Four-color multiplex real-time PCR assay prototype targeting azithromycin resistance mutations in Mycoplasma genitalium

BMC Infect Dis. 2019 Sep 23;19(1):827. doi: 10.1186/s12879-019-4424-2.

Abstract

Background: The worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient's antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician.

Methods: Azithromycin resistance mutations in MG are targeted using a four-color multiplex real-time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples.

Results: The analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing.

Conclusions: A new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen.

Keywords: Azithromycin resistance; Diagnostic; Mycoplasma genitalium; Real-time PCR.

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Azithromycin / pharmacology*
  • Drug Resistance, Bacterial / genetics*
  • Humans
  • Multiplex Polymerase Chain Reaction / methods*
  • Mutation*
  • Mycoplasma Infections / drug therapy
  • Mycoplasma Infections / microbiology
  • Mycoplasma genitalium / genetics*
  • Real-Time Polymerase Chain Reaction / methods*
  • Substrate Specificity

Substances

  • Anti-Bacterial Agents
  • Azithromycin