Abstract. We developed a TaqMan-based real-time quadriplex polymerase chain reaction (PCR) to simultaneously detect Treponema pallidum subspecies pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum, the causative agents of venereal syphilis, yaws, and bejel, respectively. The PCR assay was applied to samples from skin ulcerations of clinically presumptive yaws cases among children on Tanna Island, Vanuatu. Another real-time triplex PCR was used to screen for the point mutations in the 23S rRNA genes that have previously been associated with azithromycin resistance in T. pallidum subsp. pallidum strains. Seropositivity by the classical syphilis serological tests was 35.5% among children with skin ulcerations clinically suspected with yaws, whereas the presence of T. pallidum subsp. pertenue DNA was only found in lesions from 15.5% of children. No evidence of T. pallidum subsp. pertenue infection, by either PCR or serology was found in~59% of cases indicating alternative causes of yaws-like lesions in this endemic area.

Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the realtime triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.

IntroductionBoth yaws and trachoma are endemic in the Pacific. Mass treatment with azithromycin is the mainstay of the WHO strategy for both the eradication of yaws and the elimination of trachoma as a public health problem, but the dose recommended for trachoma is lower than that for yaws. In countries where both diseases are endemic, there is a potential for synergy between yaws and trachoma control programs if mass treatment with the lower dose of azithromycin was shown to be effective for the treatment of yaws. In an earlier study, we demonstrated a profound reduction in the clinical and serological prevalence of yaws following a single round of mass treatment with azithromycin 20 mg/kg undertaken for the purposes of trachoma elimination.MethodsThis survey was conducted 18 months following a single round of azithromycin mass treatment in the same communities in which we had conducted our previous six-month follow-up survey. We examined children aged 1–14 years and took blood and lesion samples for yaws diagnosis using the Treponema pallidum particle agglutination assay (TPPA) and the non-treponemal Rapid Plasma Reagin (RPR) test.ResultsA total of 1,284 children were enrolled in the study. Amongst children aged 5–14 years, 223 had a positive TPPA (27.5%, 95% CI 13.6–47.7%). The TPPA seroprevalence amongst this age group did not differ significantly from either our pre-mass treatment survey or our initial follow-up survey. Thirty-five children had positive TPPA and positive RPR (4.3%, 95% CI 2.1–8.7%), and this did not differ significantly from our initial post-mass drug administration (MDA) follow-up survey (4.3% versus 3.5%, p = 0.43) but remained significantly lower than our initial pre-MDA survey (4.3% vs 21.7%, p <0.0001). Village-level MDA coverage was strongly associated with dual-seropositivity (p = 0.005). Amongst children aged 1–4 years, 16 had a positive TPPA (3.5%, 95% CI 1.6–7.1%). This did not differ significantly from the seroprevalence in this age group that had been predicted based on our previous surveys (3.5% vs 5%, p = 0.11). Fourteen children (1.1%) were considered to have a skin lesion clinically consistent with yaws, but none of these individuals was seropositive for yaws. Of nine cases where a swab could be collected for PCR, all were negative for Treponema pallidum subsp. pertenue DNA.DiscussionIn this study we have shown that the benefit of a single round of mass treatment with azithromycin 20mg/kg appears to extend to 18 months without any further intervention. The lack of a significant change in seroprevalence from 6 to 18 months after mass treatment might suggest that interventions could be spaced at yearly intervals without a significant loss of impact, and that this might facilitate integration of yaws eradication with other neglected tropical disease (NTD) control programmes. MDA coverage above 90% was associated with significantly better outcomes than coverages lower than this threshold, and strategies to improve coverage at all stages of yaws eradication efforts should be inves...

Yaws is a neglected tropical disease caused by the bacterium Treponema pallidum subspecies pertenue. The disease primarily affects children under 15 years of age living in low socioeconomic conditions in tropical areas. As a result of a renewed focus on the disease owing to a recent eradication effort initiated by the World Health Organization, we have evaluated a typing method, adapted from and based on the enhanced Centers for Disease Control and Prevention typing method for T. pallidum subsp. pallidum, for possible use in epidemiological studies. Thirty DNA samples from yaws cases in Vanuatu and Ghana, 11 DNA samples extracted from laboratory strains, and 3 published genomic sequences were fully typed by PCR/RFLP analysis of the tpr E, G, and J genes and by determining the number of 60-bp repeats within the arp gene. Subtyping was performed by sequencing a homonucleotide “G” tandem repeat immediately upstream of the rpsA gene and an 84-bp region of tp0548. A total of 22 complete strain types were identified; two strain types in clinical samples from Vanuatu (5q11/ak and 5q12/ak), nine strain types in clinical samples from Ghana (3q12/ah, 4r12/ah, 4q10/j, 4q11/ah, 4q12/ah, 4q12/v, 4q13/ah, 6q10/aj, and 9q10/ai), and twelve strain types in laboratory strains and published genomes (2q11/ae, 3r12/ad, 4q11/ad, 4q12/ad, 4q12/ag, 4q12/v, 5r12/ad, 6r12/x, 6q11/af, 10q9/r, 10q12/r, and 12r12/w). The tpr RFLP patterns and arp repeat sizes were subsequently verified by sequencing analysis of the respective PCR amplicons. This study demonstrates that the typing method for subsp. pallidum can be applied to subsp. pertenue strains and should prove useful for molecular epidemiological studies on yaws.