Overview Mycoplasma genitalium, STD and molecular diagnostics Cécile Bébéar USC Mycoplasmal and chlamydial infections in humans French National Reference Center for Chlamydia INRA - Université Bordeaux Segalen CHU de Bordeaux • Introduction • Association between M. genitalium infections and disease • Diagnosis • Antimicrobial susceptibility testing and treatment studies Mycoplasma genitalium Mycoplasma genitalium An introduction An introduction • 1980: Mycoplasma genitalium isolated from 2 of 13 men with NGU (nongonoccocal uretritis) – Very slow growth (>50 days) – Very few isolates available • Similar to M. pneumoniae – Morphology (tip structure) – Genetics • Sexually transmitted bacterium, lacks a cell wall • 1990’s: development of PCR assays, allowed study of disease association • 1995: smallest genome known (580 kbp, ≈ 480 genes) – One of the first fully sequenced (Himmelreich, 1995) – Minimal requirements of life, concept of minimal cell M. genitalium: prevalence and incidence M. genitalium: an epidemiologist’s view • Prevalence - General population • How much ? Prevalence and incidence • What ? Association with diseases 1 - 4% 1 - 6% - Clinic populations 4 - 26% 4 - 38% • Who ? Risk factors • Incidence - University women: 0.9 per 100 WY - Kenya Female sex workers: 23 per 100 WY Anagrius STI 2005, Hamasuna STI 2004, Ross STI 2009, Tosh JAH 2007, Oakeshott CID 2010, Cohen STD 2007, Pepin STI 2005, Hancock STI 2010 1 M. genitalium: disease association Men M. genitalium: disease association Women Men Women Nongonococcal Urethritis (NGU) Urethritis NGU Urethritis Epididymitis Cervicitis Epididymitis Cervicitis Prostatitis Endometritis, Salpingitis (PID) Prostatitis Endometritis, Salpingitis (PID) Ectopic pregrancy Proctitis (MSM) Ectopic pregrancy Proctitis (MSM) Preterm birth Preterm birth Infertility Infertility Association between M. genitalium and male NGU Male Urethritis Odds Ratio (95% CI) Association between M. genitalium and female disease Clinical presentation • Frequently asymptomatic • Similar to Chlamydia trachomatis with some exceptions - Mucopurulent discharge - Fewer PID symptoms • Long duration of infection - Up to 21-33 months Cazanave et al, Med Mal Infect 2012 Manhart et al, Clin Infect Dis 2011 Association between M. genitalium and female disease Cervicitis Odds Ratio (95% CI) M. genitalium and upper genital tract disease Endometritis • M. genitalium was found in 9 of 58 women (16%) with histological endometritis and in 1 of 57 women (2%) w/o endometritis (Cohen, 2002) • In the PEACH study, M. genitalium was found in 15% (CT 14%; NG 15%), (Haggerty, 2008) Endometritis/PID – M. genitalium found in the endometrium of 60% of those positive in the cervix – Pelvic pain scores, clinical symptoms, and signs were similar in MG and CT-positive women (Short et al., CID, 2009) Infertility Preterm Birth Cazanave et al, Med Mal Infect 2012 Manhart et al, Clin Infect Dis 2011 2 M. genitalium and upper genital tract disease Salpingitis The MATIST project Prevalence and risk factors associated with Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium infections in French pregnant women Bordeaux University hospital - January to June 2011: 1006 pregnant women No. specimens tested C. trachomatis M. genitalium N. gonorrhoeae 1006 2.5 % 0.8 % 0% 18-24 166 7.9 % 2.4 % 0% 25-29 317 1.3 % 0.6 % 0% ≥ 30 523 1.5 % 0.4 % 0% Age (yo) 18-44 (Møller et al., 2006) • 123 Kenian women with acute salpingitis, confirmed by laparoscopy • M. genitalium detected in the cervix and endometrium of 9 women (7%), but only in 1 specimen from fallopian tubes Prevalence of infection with Risk factors for M. genitalium infection: - Younger age (OD = 9, p = 0.01) - History of abortion (OD = 8.6, p = 0.01) - Having 1st sexual intercourse after 20 yo (OD = 7.1, p = 0.03) (Cohen et al., 2005). Risk Factors Diagnosis of M. genitalium infections • Only a direct diagnosis Nbr recent partners Smoking Short duration of Marital status stable relationship HIV infection Age at sex debut Condom use Black / Indigenous Income Chlamydia Bacterial vaginosis (1) Young age Horm contraception • Culture extremely fastidious • By nucleic acid amplification tests: - a lot of in house PCRs, real-time PCR ++ - a few monoplex (Roche, Diagenode) and multiplex tests (Bio-Rad, Seegene) commercialized - some specimens better than others: FVU> urethral swabs in men vaginal swabs >cervix>FVU in women • No rapid POC, no serology commercialized Currently commercially available mono and multiplex real-time PCR-based NAATs for M. genitalium Dx CT/NG/MG assay • 1 real-time PCR reaction, double-stranded probes method • 3 bacteria: C. trachomatis, N. gonorrhoeae, M. genitalium • 1 internal control, 96 tests • On the Dx real-time system and work station • Dx collection system - for endocervical, vaginal and anorectal swabs - for urethral swabs 3 • Clinical performances of the Bio-Rad Dx CT/NG/MG assay for M. genitalium detection Le Roy et al., J Microbiol Methods 2012 Reference method results Pos B io-R ad Dx C T/NG/MG Assay Male urines (n=259) Urethral swabs (n=7) Female urines (n=180) Female swabs (n=212) Neg Sens. (%) [95% CI]] 100 [56.5-100]] Pos 5 0 Neg 0 254 Pos 0 0 Neg 0 7 Pos 1 0 Neg 0 179 Pos 4 1 Neg 0 Mycoplasma genitalium treatment Bio-Rad D x CT/NG/MG performances Spec. (%) [95% CI]] NPV (%) PPV (%) 100 100 NA 100 100 100 100 80 100 [98.5-100]] NA 100 [64.6-100]] 100 [20.6-100]] 100 [97.6-100]] 100 [51.0-100]] 207 99.5 [97.399.9]] M. genitalium treatment M. genitalium: antibiotic susceptibility testing • Intrinsic resistance European guidelines, 2009 • Active antibiotics β-lactams and other antibiotics targeting the cell wall • Acute NGU – cervicitis or Azithromycin 1 g single dose Doxycycline 100 mg x 2, 7 d • Chronic NGU 1) Extended 1.5 g azithromycin (5 d) 2) Moxifloxacin 400 mg, 10 d • No susceptibility testing done in routine Renaudin et al , Antimicrob Agents Chemother, 1992 M. genitalium male NGU treatment studies • – Doxycycline 200 mg + 100 x 8 d: 22% cure rate (n=103) – Azithromycin 1 g x 1 d: 86% cure rate (n=56) – Azithromycin 500 mg + 250 mg x 4 d: 97% cure rate (n=60) • Response to treatment in men • Insufficient treatment leads to persisting or recurring symptoms Randomized US trial (Mena CID 2009) – Doxycycline 200 mg x 7 d: 45% cure rate (n=31) – Azithromycin 1 g x 1 d: 87% cure rate (n=23) • Clinical cure in DOX group at 2-3 weeks but subsequent recurrence • M. genitalium treatment studies Open Scandinavian multicenter trial (Björnelius 2008) Randomized US trial (Schwebke CID 2011) – Doxycycline 200 mg x 7 d: 49% cure rate (n=149) • M. genitalium clearance rate 30.8% – Azithromycin 1 g x 1 d: 43.6% cure rate (n=156) • Persistence of symptoms - Patients having Mg eradicated: 17% - Patients with Mg treatment failure: 91% (p<0.0001) (Bradshaw et al. pLoS One 2008) • Men with persistent NGU after doxycycline treatment: - 41% (32/78) were M. genitalium-positive (Wikström & Jensen, STI 2006), - 68% (61/90) were M. genitalium-positive ( Sena et al., J Infect Dis, 2012). • M. genitalium clearance rate 66.7% • Moxifloxacin 400 mg for 7-10 d in treatment failure after AZM: 100 % cure rate (Bradshaw 2006; Jernberg 2008) • Patients failing azithromycin 1g single dose cannot be treated successfully with extended 1.5 g AZM (Jernerg STI 2008) 4 M. genitalium: emergence of macrolide resistance (2) M. genitalium: emergence of macrolide resistance (1) • Mutations in domain V of 23S rRNA • 8 clinical strains AZM-R (Bradshaw EID 2006) - MIC AZM >32 mg/l, ERY >64 mg/l - mutations 2058, 2059 in 23S rRNA - A2058G/C, A2059G (E. coli numbering) - Azithromycin 1 g single dose Selection of resistant mutants during AZM treatment Therapeutic failure if patient infected with a mutated strain • 19 patients: Mg positive specimens for a strain AZM-R - mutations 2058, 2059 in domain V of 23S rRNA (Jensen CID 2008) • Azithromycin 1 g single dose -> 13 - 33 % therapeutic failures 23S rRNA (Bradshaw EID 2006, Jensen CID 2008, Ito STI 2011, Shimada EID 2011) Domain V Macrolide resistance in M. genitalium in Bordeaux, France M. genitalium: emergence of macrolide resistance (3) 20 Proportion of patients infected with a 23S rRNA mutated M. genitalium a in France • Description of macrolide resistance since 2005 for M. genitalium in Australia, Scandinavia, New-Zealand, Japan and France • According to the primary treatment used in countries - Sweden : DOX = 1st line TT for NGU and cervicitis 181 M. genitalium (+) STD-clinic attendees 3 (1.6%) had 23S rRNA mutations - Danemark : AZM = 1st line TT for NGU and cervicitis 415 M. genitalium (+) GP and STD-clinic attendees 162 (39%) had 23S rRNA mutations - France : AZM and DOX = 1st line TT for NGU and cervicitis 115 M. genitalium (+) STD-clinic attendees 13 (11.3%) had 23S rRNA mutations 18 16 13.3% (2/15) 14 15.4% (2/13) 14.3% (3/21) 12.8% (5/39) 10% (1/10) 12 10 8 6 4 2 0 0% (0/1) 0% (0/10) 0% (0/6) 2003 2004 2005 2006 2007 2008 2009 2010 Years of specimen collection • Detection of mutations associated to macrolide resistance in M. genitalium since 2006 • Annual prevalence: 10-15.4% Chrisment et al, J Antimicrob Chemother 2012 M. genitalium : acquired resistance to other antimicrobials • Acquired resistance to fluoroquinolones - Few reports, Japan ++ - Target mutations (gyrase and topo IV) • Description of multidrug resistance - Resistance to macrolides and fluoroquinolones - 1st strain described for a Chinese patient: AZM and MXF MIC >16 mg/l - Few other cases described in Australia and Norway Conclusion (1) • M. genitalium, chlamydia ? emerging STI pathogen, a new • An accepted cause of male NGU and female cervicitis • Probably associated with sequelae in women - PID Infertility Preterm birth ? • Relatively low general population prevalence - screening programs not appropriate - Testing and treating in high risky STI populations 5 Conclusion (2) Acknowledgments USC EA 3671 • Commercially available nucleic acid amplification tests, multiplex PCRs for detection of STI pathogens • Treatment of M. genitalium infections - Tetracyclines not useful, AZM single dose better Extended 1.5 g AZM 85 to 95% effective - Emergence of resistance to macrolides Huge local differences in resistance rates - Moxifloxacin 10 d in case of AZM failure Manhart et al, Clin Infect Dis 2011 Taylor-Robinson and Jensen, Clin Microbiol Rev 2011 Sabine Pereyre Charles Cazanave Delphine Chrisment Alain Charron Hélène Renaudin Bertille de Barbeyrac Olivia Peuchant Chloé Le Roy Statens Serum Institut, Denmark Jorgen J. Jensen University of Washington, USA Lisa L. Manhart Gynecology clinics, CHU de Bordeaux Dominique Dallay Jacques Horowitz USMR, CHU de Bordeaux Geneviève Chêne Conflict of Interest Bio-Rad Roche Diagnostics Diagenode 6