Background To develop a TaqMan-based real-time allelic discrimination assay for the simultaneous detection of two point mutations (A2058G and A2059G) in the 23S rRNA gene of T pallidum that have been associated with azithromycin treatment failures.

Methods Initially, two TaqMan-based real-time duplex PCR assays were used to detect the A2058G and A2059G point mutations within the 23S rRNA gene of T pallidum. Genotyping results from these assays were then compared to a previously described real-time PCR assay using fluorescence resonance energy transfer (FRET) probes and melting curve analysis that is specific for the detection of the A2058G point mutation. Subsequently, a real-time triplex PCR was developed to distinguish the A2058G from the A2059G point mutation in a single assay and the results were confirmed by pyrosequencing.

Results Sixteen of 67 (23.9%) T pallidum-positive specimens collected from patients with genital ulcer disease in the US were found to have the A2059G point mutation. These strains were previously characterised as having azithromycin-susceptible genotypes (no point mutations in the 23S rRNA gene). The A2059G mutation was confirmed by a real-time duplex PCR assay containing the TaqMan probe specific for the mutation and by pyrosequencing. None of the T pallidum strains examined to date had both point mutations. The real-time triplex PCR assay was able to differentiate azithromycin-susceptible genotypes from resistant genotypes containing either the A2058G or A2059G point mutation in a single assay. The limit of detection of the assay was 1–10 copies using 23S rRNA gene fragments that were cloned into a plasmid.

Conclusions The previously reported real-time PCR detection platforms were designed to detect only the A2058G point mutation and were unable to differentiate T pallidum strains with susceptible genotypes from resistant genotypes with the A2059G mutation. The new TaqMan-based real-time allelic discrimination assay offers an alternative to the previously described PCR/RFLP method to rapidly detect both point mutations associated with azithromycin resistance in T pallidum. The prevalence of T. pallidum strains that harbour point mutations in the 23S rRNA gene associated with resistance to azithromycin might be much higher than previously estimated.