PLoS ONE plos plosone PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0196217 PONE-D-17-31825 Research Article Biology and life sciencesAnatomyBody fluidsUrineMedicine and health sciencesAnatomyBody fluidsUrineBiology and life sciencesPhysiologyBody fluidsUrineMedicine and health sciencesPhysiologyBody fluidsUrineBiology and life sciencesCell biologyCellular typesAnimal cellsBlood cellsWhite blood cellsBiology and life sciencesCell biologyCellular typesAnimal cellsImmune cellsWhite blood cellsBiology and life sciencesImmunologyImmune cellsWhite blood cellsMedicine and health sciencesImmunologyImmune cellsWhite blood cellsMedicine and health sciencesInfectious diseasesCo-infectionsMedicine and health sciencesInfectious diseasesSexually transmitted diseasesEarth sciencesGeologyPetrologySedimentEarth sciencesGeologySedimentary geologySedimentBiology and life sciencesOrganismsBacteriaMollicutesMycoplasmaPeople and placesPopulation groupingsSexuality groupingsMen who have sex with menPeople and placesGeographical locationsEurope Prevalence of Mycoplasma genitalium in men with urethritis in a large public hospital in Brussels, Belgium: An observational, cross-sectional study Prevalence of Mycoplasma genitalium in men with urethritis http://orcid.org/0000-0002-6078-878X Libois Agnès Conceptualization Data curation Funding acquisition Investigation Methodology Project administration Supervision Writing – original draft Writing – review & editing 1 * Hallin Marie Conceptualization Data curation Investigation Resources Visualization Writing – original draft Writing – review & editing 2 http://orcid.org/0000-0002-2235-6038 Crucitti Tania Data curation Investigation Resources Writing – original draft Writing – review & editing 3 Delforge Marc Data curation Formal analysis Methodology Software 1 http://orcid.org/0000-0002-8079-6170 De Wit Stéphane Supervision Writing – original draft Writing – review & editing 1 Department of Infectious Diseases, CHU Saint-Pierre, Université Libre de Bruxelles (ULB), Brussels, Belgium Center for Molecular Diagnostics, Laboratoire Hospitalier Universitaire de Bruxelles, Brussels, Belgium Department of Clinical Sciences, Institute of Tropical Medicine (ITM), Antwerp, Belgium Bruisten Sylvia Editor GGD Amsterdam, NETHERLANDS

The authors have declared that no competing interests exist.

 * E-mail: agnes_libois@stpierre-bru.be 26 4 2018 2018 13 4 e0196217 30 8 2017 9 4 2018 2018 Libois et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background

Mycoplasma genitalium (MG) is a cause of urethritis. While resistance to azithromycin is increasing, routine detection of MG is not performed in Belgium, where its prevalence is unknown. The aim of this study is to determine prevalence of MG in men with urethritis.

 Method and findings

An “in-house” amplification assay detecting MG was performed on urine of men with complaints of urethritis who consulted the emergency unit or the Sexually Transmitted Infection clinic of our public hospital in Brussels. Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) were tested on the same sample. A total of 187 men were tested. Prevalence of MG was 9% (95% Confidence Interval: 5 to 13.2%). CT was detected in 20%, NG in 22% and 56% of samples were negative for these three pathogens. Neither age, ethnic origin, sexual orientation nor HIV infection were associated with MG urethritis.

 Conclusion

M. genitalium was identified in 9% of men with complaints of urethritis indicating that amplification assay detecting MG should be implemented in routine testing for those patients.

 Bourse Vésale http://orcid.org/0000-0002-6078-878X Libois Agnès This work was supported by Association d'Aide à la Recherche Médicale André Vésale - www.associationvesale.be (Bourse de la Loterie Nationale Juin 2012, no grant number). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability All relevant data are within the paper and its Supporting Information files. Introduction

Mycoplasma genitalium (MG) is a cause of urethritis in men and is associated in women with cervicitis, pelvic inflammatory disease (PID) and infertility. [1]

Prevalence of MG in the general population is low, ranging from 0.8% in clinic based samples from asymptomatic patients to 3.2% in MSM in the community; while in men with nonchlamydial-nongonococcal urethritis (NCNGU), it varies between studies from 15% to 35%. [2, 3]

In Europe, in men with urethritis, prevalence varies between 6% and 16.7%. [4, 5]

Due to the increasing resistance of MG to azithromycin (40% in London and Denmark, 14% in France) [2,4,6], azithromycin 1 g is replaced by 1 week of doxycycline as first-line therapy for nongonococcal urethritis (NGU) in the 2016 European guideline. [7] Doxycycline, however, has poor efficacy on MG with cure rates of 31% to 45%. [1] Moxifloxacin is recommended after a treatment failure with azithromycin but resistance is also increasing (reaching 13% in Australia). [1,4,8]

Nucleic acid amplification is the only clinically useful method to detect MG. No FDA-approved test is available yet. Therefore, detection of MG is not recommended by US guidelines and still not routinely performed. [9] However, the European guideline recommends these tests in men with urethritis, when available. [7,10] New tests including detection of azithromycin resistance in MG were recently CE labeled. [11,12]

The high prevalence of MG among NGU and its capacity to develop resistance are likely to worsen in the future, jeopardizing empirical treatment options for NGU. In Belgium, the prevalence of MG is unknown. Our aim was to determine its prevalence in men with complaints of urethritis in a large public urban hospital in Brussels.

 Methods Patients

This was an observational, cross-sectional study targeting men with complaints of urethritis (dysuria, urethral discomfort and/or urethral discharge) presenting at the emergency unit or at the Sexually Transmitted Infection clinic of University Saint-Pierre hospital in Brussels, between 1/10/2012 and 11/02/2014.

Patients reporting any antibiotic treatment in the previous 30 days were excluded.

Pathogen detection and urine leucocytes count

First-void urines were collected and split into two aliquots: the first, inoculated in a sterile tube without conservative, was used for urinary sediment analysis and dipstick test; the second was inoculated into a multi-Collect transport medium tube (Abbott, Maidenhead, UK) for C. trachomatis (CT), N. gonorrhoeae (NG) and MG detection.

Urine sediment analysis was performed using the flow cytometer Sysmex UF-100. Tubes were then used for a dipstick test on the Aution Max AX-4280 (Menarini). CT and NG were tested using the Abbott RealTime CT/NG assay on the Abbott m2000 automated platform. Samples were then stored at -20°C. The amplification assay detecting MG was performed by series: DNA was extracted using the Abbott platform according to the manufacturer’s instructions. A validated in-house real-time PCR amplifying a fragment of the pdhD gene was then performed. The in house real-time PCR for detection of M. genitalium was performed based on the assay described by Müller et al. [13] and according to the procedure summarized elsewhere [14].

As MG identification was performed by testing series of samples, physicians did not receive the result, treatment of patients was thus not influenced by the presence or not of M. genitalium infection in the urine test.

 Definition

Objective urethritis was defined as a discharge on exam and/or urine leucocytes ≥35cells/μl and/or a positive leucocyte esterase. A leucocytes count ≥35cells/μl corresponds to 10 leucocytes per high power field using microscopic examination of urine after centrifugation, the recommended limit value to document urethritis according to US guidelines. [9,15]

 Statistical analyses

Simple descriptive statistics were used for summarized characteristics.

For comparison, Mann-Whitney U test was used for continuous data and Fisher’s exact test for categorical data. All reported p-values are two-sided. Analyses were produced using the SAS statistical software (version 9.4; SAS Institute, Cary, NC, USA).

 Ethics

The study was approved by the local Ethics Committee (OM007, study B076201214623) and the Institutional Review Board of Institute of Tropical Medicine. An oral inform consent was given by all participants.

 Results

During a 16 months period, 187 men with complaints of urethritis were included. Patients characteristics and results of urine tests are shown in Table 1.

10.1371/journal.pone.0196217.t001 Demographics, clinical features and results of urine samples among 187 patients with complaints of urethritis.
Patients with complaints of urethritisN = 187 Patients with objective urethritisN = 93 Patients with complaints but no objective urethritisN = 43 Patients with complaints but incomplete dataN = 51
Age (years), median (interquartile range) 30 (26–38) 29 (23–37) 31 (26–39) 31 (27–39)
Caucasians 81% 73% 93% 88%
Heterosexual 64% 65% 64% 64%
Men who have sex with men 36% 35% 36% 36%
HIV positive 7% 4% 12% 8%
Urine PCR-NG positive. n(%) 42 (22%) 38 (41%) 1 (2%) 3 (6%)
Urine PCR-CT positive. n (%) 37 (20%) 29 (31%) 1 (2%) 7 (14%)
Urine PCR-M.genitalium positive. n (%) 17 (9%) 10 (11%) 1 (2%) 6 (12%)
No pathogen found. n (%) 104 (56%) 29 (31%) 40 (93%) 35 (69%)

Prevalence of M. genitalium was 9% (95% Confidence Interval (CI): 5 to 13.2%) on the whole cohort, 11% in NGU (95% CI: 4.5 to 17) and 13% in NCNGU (95% CI: 2.9 to 20.6), excluding coinfection (MG and infection with CT and/or NG). Nine men had a NG-CT co-infection, two had a CT-MG co-infection and one a NG-CT-MG co-infection. No pathogen was found in 56% of samples. Men ≥30 years were more at risk of negative results for the 3 bacteria tested, whatever their sexual orientation (65% vs 45%, p = 0.009)

Diagnosis of M. genitalium or N.gonorrhoeae infection were not significantly associated with age, ethnicity, sexual orientation or HIV infection.

CT was more frequently identified in men <30 years (27% versus 14% in men >30 years, p = 0.0415) and in heterosexual men (27% vs 8% in MSM, p = 0.0066, also statistically significant in multivariate analysis).

Half of the men (93/187) had an objective urethritis: urine leucocytes ≥35/μl in 54 of 104 urine sediment performed, six patients had leucocytes <35/μl but a positive leucocyte esterase and 33 had a discharge on exam and no urine sediment analysis performed.

Among patients with objective urethritis, MG was present in 11%, 16% of NGU and 27% of NCNGU. The three patients with a coinfection with MG (two with CT and one with CT and NG) had an objective urethritis. No pathogen was found in 31% of samples. This percentage was also higher in men ≥30 years in this subgroup of patients with objective urethritis (42% vs 21%, p = 0.026).

Among the 43 men with complaints but no objective urethritis, only 3 had a bacteria detected (1 MG, 1 CT, 1 NG).

Finally, 51 men presented complaints but no objective discharge and due to missing data (urine leucocytes or leucocytes esterase were not performed), they could not be classified as having or not having an objective urethritis.

 Discussion

In our population of men with complaints of urethritis, prevalence of MG was 9% on the whole cohort and 11% in patients with objective urethritis. To our knowledge, these are the first Belgian data on MG in men.

These prevalence rates are similar to those found in men with objective NGU from recent series: 16.7% in London [4] and 11.9% in Tel Aviv [16]. In Amsterdam, prevalence in men reporting symptoms of urethritis was 5.9%. [5] The high prevalence we found and the increase of azithromycin resistance in numerous studies confirms the need for routine detection of MG in patients with urethritis, preferably combined with resistance testing.

Introducing these tests in daily practice in Belgium is difficult as they are not reimbursed by the social security and there is no FDA approved, standardized or well validated commercially available test (CE labeled kits suffer from limited validation). [10]

A limitation of our study is that a substantial number of patients did not carry out all tests necessary to objectify urethritis.

No control group was included in our study but prevalence is known to be low in the general population including in clinical based samples from asymptomatic patients. [3]

This was confirmed in our setting: 77 men (with or without complaints of urethritis) were tested in our STI clinic and none was positive for M.genitalium among asymptomatic men. (Mahadeb B, unpublished data).

No resistance testing for azithromycin was performed in our study as we had no access to an assay identifying the point mutations associated with azithromycin resistance at the time of the study. A very recent Belgian study in female sex workers showed a prevalence of MG of 10.8% and macrolide-resistance associated mutations (A2058G and A2059G) in 6.5% of isolates. [17]

Further studies in Belgium should address the prevalence of macrolide and moxifloxacin resistance in men with urethritis.

In conclusion, MG was identified in 9% of men with complaints of urethritis and in 11% of men with objective urethritis. Amplification assays detecting M. genitalium should be implemented in routine testing for men with urethritis, preferably with resistance testing, allowing a more adapted antibiotic treatment.

 Supporting information <p>(XLS)</p> </caption> </supplementary-material> </sec> </body> <back> <ack> <p>We thank all doctors in emergency unit and in the Sexually Transmitted Infection clinic who have included patient in the study and our colleague Sheila Cattell for English proofreading.</p> </ack> <ref-list> <title>References Manhart LE, Jensen JS, Bradshaw CS, Golden MR, Martin DH. Efficacy of Antimicrobial Therapy for Mycoplasma genitalium Infections. Clin Infect Dis. 2015 Dec 15;61 Suppl 8:S802–817. Salado-Rasmussen K, Jensen JS. Mycoplasma genitalium testing pattern and macrolide resistance: a Danish nationwide retrospective survey. Clin Infect Dis Dis 2014 Jul 1;59(1):24–30. Baumann L, Cina M, Egli-Gany D, Goutaki M, Halbeisen FS, Lohrer GR, et al. Prevalence of Mycoplasma genitalium in different population groups: systematic review and meta-analysis. Sex Transm Infect. 2018 Feb 9. doi: 10.1136/sextrans-2017-053384 29440466 Pond MJ, Nori AV, Witney AA, Lopeman RC, Butcher PD, Sadiq ST. High Prevalence of Antibiotic-Resistant Mycoplasma genitalium in Nongonococcal Urethritis: The Need for Routine Testing and the Inadequacy of Current Treatment Options. Clin Infect Dis. 2014 Mar 1;58(5):631–7. doi: 10.1093/cid/cit752 24280088 van der Veer C, van Rooijen MS, Himschoot M, de Vries HJC, Bruisten SM. Trichomonas vaginalis and Mycoplasma genitalium: age-specific prevalence and disease burden in men attending a sexually transmitted infections clinic in Amsterdam, the Netherlands. Sex Transm Infect. 2016 Feb;92(1):83–5. doi: 10.1136/sextrans-2015-052118 26283740 Le Roy C, Hénin N, Pereyre S, Bébéar C. Fluoroquinolone-Resistant Mycoplasma genitalium, Southwestern France. Emerg Infect Dis. 2016;22(9):1677–1679. doi: 10.3201/eid2209.160446 27533360 Horner PJ, Blee K, Falk L, van der Meijden W, Moi H. 2016 European guideline on the management of non-gonococcal urethritis. Int J STD AIDS. 2016;27(11):928–37 doi: 10.1177/0956462416648585 27147267 Murray GL, Bradshaw CS, Bissessor M, Danielewski J, Garland SM, Jensen JS, et al. Increasing macrolide and fluoroquinolone resistance in Mycoplasma genitalium. Emerg Infect Dis. 2017 May;23(5):809–812. doi: 10.3201/eid2305.161745 28418319 Workowski KA, Bolan GA, Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep Morb Mortal Wkly Rep Recomm Rep Cent Dis Control. 2015 Jun 5;64(RR-03):1–137. Jensen JS, Cusini M, Gomberg M, Moi H. 2016 European guideline on Mycoplasma genitalium infections. J Eur Acad Dermatol Venereol. 2016 Oct;30(10):1650–6. doi: 10.1111/jdv.13849 27505296 Le Roy C, Pereyre S, Hénin N, Bébéar C. French Prospective Clinical Evaluation of the Aptima Mycoplasma genitalium CE-IVD Assay and Macrolide Resistance Detection Using Three Distinct Assays. J Clin Microbiol. 2017 Nov;55(11):3194–3200. doi: 10.1128/JCM.00579-17 28794183 Pitt R, Cole MJ, Fifer H, Woodford N. Evaluation of theMycoplasma genitalium Resistance Plus kit for the detection of M. Genitalium and mutations associated with macrolide resistance. Sex Transm Infect. 2017 Nov 3. doi: 10.1136/sextrans-2017-053366 29101246 Müller EE, Venter JME, Magooa MP, Morrison C, Lewis DA, Mavedzenge SN. Development of a rotor-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium. J Microbiol Methods. 2012 Feb;88(2):311–5 doi: 10.1016/j.mimet.2011.12.017 22230235 Ursi D, Crucitti T, Smet H, Ieven M. Evaluation of the Bio-Rad Dx CT/NG/MG® assay for simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in urine. Eur J Clin Microbiol Infect Dis. 2016 Jul;35(7):1159–63. doi: 10.1007/s10096-016-2646-4 27116008 Lun A, Ziebig R, Hammer H, Otting U, Filler G, Sinha P. Reference Values for Neonates and Children for the UF-100 Urine Flow Cytometer. Clin Chem. 1999 Oct 1;45(10):1879–80. 10508143 Gottesman T, Yossepowitch O, Samra Z, Rosenberg S, Dan M. Prevalence of Mycoplasma genitalium in men with urethritis and in high risk asymptomatic males in Tel Aviv: a prospective study. Int J STD AIDS. 2017 Feb;28(2):127–132 doi: 10.1177/0956462416630675 26826161 Coorevits L, Traen A, Bingé L, Descheemaeker P, Boelens J, Reynders M, et al. Macrolide resistance in Mycoplasma genitalium from female sex workers in Belgium. J Glob Antimicrob Resist. 2017 Oct 5;12:149–152. doi: 10.1016/j.jgar.2017.09.018 28988903