovirus are also responsible for a small proportion of cases.1 Phylogenetic analyses have demonstrated that Ureaplasma urealyticum, a long-suspected causative agent of NGU, is actually two distinct species: U. urealyticum biovar 2 (UU-2) and Ureaplasma parvum (UP).2 ,3 Studies differentiating between these two species have found that UU-2 is associated with 16–26% of NGU cases,4–6 though this is not always the case.7 UP has not been associated with NGU,4–6 but has been associated with preterm birth8 and an increased intrauterine inflammatory response,9 suggesting that it may be pathogenic in the female reproductive tract.

    Standard therapy for men with NGU consists of either 7 days of doxycycline (100 mg twice daily) or a single 1 g dose of azithromycin.1 These two therapies were similarly efficacious in the treatment of undifferentiated U. urealyticum in a trial of men with NGU conducted in the mid-1990s,10 but no studies have prospectively assessed the efficacy of these therapies separately for differentiated Ureaplasma. In clinical isolates obtained from women, the two species had different doxycycline resistance profiles,11 ,12 suggesting that UU-2 and UP may respond differently to antimicrobial agents. Whether these differential susceptibilities translate to clinical outcomes and eradication of organisms in other settings remains unknown.

    In our recent randomised trial of men with NGU,13 microbiological cure rates (eradication of the organism) were not significantly different for UU-2-infected men treated with azithromycin or doxycycline. However, we did not evaluate the efficacy of these two therapies for UP, nor did we assess treatment outcomes among men who received additional antimicrobials after initial treatment failure. In the current study, we sought to (1) compare the efficacy of azithromycin versus doxycycline in persistently positive men with UU-2 and UP; and (2) determine whether persistence of UU-2 was associated with persisting clinical signs and symptoms of NGU.

    Methods

    Study design and population

    Details of the study design, population and data collection methods have been previously described.13 Briefly, from January 2007 to July 2011, men presenting to a Seattle, WA, STD clinic were recruited into a double-blind, randomised treatment trial for NGU. Eligible participants had NGU, defined as visible urethral discharge or ≥5 polymorphonuclear leucocytes (PMNs) per high-powered field (HPF), were ≥16 years of age and reported no antibiotic use in the previous month. Men were randomised 1:1 to receive one of two prepackaged treatments: (1) doxycycline, 100 mg administered orally twice daily for 7 days and azithromycin placebo, single dose (2–4 tablets formulated to look identical to 1 g azithromycin), administered orally; or (2) azithromycin, 1 g as a single dose (two 500 mg or four 250 mg tablets), administered orally and doxycycline placebo administered orally twice daily for 7 days (14 capsules formulated to look identical to the active doxycycline capsules). Clinical and sexual history data collected at enrolment were obtained by a single study clinician (MSL). A computer-assisted self-interview (CASI) collected additional demographic and behavioural data. At enrolment and each subsequent visit at which men received therapy, the study clinician counselled men to abstain from sexual activity for 7 days. Men were also advised to use condoms with all sex partners for the duration of the study during periods when they were not abstinent. Per clinic protocol, participants with NGU were advised to refer their sex partners to the clinic for appropriate treatment.

    At enrolment, all participants were tested for M. genitalium, C. trachomatis, T. vaginalis and, beginning in May 2007, for Ureaplasma spp. All microbiological tests were performed on first-void urine. We used the APTIMA transcription-mediated amplification assay to detect C. trachomatis and analyte-specific reagents on the same platform for T. vaginalis (GenProbe, San Diego, California, USA). M. genitalium was assessed by in-house PCR.14 Viable Ureaplasma were detected in 4.5 mL U broth15 inoculated with 0.5 mL fresh urine. The tubes were observed for up to 1 week for a colour change from yellow to red.16 Because viable Ureaplasma could not be recovered after exhaustion of urea from the medium, Ureaplasma cultures were obtained by inoculating an aliquot of the original Ureaplasma-positive frozen (−80°C) urine in serial 10-fold dilutions of U broth. Cultures in the dilution tube that were starting to turn colour plus the next higher dilution tube were combined and frozen. After follow-up was completed, species-specific PCR assays were applied to recovered Ureaplasma cultures to identify UU-2 and UP6 and for the minimum inhibitory concentration (MIC) assays.

    Men infected with Ureaplasma at enrolment returned for up to three additional follow-up visits, each of which included a clinical exam, repeat specimen testing, counselling and completion of a follow-up CASI to obtain sexual behaviour data. The first follow-up visit was scheduled 3 weeks postenrolment (allowable window=2–5 weeks). At this 3-week follow-up visit, men with recurrent/persistent NGU or a repeat positive Ureaplasma culture received ‘reverse therapy’: men initially randomised to active azithromycin/placebo doxycycline were given active doxycycline/placebo azithromycin and vice versa. All men were scheduled for an additional 6-week follow-up visit (allowable window=2–5 weeks), at which men with recurrent/persistent NGU or a repeat positive Ureaplasma culture received moxifloxacin (400 mg/day for 7 days). Men receiving moxifloxacin were scheduled for a final test-of-cure visit 3 weeks later (9 weeks postenrolment; allowable window=2–5 weeks). Men who did not have NGU at the 3-week or 6-week follow-up visits but tested culture-positive for Ureaplasma were asked to return to the clinic to receive the appropriate therapy. Any men with persistent detection of Ureaplasma after the 9-week visit were followed under clinical standard of care. We defined microbiological treatment failure (henceforth referred to as persistent detection of the organism) as a positive Ureaplasma culture 3 weeks after therapy that was subsequently confirmed via PCR as the same species that was present at enrolment. Clinical treatment failure was defined as self-reported urethral symptoms (dysuria, discharge, itching, tingling) and ≥5 PMNs/HPF or visible urethral discharge at the clinic visit 3 weeks following the receipt of therapy.

    Antimicrobial susceptibility testing was performed in triplicate on Ureaplasma culture-positive isolates collected at enrolment from June 2007 to May 2008. MIC of each antibiotic was determined by the agar dilution method using a Steers replicator to inoculate agar plates with logarithmically growing Ureaplasma cultures (revived from frozen aliquots).17 MICs were defined as the concentration of antibiotic that inhibited growth by 99%. Twofold dilutions of antibiotics tested ranged from 0.065 to 8.0 µg/mL for azithromycin, doxycycline and moxifloxacin.

    The analytic sample includes men who met a more stringent definition of NGU (self-reported urethral symptoms or clinical signs of visible urethral discharge plus ≥5 PMNs/HPF), who tested positive for UU-2 or UP at enrolment, and who returned for at least the 3-week follow-up visit. Men who were positive for both species were included in each species-specific analysis. We excluded men with positive Ureaplasma cultures but negative species-specific PCR tests for both UU-2 and UP.

    Statistical analysis

    We summarised demographic, behavioural and clinical characteristics by infecting organism at enrolment (UU-2 or UP). Statistically significant differences in characteristics among men with UU-2 versus UP were assessed with Pearson's χ2 tests for categorical variables and t tests for continuous variables.

    To compare the efficacy of azithromycin and doxycycline for each species, we used Fisher's exact test to test for significant differences in the proportion of men who had persistent detection of the organism by therapy received, at each follow-up visit. We also evaluated these associations in two subpopulations: men who denied any unprotected sex between follow-up visits (ie, men who had no sex or who used condoms for each penetrative vaginal, anal or oral sex act with all partners) and men who had no coinfections at enrolment or follow-up (ie, negative tests for M. genitalium, C. trachomatis, T. vaginalis and UU-2 or UP—depending on the baseline infection).

    To examine the association between clinical and microbiological outcomes, we used Fisher's exact test to compare the proportion of men who experienced clinical treatment failure among those who did or did not have persistent detection of the organisms. We examined this association for each follow-up visit among men without coinfections at enrolment or follow-up, among men who denied unprotected sex between follow-up visits and among men with <10 lifetime vaginal partners since the association between UU-2 and NGU was previously found to be the strongest in this subgroup.5

    For susceptibility testing, we report the MIC value from the first of the three assays. Isolates from men who were infected with both UU-2 and UP were excluded since MIC values from positive cultures of these men could not be attributed to one species.

    All analyses were performed using Stata statistical software (V.13.0; StataCorp, College Station, Texas, USA). Two-sided tests were performed at a significance level (α) of 0.05. All study procedures and analyses were approved by the University of Washington Institutional Review Board.

    Results

    A total of 606 men with NGU were enrolled in the parent trial, of whom 567 (94%) were tested for Ureaplasma. Eighty-six per cent (490 of 567) met the revised definition of NGU. Of these, 126 (25.7%) tested positive for UU-2 and 69 (14.1%) tested positive for UP. Twenty-one men were Ureaplasma-positive by culture at the enrolment visit but could not be speciated at either the enrolment or follow-up visit. These men were excluded from species-specific analyses. Eighty-five per cent (107 of 126) and 87% (60 of 69) of UU-2 and UP-positive men, respectively, returned for the 3-week follow-up visit and were included in this analysis.

    The mean age of included cases was 33 years, approximately one-half were white race and most (90%) had visible urethral discharge on exam (table 1). Consistent with our previous report of this study population at baseline,5 UP-infected men were significantly more likely to report only female sex partners than men with UU-2 (97% vs 69%, p<0.001). Men with UU-2 reported more sex partners in the past 12 months compared with men with UP (5.4 vs 3.2, p=0.01) and were more likely to report symptoms of urethral discharge and dysuria than men with UP. Roughly similar proportions of men with UU-2 and UP were coinfected with at least one other organism and four men (2.5%) were coinfected with both Ureaplasma spp. There were no statistically significant differences in demographic, clinical or behavioural characteristics among men who received azithromycin compared with those who received doxycycline, for men infected with either UU-2 or UP (data not shown).

    View this table:
    Table 1

    Baseline characteristics of men infected with Ureaplasma urealyticum biovar 2 or Ureaplasma parvum*

    Efficacy of therapies

    Persistent detection of Ureaplasma spp. at the 3-week visit was 34.8% and did not differ for men who received azithromycin or doxycycline (34.7% vs 34.8%; p=1.00).

    As previously reported, among men with UU-2, persistent detection at 3 weeks was similar in men treated with azithromycin and doxycycline (25.0% vs 30.9%, respectively; p=0.53).13 Of those with persistent detection of UU-2 after initial treatment, over one-half had persistent detection of the organism after re-treatment with the alternate therapy (figure 1). Among men who denied unprotected sex between visits, 16% (5/32) who received azithromycin and 29% (7/24) who received doxycycline had persistent UU-2 detection at 3-week follow-up (p=0.33). At 6 weeks, the comparable proportions were 33% (1/3) and 60% (3/5), respectively (p=1.0). These relationships were similar when analyses were restricted to men with no coinfecting organisms (data not shown). Eleven men with persistent UU-2 infection received moxifloxacin and returned for 9-week follow-up; of these, four (36%) again had persistent detection of UU-2.

    Figure 1
    Figure 1

    Persistent detection of the organism (microbiological treatment failure) at each of three follow-up visits, among all men infected with  Ureaplasma urealyticum biovar 2 or Ureaplasma parvum participating in a randomised, controlled trial (N=107 for U. urealyticum biovar 2 and N=60 for U. parvum at enrolment). RX, treatment. *p Values derived from Fisher's exact test. †Reverse therapy: men initially randomised to active azithromycin/placebo doxycycline were given active doxycycline/placebo azithromycin and vice versa. ‡Only men who received moxifloxacin were asked to return for a test of cure at 9 weeks.

    Among UP-infected men, persistent detection of UP at 3 weeks occurred somewhat more often after initial azithromycin therapy than after doxycycline therapy (45.2% vs 24.1%, respectively; p=0.11; figure 1). Persistent detection after re-treatment with doxycycline and azithromycin at the 6-week visit was high (figure 1). Men who denied unprotected sex between visits also had a high proportion of persistent UP detection at 3 weeks (azithromycin=43% [9/21] vs doxycycline=19% [4/21]; p=0.18) and 6 weeks (57% [4/7] vs 100% [1/1], respectively; p=1.00). This finding was not altered when analyses were restricted to men who had no coinfections. Also, 3 of 10 (30%) UP-positive men remained persistently positive after moxifloxacin treatment.

    We performed antimicrobial susceptibility testing for 22 UU-2 and 8 UP specimens obtained at enrolment (table 2). MICs did not exceed 2 μg/mL for either species.

    View this table:
    Table 2

    In vitro susceptibilities of Ureaplasma urealyticum biovar 2 and Ureaplasma parvum to three antibiotics*

    Association between clinical and microbiological outcomes

    Among men with no coinfections at enrolment or follow-up, persistent detection of the organism was not associated with clinical treatment failure at 3, 6 or 9 weeks for men with UU-2 or men with UP (table 3). Four men remained persistently positive for UU-2 or UP after treatment with moxifloxacin, but all experienced resolution of signs/symptoms. There was also no association between clinical failure and persistent detection of the organism among men with <10 lifetime vaginal sex partners or among men who had no unprotected sex between visits (data not shown).

    View this table:
    Table 3

    Association between clinical failure (presence of signs/symptoms) and microbiological failure (persistence of the organism) at each of three follow-up visits among men infected with Ureaplasma urealyticum or Ureaplasma parvum (UP) at time of initial diagnosis of urethritis who had no coinfections at enrolment or any follow-up visit*

    Discussion

    In this population of men with NGU, infection with UU-2 was more common than infection with UP (25% vs 14%, respectively). Approximately 24–45% of all men, and 16–43% of men who denied unprotected sex between visits, had persistent detection of Ureaplasma after initial treatment with azithromycin or doxycycline. Re-treatment with the alternative therapy was unsuccessful in eradicating UU-2 and UP in many cases, irrespective of the regimen used. These high rates of treatment failure did not appear to be explained by antimicrobial resistance, as all tested clinical isolates demonstrated relatively low MICs to the three therapies. Somewhat surprisingly, there was no association between persistence of UU-2 and persistent signs/symptoms of NGU.

    The rates of persistent detection among men with UU-2 and UP treated with azithromycin (25% and 45%, respectively) or doxycycline (31% and 24%, respectively) were lower than that observed by Stamm et al10 among men with NGU with undifferentiated Ureaplasma infection (55% vs 53%, for azithromycin and doxycycline, respectively). However, our findings are similar to a more recent trial of men with prostatitis attributed to undifferentiated Ureaplasma, with rates of persistent detection of 22% and 26%, respectively.18 Consistent with both previous studies, we found little difference in the efficacy of the two therapies for UU-2, suggesting that either treatment is an appropriate first-line regimen for men with UU-2-associated NGU. However, among men with UP, persistent detection was somewhat more common among men who received azithromycin compared with those that received doxycycline.

    Re-treatment of men infected with either UU-2 or UP after initial persistent detection was relatively unsuccessful, irrespective of the antibiotic used. These high rates of persistent detection, even among men who denied unprotected sex between visits, contrast with the apparent in vitro susceptibility of UU-2 and UP isolates that we observed. Consistent with our findings, studies examining Ureaplasma susceptibility to azithromycin19 and moxifloxacin17 ,19–21 have generally observed isolates in the susceptible range and have not noted differences in susceptibility patterns by species. However, the universal in vitro susceptibility to doxycycline in this study is in contrast to studies conducted in Europe and West Africa, where 18–37% of Ureaplasma isolates demonstrated in vitro resistance to doxycycline.11 ,12 ,20 Given the high failure rates that we observed in the presence of low MICs, other factors likely influence persistent infection with Ureaplasma. For example, the bacteriostatic nature of doxycycline may allow their persistence during treatment and subsequent regrowth. Alternatively, the penetrance and activity of these antibiotics in the urogenital tract may affect their treatment efficacy.

    Despite moderate to high rates of persistent detection, the persistence of signs/symptoms of infection was not associated with persistent detection of the organism. There are two key implications of this finding. First, it suggests that infection with UU-2 may not necessarily lead to urethritis. If UU-2 causes urethritis, we would expect some relationship between presence of the organism and signs and symptoms of disease. Though several studies have demonstrated an association between UU-2 and NGU,4–6 this association was not found in the largest study to date7 and the topic remains controversial. If UU-2 does cause urethritis, the clinical syndrome may be self-limited and symptoms may resolve over several weeks, with or without effective therapy. Second, the absence of any association between persisting signs and symptoms and persisting organisms calls into question the necessity to re-treat persistently positive men. While there are currently no recommendations for diagnostic testing for Ureaplasma, some clinicians opt to do this. Although few would argue against re-treating symptomatic men, the decision to treat asymptomatic men should balance the benefits of eradicating the organism (eg, disrupting transmission to female sexual partners) with the drawbacks of side effects and potential expansion of antibiotic resistance.

    This study has a number of strengths, including its randomised design, use of sensitive species-specific PCR assays and pairing of in vitro antimicrobial susceptibility testing with clinical outcomes. This study also has several limitations that merit discussion. First, because this analysis only included persistently positive men, our sample sizes at later follow-up visits were small, which limited our ability to detect statistically significant differences in the proportion of treatment failures. Second, we ceased antimicrobial susceptibility testing after the first year of the study and it is possible that isolates from men enrolled later in the trial had a different susceptibility profile than the ones included here. Third, we did not have data on bacterial load at follow-up, which prevented us from assessing the association between UU-2 organism burden and clinical outcomes. It is possible that asymptomatic men with persistent detection of UU-2 had the bacteria present at levels too low to elicit symptoms. Fourth, our strategy of speciating Ureaplasma on cultured specimens may have produced different results had we performed PCR directly from urine specimens. Fifth, data on sexual exposures between visits were self-reported and are limited by recall bias and social desirability bias. Finally, resistance patterns vary geographically and the extent to which our findings may generalise to other settings is unknown.

    In conclusion, azithromycin and doxycycline were similarly efficacious in the re-treatment of UU-2 among men with NGU who experienced initial persistent detection of UU-2 (ie, microbiological treatment failure), but doxycycline may be more effective against UP. Persistent detection of UU-2 was common among the subset of men who required re-treatment, but was not associated with persistent clinical urethritis. Several commercial laboratories in the USA and the UK offer tests for Ureaplasma. However, this absence of an association between persistent UU-2-infection and clinical signs, coupled with the difficulty in eradicating Ureaplasma after initial treatment failure, suggests that individuals with asymptomatic UU-2 infections do not benefit from nor require repeat testing or ongoing antimicrobial therapy.

    Key messages

    • In total, 24–45% of men with non-gonococcal urethritis (NGU) infected with Ureaplasma urealyticum biovar 2 or Ureaplasma parvum had persistent detection of the organism after receiving azithromycin or doxycycline

    • In most cases, re-treatment with the alternative therapy and moxifloxacin was unsuccessful in clearing the organism, despite apparent in vitro susceptibility to the three antibiotics

    • Detection of U. urealyticum biovar 2 at follow-up was not associated with persistent signs/symptoms of NGU.

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    Footnotes

    • Handling editor Jackie A Cassell

    • Acknowledgements The authors would like to thank the men who participated in the trial, as well as the clinicians and staff in the Public Health—Seattle & King County STD Clinic (Yolanda Bantolino, Sylvia Berry, Irene King, Eduardo Muñoz, Victory Murphy, Sally Pendras, Sue Szabo, Michael Verdon, Fred Koch, Roxanne Kerani, Barbara Krekeler); study staff (Sarah McDougal, Noa Kay, Dwyn Dithmer-Schreck); Sabina Astete, Lisa Lowenstein, and Linda Arnesen in the Totten Laboratory; Linda Cles in the UW Chlamydia Laboratory; Gen-Probe for reagents; Ana-Maria Xet-Mull and William Whittington for Trichomonas testing at the University of Washington; HMC IDS (Jeffrey Purcell, Bao Chau Vo, Asaad Awan, Kelly Nguyen); and the data safety and monitoring board (Edward W. Hook III, David H. Martin, H. Hunter Handsfield, Sarah Holte). We also thank Carolyn Deal, Elizabeth Rogers and Peter Wolff at the Division of Microbiology and Infectious Diseases at the National Institutes of Health and Pfizer for supplying study drugs.

    • Contributors Clinical trial concept and design: LEM, PAT and MRG. Conduct of clinical trial: LEM, CWG, MSL and NLJ. Study design: LEM, CMK, PAT and GEK. Analysis and interpretation of data: CMK, LEM, CWG, PAT, MRG and GEK. Drafting of the manuscript: CMK. Critical revision of the manuscript: CMK, LEM, CWG, MSL, MRG, NLJ, GEK and PAT.

    • Funding This work was supported by the University of Washington (UW) Sexually Transmitted Infections and Topical Microbicides Cooperative Research Center (NIH/NIAID U19 AI31448), the Center for AIDS Research (P30 AI027757) and by a grant from the National Institutes of Health (NIH/NIAID R01 AI072728). CWG and CMK were supported by the UW STD/AIDS Research Training Fellowship programme (NIH/NIAID T32 AI07140). Pfizer provided study drugs (active azithromycin, active doxycycline and placebo azithromycin). Harborview Investigational Drug Service provided placebo doxycycline.

    • Competing interests None.

    • Ethics approval University of Washington Institutional Review Board.

    • Provenance and peer review Not commissioned; externally peer reviewed.