Resurgence of yaws cases and bacterial drug resistance after mass azithromycin treatment: a genomic epidemiology investigation. Treponema pallidum subsp pertenue

Background: Yaws, caused by the bacterium Treponema pallidum subspecies pertenue (TPE), is the target of a current WHO eradication campaign using single-dose azithromycin mass drug administration (MDA). Longitudinal monitoring of patients in an early trial showed a resurgence of yaws cases with dominance of a single molecular type JG8 after MDA and the first cases of macrolide resistant TPE. We analysed whole genome sequences along with epidemiological data to determine the source of newly incident cases and to investigate the cluster of cases with macrolide-resistance. We also assessed the presence of minority drug-resistant bacterial variants in the overall population.Methods: We identified patients with PCR-confirmed yaws during an eradication program in Papua New Guinea between Apr 1, 2013, and Nov 1, 2016. We used whole genome sequencing performed directly on DNA extracted from patient swabs to enable phylogenomic reconstruction of strains and correlated these data with temporo-spatial information. We used deep amplicon sequencing of pre-treatment samples to assess the presence of minority macrolide resistance populations.Results: We were able to recover 20 whole TPE genomes from the trial and we contextualised these with an additional 17 publicly available TPE genomes. We confirmed that the JG8 type is extremely clonal (all genomes separated by ≤ 20 SNP) but the post-MDA cases had multiple and distinct geographical, temporal and genetic properties. We recovered treponemal genomes from three out of five patients with the macrolide-resistance mutation, and found zero pairwise SNPs among them reflecting a single transmission network. We found no evidence for minority variants in the pre-treatment sample for the index patient (>99% of reads were wild type), while the samples from all resistant cases showed a fixed genotype (>99% of reads were resistant). There was also no evidence of pre-existing minority TPE drug-resistance variants in the general population.Conclusions: Re-emergence of yaws cases after mass treatment is related to multiple epidemiological routes, consistent with reactivation of multiple latent cases. Resistance evolved only once in this study and was then disseminated. Patients in treatment naïve populations do not harbour resistance mutations even at low frequency.