Genomic Epidemiology of Treponema pallidum and Circulation of Strains With Diminished tprK Antigen Variation Capability in Seattle, 2021-2022

Abstract

Background: The incidence of syphilis continues to increase in the United States, yet little is known about Treponema pallidum genomic epidemiology within American metropolitan areas.

Methods: We performed whole-genome sequencing and tprK deep sequencing of 28 T. pallidum-containing specimens, collected mostly from remnant Aptima swab specimens from 24 individuals from Seattle Sexual Health Clinic during 2021-2022.

Results: All 12 individuals infected with Nichols-lineage strains were men who have sex with men, while a specific SS14 cluster (mean, 0.33 single-nucleotide variant) included 1 man who has sex with women and 5 women. All T. pallidum strains sequenced were azithromycin resistant via 23S ribosomal RNA A2058G mutation. Identical T. pallidum genomic sequences were found in pharyngeal and rectal swab specimens taken concurrently from the same individuals. The tprK sequences were less variable between patient-matched specimens and between epidemiologically linked clusters. We detected a 528-base pair deletion in the tprK donor site locus, eliminating 9 donor sites, in T. pallidum genomes of 3 individuals with secondary syphilis, associated with diminution of TprK diversity.

Conclusions: We developed an end-to-end workflow for public health genomic surveillance of T. pallidum from remnant Aptima swab specimens. tprK sequencing may assist in linking cases beyond routine T. pallidum genome sequencing. T. pallidum strains with deletions in tprK donor sites currently circulate and are associated with diminished TprK antigenic diversity.

Keywords: Treponema pallidum; tprK; genomic epidemiology; immune evasion; syphilis.

Figure 1.

Relationship between Treponema pallidum genome copies in extracted DNA, rTMA time on Aptima assay, and ability to recover complete genome. T. pallidum genome copies, as determined by tp47 quantitative polymerase chain reaction, were highly correlated with rTMA time taken directly from the Hologic Aptima assay (r = −0.78). Complete genomes were defined as having <5.3% missing data in the genome (green), while incomplete genomes had >5.3% missing data (red). We did not attempt capture on specimens that had <10 copies/µL extracted DNA, based on prior experience (light blue). Abbreviation: TMA, transcription-mediated assay.

Figure 2.

Maximum likelihood phylogeny of Treponema pallidum strains in Seattle from 2021 to 2022. Red nodes labeled with letters indicate high-confidence bootstrap values >0.9. Strains sequenced from paired pharyngeal (beginning with P or ending with O) and rectal (R) swab specimens obtained at the same time from 3 separate individuals are highlighted in pink, green, and yellow. Node C (P−22−20006 and UW15977O) consists of paired pharyngeal swab samples from the same individual on the same day and is highlighted in purple. Samples collected for study 1 begin with UW, and those collected for study 2 begin with P, R, or V. Reference sequences for cultured strains Nichols (NC_021490) and SS14 (NC_021508) are used for orientation. Abbreviations: AZ, azithromycin; MSM, men who have sex with men; NA, not applicable; SNVs, single-nucleotide polymorphisms.

Figure 3.

tprK V region sequence proportion correlations from strains in this study. A, tprK V region sequences and proportions were compared pairwise, and Pearson correlation coefficients were determined. More similar patterns of V region use result in higher coefficients. The outline of the phylogeny from Figure 2 (slightly modified to include only strains yielding tprK sequence data) is shown for orientation. Patient-matched samples are shown in the same color. B, Pearson coefficients are significantly different among patient-matched, node F cluster–associated, and within the same or opposite lineages. *P < .05; ***P < .001 (1-way analysis of variance with the Tukey honestly significant difference test).

Figure 4.

Nichols-lineage strain with a 528–base pair (bp) deletion in tprK donor site locus. Sequencing read depth is depicted across the tprK donor site locus for 3 T. pallidum strains sequenced in this study, using SS14 genome reference NC_021508.1. The 528-bp deletion that removes 9 donor sites is denoted by red dotted lines in strain P-22-20168. A 51-bp deletion present in all sequenced Nichols strains is depicted in strain P-21-20135. SS14-lineage strains have neither deletion, as indicated by sequencing depth for strain UW15970L.

Figure 5.

tprK variable region composition and donor site usage in Treponema pallidum strains with donor site deletion. A, Among Nichols-lineage strains isolated from secondary syphilis lesions, diversity, as measured by the total number of unique V regions detected, is lower in strains bearing a deletion of 9 donor sites (P < .05; Welch's t test). B, As a proportion of total V regions, tprK V regions that incorporate the deleted donor sites are less abundant in strains with a deletion of 9 donor sites. C, Map of predicted donor site usage by strain. Donor sites are arranged relative to genomic coordinates; the x-axis is not to scale. Seven donor sites not found in any strain are excluded from the visualization. Green squares indicate the number of tprK V region sequences in which each donor site is found. Gray squares indicate the sum of use of each donor site across all samples, with darker gray indicating higher frequency. The deleted region is boxed in red in the affected strains.