title: Transmission and selection of macrolide resistant Mycoplasma genitalium infections detected by rapid high resolution melt analysis.
abstract: BACKGROUND
  Mycoplasma genitalium (MG) causes urethritis, cervicitis and pelvic inflammatory disease. The MG treatment failure rate using 1 g azithromycin at an Australian Sexual Health clinic in 2007-9 was 31% (95%CI 23-40%). We developed a rapid high resolution melt analysis (HRMA) assay targeting resistance mutations in the MG 23S rRNA gene, and validated it against DNA sequencing by examining pre- and post-treatment archived samples from MG-infected patients.
  
  
  RESULTS
  Available MG-positive pre-treatment (n = 82) and post-treatment samples from individuals with clinical treatment failure (n = 20) were screened for 23S rRNA gene mutations. Sixteen (20%) pre-treatment samples possessed resistance mutations (A2058G, A2059G, A2059C), which were significantly more common in patients with symptomatic azithromycin-treatment failure (12/26; 44%) than in those clinically cured (4/56; 7%), p<0.001. All 20 patients experiencing azithromycin-failure had detectable mutations in their post-treatment samples. In 9 of these cases, the same mutational types were present in both pre- and post-treatment samples indicating transmitted resistance, whilst in 11 of these cases (55%), mutations were absent in pre-treatment samples indicating likely selection of resistant isolates have occurred. HRMA was able to detect all mutational changes determined in this study by DNA sequencing. An additional HRMA assay incorporating an unlabelled probe was also developed to detect type 4 single-nucleotide polymorphisms found in other populations, with a slightly lower sensitivity of 90%.
  
  
  CONCLUSIONS
  Treatment failure is associated with the detection of macrolide resistance mutations, which appear to be almost equally due to selection of resistant isolates following exposure to 1 g azithromycin and pre-existing transmitted resistance. The application of a rapid molecular assay to detect resistance at the time of initial detection of infection allows clinicians to shorten the time to initiate effective second line treatment. This has the potential to reduce transmission of resistant strains and to avoid sequelae associated with persistent untreated infection.
authors.affiliation: Department of Microbiology and Infectious Diseases, The Royal Women's Hospital, Melbourne, Australia. jimmy.twin@mcri.edu.au
authors.forename: Jimmy
authors.initials: J
authors.lastname: Twin
authors.forename: Jorgen S
authors.initials: JS
authors.lastname: Jensen
authors.forename: Catriona S
authors.initials: CS
authors.lastname: Bradshaw
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Garland
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Fairley
authors.forename: Lim Yi
authors.initials: LY
authors.lastname: Min
authors.forename: Sepehr N
authors.initials: SN
authors.lastname: Tabrizi
mesh_terms: D000900:Anti-Bacterial Agents / Q000627:therapeutic use*; D001315:Australia; D017963:Azithromycin / Q000627:therapeutic use*; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D006801:Humans; D009175:Mycoplasma Infections / Q000188:drug therapy / Q000635:transmission*; D045704:Mycoplasma genitalium / Q000235:genetics* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000235:genetics; D017422:Sequence Analysis, DNA; D017211:Treatment Failure
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D017963:Azithromycin