Immunization with a tri-antigen syphilis vaccine significantly attenuates chancre development, reduces bacterial load, and inhibits dissemination of Treponema pallidum.

Syphilis continues to be a significant public health concern worldwide. The disease is endemic in many low- and middle-income countries, and rates have risen sharply in high-income countries over the last decade. The continued prevalence of infectious and congenital syphilis worldwide highlights the need for the development of an effective syphilis vaccine to complement public health measures for syphilis control. The complex, multi-stage course of syphilis infection necessitates a holistic approach to the development of an effective vaccine, in which immunization prevents both the localized stage of infection (typified by the highly infectious chancre) and the disseminated stages of infection (typified by the secondary rash, neurosyphilis, and destructive tertiary lesions, as well as congenital syphilis). Inhibiting development of the infectious chancre would reduce transmission thus providing community- level protection, while preventing dissemination would provide individual-level protection by reducing serious sequelae and may also provide community level protection by reducing shedding during secondary syphilis. In the current study we build upon prior investigations which demonstrated that immunizations with individual, well characterized T. pallidum TprK, TprC, and Tp0751 peptides elicits partial protection against infection in the animal model. Specifically, we show here that immunization with a TprC/TprK/Tp0751 tri-antigen cocktail protects animals from progressive syphilis lesions and substantially inhibits dissemination of the infection.

High-throughput nanopore sequencing of Treponema pallidum tandem repeat genes arp and tp0470 reveals clade-specific patterns and recapitulates global whole genome phylogeny.

Sequencing of most Treponema pallidum genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modeling of TP0470 suggested that the eight residue repeats form an extended α-helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged ß-sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

B-Cell Epitope Mapping of TprC and TprD Variants of Treponema pallidum Subspecies Informs Vaccine Development for Human Treponematoses.

Several recent studies have focused on the identification, functional analysis, and structural characterization of outer membrane proteins (OMPs) of Treponema pallidum (Tp). The Tp species encompasses the highly related pallidum, pertenue, and endemicum subspecies of this pathogen, known to be the causative agents of syphilis, yaws, and bejel, respectively. These studies highlighted the importance of identifying surface-exposed OMP regions and the identification of B-cell epitopes that could be protective and used in vaccine development efforts. We previously reported that the TprC and TprD OMPs of Tp are predicted to contain external loops scattered throughout the entire length of the proteins, several of which show a low degree of sequence variability among strains and subspecies. In this study, these models were corroborated using AlphaFold2, a state-of-the-art protein structure modeling software. Here, we identified B-cell epitopes across the full-length TprC and TprD variants using the Geysan pepscan mapping approach with antisera from rabbits infected with syphilis, yaws, and bejel strains and from animals immunized with refolded recombinant TprC proteins from three syphilis strains. Our results show that the humoral response is primarily directed to sequences predicted to be on surface-exposed loops of TprC and TprD proteins, and that the magnitude of the humoral response to individual epitopes differs among animals infected with various syphilis strains and Tp subspecies. Rather than exhibiting strain-specificity, antisera showed various degrees of cross-reactivity with variant sequences from other strains. The data support the further exploration of TprC and TprD as vaccine candidates.

Previous Syphilis Alters the Course of Subsequent Episodes of Syphilis.

BACKGROUND: The influence of previous syphilis on the course of a subsequent episode is unknown. METHODS: Individuals enrolled in a study of cerebrospinal fluid (CSF) abnormalities in syphilis were allowed to enroll in the study again with subsequent syphilis. For each participant, the index episode was defined as the most recent syphilis episode for which the study entry visit was performed within 30 days of the syphilis diagnosis date. Venipuncture and lumbar puncture (LP) were performed. Total number of syphilis episodes was determined by review of medical and public health records. T. pallidum DNA in blood and rRNA in CSF were detected by polymerase chain reaction (PCR) and reverse transcriptase PCR. Odds ratios (ORs) with 95% confidence intervals (95% CI) were determined by logistic regression. RESULTS: 651 individuals had one (n = 482), two (n = 121) or three or more (n = 48) episodes of syphilis. The proportion of individuals whose index episode was early latent stage was significantly higher in those with ≥3 syphilis episodes; this relationship was reduced to a trend when rate of testing was taken into account. Adjusted odds (aOR) of detection of T. pallidum DNA in blood or rRNA in CSF at the index episode were significantly lower in those with previous syphilis (0.17 [95% CI, 0.09-0.31] and 0.15 [95% CI, 0.07-0.35]). The aOR for neurosyphilis at the index episode was also significantly lower in individuals with previous syphilis (0.54 [95% CI, 0.34-0.87]). CONCLUSIONS: Previous syphilis attenuates the manifestations of subsequent infection with T. pallidum.

Treponema pallidum genome sequencing from six continents reveals variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar.

In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains-including 191 T. pallidum subsp. pallidum-sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure.

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.

Analytical Performance Characteristics of a New Transcription-Mediated Amplification Assay for Treponema pallidum.

This study evaluated the performance characteristics of a new research-use-only transcription-mediated amplification (TMA) assay for the detection of rRNA from Treponema pallidum. Analytical sensitivity determined using dark-field microscopy-quantitated T. pallidum was 1.4 organisms/ml (95% confidence interval [CI], 0.7 to 6.33 organisms/ml). Dilution of in vitro-transcribed (IVT) T. pallidum RNA in Aptima sample transport medium (STM) yielded 100% positivity (n = 3) at 10 copies/ml (4 copies/reaction). Analytical specificity testing of nontarget microorganisms (n = 59), including the closely related nonsyphilis treponemes Treponema denticola and Treponema phagedenis, yielded 0% positivity. TMA testing of mucosal swab specimens collected from men who have sex with men (MSM) attending a sexually transmitted disease clinic yielded 1.8% (17/944) positive results. A collection of 56 serum specimens obtained from a separate cohort of patients with known rapid plasma reagin (RPR) statuses and clinical diagnoses of syphilis was 19.6% (11/56) TMA positive overall and 29.7% (11/37) positive among subjects with syphilis diagnoses, including 8 (36.3%) of 22 persons with primary or secondary syphilis, 2 (20%) of 10 persons with early latent syphilis, and 1 (20%) of 5 persons with late latent or unstaged syphilis. None (0%) of the 18 RPR-positive sera from patients with histories of treated syphilis were TMA positive. These results show that TMA is an analytically sensitive and specific method for the detection of T. pallidum rRNA and is compatible with serum specimens in addition to pharyngeal and rectal mucocutaneous swab specimens. Automated real-time TMA testing for T. pallidum may be useful as an adjunctive method with serology for screening and diagnostic testing of selected patient populations for syphilis.

Streptococcus pyogenes Is Associated with Idiopathic Cutaneous Ulcers in Children on a Yaws-Endemic Island.

Exudative cutaneous ulcers (CU) in yaws-endemic areas are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD), but one-third of CU cases are idiopathic (IU). Using mass drug administration (MDA) of azithromycin, a yaws eradication campaign on Lihir Island in Papua New Guinea reduced but failed to eradicate yaws; IU rates remained constant throughout the campaign. To identify potential etiologies of IU, we obtained swabs of CU lesions (n = 279) and of the skin of asymptomatic controls (AC; n = 233) from the Lihir Island cohort and characterized their microbiomes using a metagenomics approach. CU bacterial communities were less diverse than those of the AC. Using real-time multiplex PCR with pathogen-specific primers, we separated CU specimens into HD-positive (HD+), TP+, HD+TP+, and IU groups. Each CU subgroup formed a distinct bacterial community, defined by the species detected and/or the relative abundances of species within each group. Streptococcus pyogenes was the most abundant organism in IU (22.65%) and was enriched in IU compared to other ulcer groups. Follow-up samples (n = 31) were obtained from nonhealed ulcers; the average relative abundance of S. pyogenes was 30.11% in not improved ulcers and 0.88% in improved ulcers, suggesting that S. pyogenes in the not improved ulcers may be azithromycin resistant. Catonella morbi was enriched in IU that lacked S. pyogenes As some S. pyogenes and TP strains are macrolide resistant, penicillin may be the drug of choice for CU azithromycin treatment failures. Our study will aid in the design of diagnostic tests and selective therapies for CU.IMPORTANCE Cutaneous ulcers (CU) affect approximately 100,000 children in the tropics each year. While two-thirds of CU are caused by Treponema pallidum subspecies pertenue and Haemophilus ducreyi, the cause(s) of the remaining one-third is unknown. Given the failure of mass drug administration of azithromycin to eradicate CU, the World Health Organization recently proposed an integrated disease management strategy to control CU. Success of this strategy requires determining the unknown cause(s) of CU. By using 16S rRNA gene sequencing of swabs obtained from CU and the skin of asymptomatic children, we identified another possible cause of skin ulcers, Streptococcus pyogenes Although S. pyogenes is known to cause impetigo and cellulitis, this is the first report implicating the organism as a causal agent of CU. Inclusion of S. pyogenes into the integrated disease management plan will improve diagnostic testing and treatment of this painful and debilitating disease of children and strengthen elimination efforts.

Archaeogenetics: What Can Ancient Genomes Tell Us about the Origin of Syphilis?

The origin of syphilis has been hotly debated for decades. Ancient pathogen DNA may provide new evidence to redefine our understanding of this mystery, but is the mystery itself flawed in its assumptions?

Advancing the understanding of treponemal disease in the past and present.

Syphilis was perceived to be a new disease in Europe in the late 15th century, igniting a debate about its origin that continues today in anthropological, historical, and medical circles. We move beyond this age-old debate using an interdisciplinary approach that tackles broader questions to advance the understanding of treponemal infection (syphilis, yaws, bejel, and pinta). How did the causative organism(s) and humans co-evolve? How did the related diseases caused by Treponema pallidum emerge in different parts of the world and affect people across both time and space? How are T. pallidum subspecies related to the treponeme causing pinta? The current state of scholarship in specific areas is reviewed with recommendations made to stimulate future work. Understanding treponemal biology, genetic relationships, epidemiology, and clinical manifestations is crucial for vaccine development today and for investigating the distribution of infection in both modern and past populations. Paleopathologists must improve diagnostic criteria and use a standard approach for recording skeletal lesions on archaeological human remains. Adequate contextualization of cultural and environmental conditions is necessary, including site dating and justification for any corrections made for marine or freshwater reservoir effects. Biogeochemical analyses may assess aquatic contributions to diet, physiological changes arising from treponemal disease and its treatments (e.g., mercury), or residential mobility of those affected. Shifting the focus from point of origin to investigating who is affected (e.g., by age/sex or socioeconomic status) and disease distribution (e.g., coastal/ inland, rural/urban) will advance our understanding of the treponemal disease and its impact on people through time.

Yaws re-emergence and bacterial drug resistance selection after mass administration of azithromycin: a genomic epidemiology investigation.

BACKGROUND: In a longitudinal study assessing the WHO strategy for yaws eradication using mass azithromycin treatment, we observed resurgence of yaws cases with dominance of a single JG8 sequence type and emergence of azithromycin-resistant Treponema pallidum subspecies pertenue (T p pertenue). Here, we analyse genomic changes in the bacterial population using samples collected during the study. METHODS: We did whole bacterial genome sequencing directly on DNA extracted from 37 skin lesion swabs collected from patients on Lihir Island, Papua New Guinea, between April 1, 2013, and Nov 1, 2016. We produced phylogenies and correlated these with spatiotemporal information to investigate the source of new cases and the emergence of five macrolide-resistant cases. We used deep amplicon sequencing of surveillance samples to assess the presence of minority macrolide-resistant populations. FINDINGS: We recovered 20 whole T p pertenue genomes, and phylogenetic analysis showed that the re-emerging JG8 sequence type was composed of three bacterial sublineages characterised by distinct spatiotemporal patterns. Of five patients with resistant T p pertenue, all epidemiologically linked, we recovered genomes from three and found no variants. Deep sequencing showed that before treatment, the index patient had fixed macrolide-sensitive T p pertenue, whereas the post-treatment sample had a fixed resistant genotype, as did three of four contact cases. INTERPRETATION: In this study, re-emergence of yaws cases was polyphyletic, indicating multiple epidemiological sources. However, given the genomic and epidemiological linkage of resistant cases and the rarity of resistance alleles in the general population, azithromycin resistance is likely to have evolved only once in this study, followed by onward dissemination. FUNDING: Wellcome and Provincial Deputation of Barcelona.

Previous Syphilis Alters the Course of Subsequent Episodes of Syphilis.

BACKGROUND: Individuals with previous syphilis may be more likely to be asymptomatic when they are reinfected with Treponema pallidum. METHODS: Individuals enrolled in a study of cerebrospinal fluid (CSF) abnormalities in syphilis were allowed to enroll in the study again with subsequent syphilis. For each participant, the index episode was defined as the most recent syphilis episode for which the study entry visit was performed within 30 days of the syphilis diagnosis date. Venipuncture and lumbar puncture were performed. The total number of syphilis episodes was determined by review of medical and public health records. Treponema pallidum DNA in blood and rRNA in CSF were detected using polymerase chain reaction (PCR) and reverse transcriptase PCR. Odds ratios (ORs) with 95% confidence intervals (CIs) were determined using logistic regression. RESULTS: 701 individuals had 1 (n = 478), 2 (n = 155), or ≥3 (n = 68) episodes of syphilis. The proportion of individuals whose index episode was asymptomatic significantly increased with increased number of syphilis episodes (P < .001). This difference was not explained by frequency of serological tests. Adjusted ORs (aORs) of detection of T. pallidum DNA in blood or rRNA in CSF at the index episode were significantly lower in those with previous syphilis (0.13; 95% CI, .08-.23, and 0.06, 95% CI, .02-.17). The aOR of neurosyphilis at the index episode was also significantly lower in individuals with previous syphilis (0.43; 95% CI, .27-.68). CONCLUSIONS: Previous syphilis attenuates clinical and laboratory manifestations of infection with T. pallidum.

Genomic epidemiology of syphilis reveals independent emergence of macrolide resistance across multiple circulating lineages.

Syphilis is a sexually transmitted infection caused by Treponema pallidum subspecies pallidum and may lead to severe complications. Recent years have seen striking increases in syphilis in many countries. Previous analyses have suggested one lineage of syphilis, SS14, may have expanded recently, indicating emergence of a single pandemic azithromycin-resistant cluster. Here we use direct sequencing of T. pallidum combined with phylogenomic analyses to show that both SS14- and Nichols-lineages are simultaneously circulating in clinically relevant populations in multiple countries. We correlate the appearance of genotypic macrolide resistance with multiple independently evolved SS14 sub-lineages and show that genotypically resistant and sensitive sub-lineages are spreading contemporaneously. These findings inform our understanding of the current syphilis epidemic by demonstrating how macrolide resistance evolves in Treponema subspecies and provide a warning on broader issues of antimicrobial resistance.

Primary Syphilis in the Male Urethra: A Case Report.

We documented urethral Treponema pallidum infection in a man with nongonococcal urethritis and a negative syphilis serology using broad-range bacterial polymerase chain reaction (PCR) and sequencing, targeted PCR, and immunofluorescence microscopy. He subsequently seroconverted for syphilis. Early syphilis may present as urethritis. Urethral T. pallidum shedding can occur before seroconversion.

New Tools for Syphilis Research.

Syphilis research has been severely limited by the necessity to propagate Treponema pallidumin vivo in rabbits. After decades of erroneous or irreproducible reports of cultivation of T. pallidum, the recent very convincing report of its successful long-term in vitro propagation opens numerous opportunities for development of genetic tools for studying pathogenesis and protein function, antigenic variation, and surface exposure of antigens. The possibility of more rapid isolation of new strains will expand our knowledge of this organism beyond the century-old Nichols strain.

Biomedical Research Priorities for Modern Syphilis Clinical Management, Diagnosis, and Vaccines: Overview and Commentary for Unit 1.

The first session at the 2016 Syphilis Summit provided an opportunity for laboratory researchers and clinicians to comment on gaps in biomedical knowledge and technologies. Predominant themes in the presentations and discussion included the need for optimization of existing diagnostic tests, commercial availability, and Food and Drug Administration approval of nucleic acid amplification tests for primary and secondary syphilis, development of sensitive and specific new blood tests for diagnosis of active (vs treated) syphilis infection, clarification of the best measures for adequacy of response to treatment, continued study of complications of syphilis, including neurosyphilis and ocular syphilis, and development of a safe and effective vaccine that will protect against transmission and complications of disseminated infection (including congenital and neurosyphilis). Renewed and sustained support of biomedical syphilis research and an influx of talent could move the needle in the fight against this reemerging ancient disease.

Re-emergence of yaws after single mass azithromycin treatment followed by targeted treatment: a longitudinal study.

BACKGROUND: Yaws is a substantial cause of chronic disfiguring ulcers in children in at least 14 countries in the tropics. WHO's newly adopted strategy for yaws eradication uses a single round of mass azithromycin treatment followed by targeted treatment programmes, and data from pilot studies have shown a short-term significant reduction of yaws. We assessed the long-term efficacy of the WHO strategy for yaws eradication. METHODS: Between April 15, 2013, and Oct 24, 2016, we did a longitudinal study on a Papua New Guinea island (Lihir; 16 092 population) in which yaws was endemic. In the initial study, the participants were followed for 12 months; in this extended follow-up study, clinical, serological, and PCR surveys were continued every 6 months for 42 months. We used genotyping and travel history to identify importation events. Active yaws confirmed by PCR specific for Treponema pallidum was the primary outcome indicator. The study is registered with ClinicalTrials.gov, number NCT01955252. FINDINGS: Mass azithromycin treatment (coverage rate of 84%) followed by targeted treatment programmes reduced the prevalence of active yaws from 1·8% to a minimum of 0·1% at 18 months (difference from baseline -1·7%, 95% CI, -1·9 to -1·4; p<0·0001), but the infection began to re-emerge after 24 months with a significant increase to 0·4% at 42 months (difference from 18 months 0·3%, 95% CI 0·1 to 0·4; p<0·0001). At each timepoint after baseline, more than 70% of the total community burden of yaws was found in individuals who had not had the mass treatment or as new infections in non-travelling residents. At months 36 and 42, five cases of active yaws, all from the same village, showed clinical failure following azithromycin treatment, with PCR-detected mutations in the 23S ribosomal RNA genes conferring resistance to azithromycin. A sustained decrease in the prevalence of high-titre latent yaws from 13·7% to <1·5% in asymptomatic children aged 1-5 years old and of genetic diversity of yaws strains from 0·139 to less than 0·046 between months 24 and 42 indicated a reduction in transmission of infection. INTERPRETATION: The implementation of the WHO strategy did not, in the long-term, achieve elimination in a high-endemic community mainly due to the individuals who were absent at the time of mass treatment in whom yaws reactivated; repeated mass treatment might be necessary to eliminate yaws. To our knowledge, this is the first report of the emergence of azithromycin-resistant T p pertenue and spread within one village. Communities' surveillance should be strengthened to detect any possible treatment failure and biological markers of resistance. FUNDING: ISDIN laboratories, Newcrest Mining Limited, and US Public Health Service National Institutes of Health.

Diagnostics for Yaws Eradication: Insights From Direct Next-Generation Sequencing of Cutaneous Strains of Treponema pallidum.

Background: Yaws-like chronic ulcers can be caused by Treponema pallidum subspecies pertenue, Haemophilus ducreyi, or other, still-undefined bacteria. To permit accurate evaluation of yaws elimination efforts, programmatic use of molecular diagnostics is required. The accuracy and sensitivity of current tools remain unclear because our understanding of T. pallidum diversity is limited by the low number of sequenced genomes. Methods: We tested samples from patients with suspected yaws collected in the Solomon Islands and Ghana. All samples were from patients whose lesions had previously tested negative using the Centers for Disease Control and Prevention (CDC) diagnostic assay in widespread use. However, some of these patients had positive serological assays for yaws on blood. We used direct whole-genome sequencing to identify T. pallidum subsp pertenue strains missed by the current assay. Results: From 45 Solomon Islands and 27 Ghanaian samples, 11 were positive for T. pallidum DNA using the species-wide quantitative polymerase chain reaction (PCR) assay, from which we obtained 6 previously undetected T. pallidum subsp pertenue whole-genome sequences. These show that Solomon Islands sequences represent distinct T. pallidum subsp pertenue clades. These isolates were invisible to the CDC diagnostic PCR assay, due to sequence variation in the primer binding site. Conclusions: Our data double the number of published T. pallidum subsp pertenue genomes. We show that Solomon Islands strains are undetectable by the PCR used in many studies and by health ministries. This assay is therefore not adequate for the eradication program. Next-generation genome sequence data are essential for these efforts.

Development of a Multilocus Sequence Typing (MLST) scheme for Treponema pallidum subsp. pertenue: Application to yaws in Lihir Island, Papua New Guinea.

BACKGROUND: Yaws is a neglected tropical disease, caused by Treponema pallidum subsp. pertenue. The disease causes chronic lesions, primarily in young children living in remote villages in tropical climates. As part of a global yaws eradication campaign initiated by the World Health Organization, we sought to develop and evaluate a molecular typing method to distinguish different strains of T. pallidum subsp. pertenue for disease control and epidemiological purposes. METHODS AND PRINCIPAL FINDINGS: Published genome sequences of strains of T. pallidum subsp. pertenue and pallidum were compared to identify polymorphic genetic loci among the strains. DNA from a number of existing historical Treponema isolates, as well as a subset of samples from yaws patients collected in Lihir Island, Papua New Guinea, were analyzed using these targets. From these data, three genes (tp0548, tp0136 and tp0326) were ultimately selected to give a high discriminating capability among the T. pallidum subsp. pertenue samples tested. Intragenic regions of these three target genes were then selected to enhance the discriminating capability of the typing scheme using short readily amplifiable loci. This 3-gene multilocus sequence typing (MLST) method was applied to existing historical human yaws strains, the Fribourg-Blanc simian isolate, and DNA from 194 lesion swabs from yaws patients on Lihir Island, Papua New Guinea. Among all samples tested, fourteen molecular types were identified, seven of which were found in patient samples and seven among historical isolates or DNA. Three types (JG8, TD6, and SE7) were predominant on Lihir Island. CONCLUSIONS: This MLST approach allows molecular typing and differentiation of yaws strains. This method could be a useful tool to complement epidemiological studies in regions where T. pallidum subsp. pertenue is prevalent with the overall goals of improving our understanding of yaws transmission dynamics and helping the yaws eradication campaign to succeed.