HIV Infection Modifies the Role of Prior Treponema pallidum Infection in the Clinical Presentation of Early Syphilis Among Adult Patients From Sexually Transmitted Infection Clinics in Peru.

BACKGROUND: We aimed to compare the clinical presentations (symptomatic vs. asymptomatic) with prior Treponema pallidum infection status (first infection vs. reinfection) among people with early syphilis. METHODS: We used data from PICASSO, a cohort study in Peru that enrolled people with active syphilis from May 2019 to August 2021. Study participants had early syphilis and a prior syphilis serological test result within the prior 12 months to determine prior T. pallidum infection status. We calculated prevalence ratios (PRs) of symptomatic clinical presentation (primary or secondary syphilis) by prior T. pallidum infection status, stratified by HIV infection status. In addition, we explored the association of prior T. pallidum infection status and lesion presentation, stratified by primary and secondary syphilis cases, using the Fisher exact test. RESULTS: We include 84 T. pallidum reinfection cases and 61 first infection cases. We found increased frequency of symptomatic clinical presentation among first-infection cases (39% vs. 20%; PR, 1.94; P = 0.014). This association was stronger among persons living without HIV infection (38% vs. 7%; adjusted PR, 6.63; P = 0.001) in comparison to those living with HIV infection (45% vs. 34%; adjusted PR, 1.38; P = 0.458). Among secondary syphilis cases, more participants from the reinfection group reported that their lesions improved 1 week after treatment (100% vs. 29%, P = 0.045) compared with those with a first infection. Among the primary syphilis cases, all participants reported that their lesions improved 1 week after treatment. CONCLUSIONS: Prior syphilis was associated with a decreased prevalence of symptomatic reinfection, especially among persons not living with HIV infection.

Quality control and external quality assessment for the independent clinic-based evaluation of point-of-care testing to detect Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis in eight countries.

BACKGROUND: Sexually transmitted infections caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) remain significant global health problems. The World Health Organization (WHO) has recently conducted a multi-faceted, multi-country validation study (ProSPeRo), which included an evaluation of the Xpert CT/NG and Xpert TV assays on the GeneXpert system (Cepheid, Sunnyvale, Ca., USA) in clinic-based settings across eight countries. To support the study, a training and quality management system was implemented and evaluated. METHODS: A comprehensive training program for the study was developed. Quality control (QC) and external quality assessment (EQA) samples were provided by an accredited quality assurance provider. QC testing was conducted at 14 point-of-care testing (POCT) clinics, while EQA samples were tested by the POCT sites and a reference laboratory supporting each clinic. RESULTS: For QC testing, concordance with the expected results for CT and NG was > 99% and rates of unsuccessful tests were < 4%. For TV testing, concordance was similar (97%), but rates of unsuccessful tests were high (18%), particularly in the 'TV negative' sample. For EQA testing initially conducted in 2018, concordance was 100% for CT and NG, and 90% for TV for the reference laboratory group (which used non-GeneXpert systems). Concordance for the POCT group was also high (> 94%) for all analytes, but this cohort (which used GeneXpert systems) exhibited a high rate of unsuccessful TV tests. All but one of these unsuccessful tests was subcategorised as 'invalid'. CONCLUSIONS: The high level of concordance for QC and EQA testing confirm that the trained operators at the POC clinical sites were competent to conduct POC testing and that the training and quality systems implemented for the ProSPeRo study were effective. The quality materials used were satisfactory for CT and NG but exhibited poor performance for TV testing on the GeneXpert system. The WHO should continue to work with industry and EQA providers to provide improved materials that are reliable, stable and cost effective for quality management, as it seeks to rollout molecular-based STI POCT in non-laboratory-based settings. TRIAL REGISTRATION: Ethics approval to conduct the ProSPeRo study was granted by the WHO Ethics Review Committee.

External quality assessment to support the WHO ProSPeRo study for the evaluation of two dual HIV/syphilis point-of-care tests in seven countries.

BACKGROUND: Sexually transmitted infections (STIs) such as syphilis and HIV remain to be a significant public health issue worldwide. Dual rapid point-of-care tests (POCTs) have shown promise for detecting antibodies to HIV and syphilis but have not been fully evaluated in the field. Our study supported the WHO ProSPeRo study on Sexually Transmitted Infection Point-of-Care Testing (STI POCT) by providing external quality assessment (EQA) for HIV and syphilis testing in reference laboratories and their associated clinical sites in seven countries. METHODS: HIV/syphilis serum liquid and dried tube specimen (DTS) panels were prepared by CDC. Liquid panels were distributed to the reference laboratories for three rounds of testing using commercially and locally available laboratory-based serological tests. DTS panels were sent to the clinical testing sites for 8 rounds of POC testing using the Abbott SD BIOLINE HIV/Syphilis Duo test (hereafter referred to as SD BIOLINE) and the Chembio Dual Path Platform (DPP) HIV-Syphilis assay. EQA panels were tested at CDC using the Rapid Plasma Reagin (RPR) test and the Treponema pallidum Particle Agglutination assay (TP-PA) for syphilis antibodies. Genetic Systems HIV-1/HIV-2 Plus O EIA, Geenius HIV Supplemental Assay and the Oraquick Advance HIV test were used to detect HIV antibodies in the EQA panels. Results from the reference laboratories and POCT sites were compared to those obtained at the CDC and a percentage agreement was calculated. RESULTS: Qualitative RPR and TP-PA performed at the reference laboratories demonstrated 95.4-100% agreement with CDC results while quantitative RPR and TP-PA tests demonstrated 87.7% and 89.2% agreement, respectively. A 93.8% concordance rate was observed for qualitative HIV testing in laboratories. EQA testing at clinical sites using dual tests showed 98.7% and 99.1% agreement for detection of HIV antibodies and eight out of 10 sites had > 95.8% agreement for syphilis testing. However, two clinical sites showed only 65.0-66.7% agreement for SD BIOLINE and 84.0-86.7% for DPP, respectively, for syphilis testing. CONCLUSIONS: Overall, laboratories demonstrated high EQA performance in this study. Both HIV/syphilis POCTs gave expected results in the clinic-based evaluations using DTS. However, testing errors were identified in a few testing sites suggesting the necessity for continuous training and monitoring the quality of POC testing.

Independent clinic-based evaluation of dual POCTs for screening for HIV and syphilis in men who have sex with men in Italy, Malta, Peru, and the United Kingdom.

INTRODUCTION: Globally, the incidence of HIV and syphilis can be reduced by the use of validated point of care tests (POCTs). As part of the WHO PRoSPeRo Network, we aimed to evaluate the performance, acceptability, and operational characteristics of two dual HIV/syphilis POCTs (Bioline HIV/Syphilis Duo (Abbott) and DPP® HIV-Syphilis assay (Chembio) for the screening of HIV and syphilis amongst men who have sex with men (MSM). METHOD AND ANALYSES: A cross sectional study of 2,577 MSM in Italy, Malta, Peru, and the United Kingdom (UK) presenting to seven clinic sites, were enrolled. Finger prick blood was collected to perform POCTs and results compared with standard laboratory investigations on venepuncture blood. Acceptability and operational characteristics were assessed using questionnaires. Diagnostic meta-analysis was used to combine data from the evaluation sites. RESULTS: Based on laboratory tests, 23.46% (n = 598/2549) of participants were confirmed HIV positive, and 35.88% of participants (n = 901/2511) were positive on treponemal reference testing. Of all participants showing evidence of antibodies to Treponema pallidum, 50.56% (n = 455/900) were Rapid Plasma Reagin (RPR) test reactive. Of HIV positive individuals, 60.62% (n = 354/584) had evidence of antibodies to T. pallidum, and of these 60.45% (n = 214/354) exhibited reactive RPR tests indicating probable (co)infection. For Bioline POCT, pooled sensitivities and specificities for HIV were 98.95% and 99.89% respectively, and for syphilis were 73.79% and 99.57%. For Chembio pooled sensitivities and specificities for HIV were 98.66% and 99.55%, and for syphilis were 78.60% and 99.48%. Both tests can detect greater than 90% of probable active syphilis cases, as defined by reactive RPR and treponemal test results. These dual POCTs were preferred by 74.77% (n = 1,926) of participants, due to their convenience, and the operational characteristics made them acceptable to health care providers (HCPs). CONCLUSIONS: Both the Bioline and the Chembio dual POCT for syphilis and HIV had acceptable performance, acceptability and operational characteristics amongst MSM in the PRoSPeRo network. These dual POCTs could serve as a strategic, more cost effective, patient and healthcare provider (HCP) friendly alternative to conventional testing; in clinical and other field settings, especially those in resource-limited settings.

Clinic-based evaluation of the dual Xpert CT/NG assay on the GeneXpert System for screening for extragenital chlamydial and gonococcal infections amongst men who have sex with men.

BACKGROUND: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections have increased globally. Asymptomatic infections represent a significant risk of long-term complications. Men who have sex with men (MSM) are disproportionally affected, underscoring the need to offer screening programmes to this population. CT/NG Point of Care Testing (POCT) constitutes a strategic tool to improve the continuum of STI care, however extensive real-life evaluations amongst at risk populations are lacking. The aim of this study is to estimate the GeneXpert CT/NG assay performance and usability for CT and NG at genital and extragenital sites for screening amongst MSM. METHODS: This study was a multi-site sexual health clinic-based evaluation (Italy, Malta and Peru) with consecutive enrolment. A first void urine sample (divided in two aliquots), two oropharyngeal and two anorectal swabs were collected for each study participant. One specimen set (one for each anatomical site) was tested with the dual index test (Cepheid) at the clinics by the healthcare staff, the other set with FDA/CE approved Nucleic Acid Amplification Tests (NAATs) at the laboratory. Clinical sites and reference laboratories participated in an internal and external quality control programme. Sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values for each anatomical site were estimated using a meta-analytic approach. RESULTS: One thousand seven hundred two MSM were recruited across all clinical sites for a total of 5049 biological specimens. NG and CT were respectively detected in 274 and 287 of samples. Overall, the NG POCT sensitivity and specificity was 91.43% and 99.75% in urine (LR + 372.80, LR- 0.09), 89.68% and 99.55% in rectal specimens (LR + 197.30, LR- 0.10) and 75.87% and 98.77% at the pharynx respectively (LR + 61.94, LR- 0.24). The CT component of the POCT sensitivity was 84.82% and specificity 99.63% in urine (LR + 228.68, LR- 0.15), 78.07% and 99.19% respectively on rectal site (LR + 96.23, LR-0.22), 67.79% and 99.88% respectively at pharyngeal site (LR + 554.89, LR- 0.32). 95.95% of MSM reported to be willing to wait for POCT results and no provider reported difficulties in terms of performance or interpretation of the results of the Xpert CT/NG. CONCLUSION: Rapid turnaround time, ease of use and high acceptability make the Xpert CT/NG testing system a strategic tool for increasing testing frequency, reaching those not yet tested and offering the possibility of immediate treatment if needed. The assay showed good negative likelihood ratios and confirms its use to rule out CT/NG infections. Sensitivity varied across sites and pathogens. Periodic staff training at the testing sites should be mandatory.

Evaluation of a minimal array of Treponema pallidum antigens as biomarkers for syphilis diagnosis, infection staging, and response to treatment.

IMPORTANCE: This manuscript explores the host humoral response to selected antigens of the syphilis agent during infection to evaluate their potential use as diagnostic tests and markers for treatment.

Does genital herpes symptom history predict herpes simplex virus type 2 antibody positivity?

BACKGROUND: Sexually transmitted infections (STIs) associated with genital ulcer disease due to herpes simplex virus-2 (HSV-2) are a prominent cause of morbidity and mortality. Serologic screening for HSV-2 is recommended only for individuals with genital herpes symptom history. However, no validated symptom screening tool currently exists. METHODS: Currently asymptomatic adults presenting for routine care at STI clinics in Lima, Peru completed a survey of prior genital herpes symptoms and received HSV-2 serological testing with the Euroimmun Anti-HSV-2 (gG2) ELISA IgG (Lubeck, Germany). A sub-sample of indeterminate results were sent for Western blot confirmatory testing. We assessed associations between past symptoms and anti-HSV-2 positivity and corrected the HSV-2 prevalence by re-classifying indeterminates per Western Blot results. RESULTS: We enrolled 131 participants between July and October 2022. HSV-2 antibody test results found 21.4% positive, 41.2% indeterminate, and 37.4% negative. Excluding indeterminate results, 36.4% were positive. Of participants with no prior symptoms 31.2% tested positive, compared to 35.7% with one prior symptom, 50.0% with 2, and 50.0% with 3+ prior symptoms. Among the sub-sample of indeterminates, 92.6% were confirmed positive by Western Blot, bringing the total estimated proportion of participants with HSV-2 antibodies to 59.5%. Either based on the original classification of HSV-2 antibody status or after incorporation of confirmatory testing results, there was no significant association between symptom history and HSV-2 antibody positivity. CONCLUSIONS: With currently available tests, recommendations to screen individuals based on genital herpes symptom history may not be useful. More discriminatory symptom screening tools or HSV-2 antibody tests with better performance are needed.

Recent HIV infection and annualized HIV incidence rates among sexual and gender minorities in Brazil and Peru (ImPrEP seroincidence study): a cross-sectional, multicenter study.

Background: HIV incidence estimation is critical for monitoring the HIV epidemic dynamics and the effectiveness of public health prevention interventions. We aimed to identify sexual and gender minorities (SGM) with recent HIV infections, factors associated with recent HIV infection, and to estimate annualised HIV incidence rates. Methods: Cross-sectional multicentre study in HIV testing services in Brazil and Peru (15 cities). Inclusion criteria: 18+ years, SGM assigned male at birth, not using pre-/post-exposure prophylaxis. We identified recent HIV infection using the Maxim HIV-1 LAg-Avidity EIA assay as part of a recent infection testing algorithm (RITA). Annualized HIV incidence was calculated using the UNAIDS/WHO incidence estimator tool. Multivariable logistic regression models were used to estimate factors associated with recent HIV infection. Trial registration: NCT05674682. Findings: From 31-Jan-2021 to 29-May-2022, 6899 individuals participated [Brazil: 4586 (66.5%); Peru: 2313 (33.5%)]; 5946 (86.2%) cisgender men, 751 (10.9%) transgender women and 202 (2.9%) non-binary/gender diverse. Median age was 27 (IQR: 23-34) years. HIV prevalence was 11.4% (N = 784/6899); 137 (2.0%) SGM were identified with recent HIV infection. The overall annualized HIV incidence rate was 3.88% (95% CI: 2.86-4.87); Brazil: 2.62% (95% CI: 1.78-3.43); Peru: 6.69% (95% CI: 4.62-8.69). Participants aged 18-24 years had higher odds of recent HIV infection compared to those aged 30+ years in both countries. Interpretation: Our results highlight the significant burden of HIV epidemic among SGM in large urban centres of Brazil and Peru. Public health policies and interventions to increase access to effective HIV prevention methods such as PrEP are urgently needed in Latin America. Funding: Unitaid, WHO (Switzerland), Ministry of Health from Brazil and Peru.

Molecular Characterization of Genital and Extragenital Lesions With the PlexPCR VHS Assay in Patients Diagnosed With Syphilis.

Background: Syphilis diagnosis relies on immunologic markers and clinical protocols. However, syphilitic lesions can be confused with other genital ulcer diseases. Methods: Using a PlexPCR VHS assay, we analyzed lesion DNA samples from 87 individuals who were clinically diagnosed with early syphilis infection and had at least 1 positive serologic test result. DNA was detected by the PlexPCR VHS multiplex assay and ß-globin genes. Results: Among the participants, 99% (86/87) had a positive rapid treponemal test result. DNA was successfully detected in 91% (79/87) of the lesion samples. PlexPCR VHS identified 5 herpes simplex virus (HSV)/Treponema pallidum coinfections (2 HSV-1 and 3 HSV-2), only T pallidum DNA in 62% (49/79), and only HSV-2 in 12.7% (10/79). While 19% (15/79) were negative for all pathogens, none were varicella zoster virus positive. The PlexPCR VHS had 68.4% agreement with the clinical diagnosis. Conclusions: Since the PlexPCR VHS detects multiple organisms simultaneously, it can help to confirm actual syphilis and identify other pathogen coinfections or the pathogen causing the ulcer.

Mycoplasma genitalium prevalence, antimicrobial resistance-associated mutations, and coinfections with non-viral sexually transmitted infections in high-risk populations in Guatemala, Malta, Morocco, Peru and South Africa, 2019-2021.

The prevalence of Mycoplasma genitalium (MG) and MG antimicrobial resistance (AMR) appear to be high internationally, however, prevalence data remain lacking globally. We evaluated the prevalence of MG and MG AMR-associated mutations in men who have sex with men (MSM) in Malta and Peru and women at-risk for sexually transmitted infections in Guatemala, South Africa, and Morocco; five countries in four WHO regions mostly lacking MG prevalence and AMR data, and estimated MG coinfections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Male urine and anorectal samples, and vaginal samples were tested for MG, CT, NG, and TV (only vaginal samples) using Aptima assays (Hologic). AMR-associated mutations in the MG 23S rRNA gene and parC gene were identified using ResistancePlus MG kit (SpeeDx) or Sanger sequencing. In total, 1,425 MSM and 1,398 women at-risk were recruited. MG was detected in 14.7% of MSM (10.0% in Malta and 20.0% Peru) and in 19.1% of women at-risk (12.4% in Guatemala, 16.0% Morocco, 22.1% South Africa). The prevalence of 23S rRNA and parC mutations among MSM was 68.1 and 29.0% (Malta), and 65.9 and 5.6% (Peru), respectively. Among women at-risk, 23S rRNA and parC mutations were revealed in 4.8 and 0% (Guatemala), 11.6 and 6.7% (Morocco), and 2.4 and 3.7% (South Africa), respectively. CT was the most frequent single coinfection with MG (in 2.6% of MSM and 4.5% of women at-risk), compared to NG + MG found in 1.3 and 1.0%, respectively, and TV + MG detected in 2.8% of women at-risk. In conclusion, MG is prevalent worldwide and enhanced aetiological MG diagnosis, linked to clinical routine detection of 23S rRNA mutations, in symptomatic patients should be implemented, where feasible. Surveillance of MG AMR and treatment outcome would be exceedingly valuable, nationally and internationally. High levels of AMR in MSM support avoiding screening for and treatment of MG in asymptomatic MSM and general population. Ultimately, novel therapeutic antimicrobials and/or strategies, such as resistance-guided sequential therapy, and ideally an effective MG vaccine are essential.

Extra-genital Neisseria gonorrhoeae infections with genetic mutations conferring ciprofloxacin resistance among men who have sex with men and transgender women in Lima, Peru.

BACKGROUND: The increasing prevalence of drug-resistant Neisseria gonorrhoeae (NG) infections has caused great concern. Ciprofloxacin remains the empiric antimicrobial recommended to treat NG infections in Peru disregarding the susceptibility profile of circulating NG strains. We report the prevalence of individuals infected with NG strains presenting mutations in the gyrA gene that confers ciprofloxacin resistance. METHODS: We conducted a descriptive study assessing extragenital swab samples collected from a cohort of men who have sex with men and transgender women in Lima, Peru. Anal and pharyngeal NG positive swabs for Aptima Combo 2 assay (Hologic Inc., USA) were used for DNA extraction. We performed TaqMan real time PCR assays to detect a point mutation at codon Ser91 of the gyrase A (gyrA) gene. RESULTS: From 156 individuals who had at least one positive sample for NG reported by the Aptima assay, 80 individuals had at least one amplified DNA for the gyrA gene. We found that 67 of them (84.0%) were infected with a gyrA-mutated NG strain at the Ser91 codon. CONCLUSIONS: We report a high prevalence of gyrA mutation conferring ciprofloxacin resistance among individuals with extragenital NG infection. Empirical treatment of NG needs to be urgently updated in Peru in concordance with international guidelines.

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.

Laboratory Evaluation of the DPP Syphilis Screen & Confirm Assay.

Because syphilis is a public health concern, new strategies and tools for detecting active syphilis cases should be evaluated for future implementation. We assessed the laboratory performance of the DPP Syphilis Screen & Confirm rapid immunodiagnostic test (Chembio Diagnostics, Medford, NY, USA), using visual reading and the manufacturer's electronic test microreader, for detection of treponemal and nontreponemal antibodies in 383 fully characterized stored serum specimens. We used the Treponema pallidum particle agglutination (TPPA) test and rapid plasma reagin (RPR) test as reference tests for the DPP Syphilis Screen & Confirm assay treponemal and nontreponemal components, respectively. The sensitivity values for treponemal antibody detection by electronic reader and visual interpretation were 83.2% and 85.9%, respectively, with 100% specificity. For nontreponemal antibody detection, the sensitivity values were 65.7% and 69.0% and the specificity values were 88.7% and 89.4% for electronic reader and visual interpretation, respectively. There was excellent correlation between visual interpretation and the microreader for either component (kappa coefficient, 0.953). When restricting the analysis to RPR titers of ≥1:8, the sensitivity was 96.9% for either reading method; numerical microreader values showed good correlation with RPR titers (Spearman rho of 0.77). The DPP Syphilis Screen & Confirm assay showed good performance, compared to reference syphilis tests, using serum. Field evaluation studies should be done to validate its use for detection of active cases and for monitoring of treated syphilis patients. IMPORTANCE Syphilis remains a public health problem; therefore, health systems must incorporate screening tools that allow a rapid and accurate diagnosis to provide adequate treatment. The DPP Syphilis Screen & Confirm Assay simultaneously detects treponemal and nontreponemal antibodies, emerging as an alternative for identifying cases in situations in which there is no infrastructure to perform conventional syphilis testing, but it is necessary to generate evidence regarding the performance of this technology in various scenarios. We found that the test performs well, compared to TPPA and RPR tests, using stored samples from participants at high risk of acquiring syphilis. Additionally, when the Chembio microreader was incorporated, similar results are obtained by the device, compared to those reported by trained laboratory professionals, and correlated with the semiquantitative results of the RPR test. We think that the use of the DPP Syphilis Screen & Confirm Assay with the microreader might help in detecting active syphilis cases and perhaps in monitoring treatment responses in the field.

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.

Transcriptional and immunological analysis of the putative outer membrane protein and vaccine candidate TprL of Treponema pallidum.

BACKGROUND: An effective syphilis vaccine should elicit antibodies to Treponema pallidum subsp. pallidum (T. p. pallidum) surface antigens to induce pathogen clearance through opsonophagocytosis. Although the combination of bioinformatics, structural, and functional analyses of T. p. pallidum genes to identify putative outer membrane proteins (OMPs) resulted in a list of potential vaccine candidates, still very little is known about whether and how transcription of these genes is regulated during infection. This knowledge gap is a limitation to vaccine design, as immunity generated to an antigen that can be down-regulated or even silenced at the transcriptional level without affecting virulence would not induce clearance of the pathogen, hence allowing disease progression. PRINCIPAL FINDINGS: We report here that tp1031, the T. p. pallidum gene encoding the putative OMP and vaccine candidate TprL is differentially expressed in several T. p. pallidum strains, suggesting transcriptional regulation. Experimental identification of the tprL transcriptional start site revealed that a homopolymeric G sequence of varying length resides within the tprL promoter and that its length affects promoter activity compatible with phase variation. Conversely, in the closely related pathogen T. p. subsp. pertenue, the agent of yaws, where a naturally-occurring deletion has eliminated the tprL promoter region, elements necessary for protein synthesis, and part of the gene ORF, tprL transcription level are negligible compared to T. p. pallidum strains. Accordingly, the humoral response to TprL is absent in yaws-infected laboratory animals and patients compared to syphilis-infected subjects. CONCLUSION: The ability of T. p. pallidum to stochastically vary tprL expression should be considered in any vaccine development effort that includes this antigen. The role of phase variation in contributing to T. p. pallidum antigenic diversity should be further studied.

Investigation of syphilis immunology and Treponema pallidum subsp. pallidum biology to improve clinical management and design a broadly protective vaccine: study protocol.

BACKGROUND: The syphilis epidemic continues to cause substantial morbidity and mortality worldwide, particularly in low- and middle-income countries, despite several recent disease control initiatives. Though our understanding of the pathogenesis of this disease and the biology of the syphilis agent, Treponema pallidum subsp. pallidum has improved over the last two decades, further research is necessary to improve clinical diagnosis and disease management protocols. Additionally, such research efforts could contribute to the identification of possible targets for the development of an effective vaccine to stem syphilis spread. METHODS: This study will recruit two cohorts of participants with active syphilis infection, one with de novo infection, one with repeat infection. Whole blood specimens will be collected from each study participant at baseline, 4, 12, 24, 36, and 48 weeks, to track specific markers of their immunological response, as well as to compare humoral reactivity to Treponema pallidum antigens between the two groups. Additionally, we will use serum specimens to look for unique cytokine patterns in participants with early syphilis. Oral and blood samples, as well as samples from any syphilitic lesions present, will also be collected to sequence any Treponema pallidum DNA found. DISCUSSION: By furthering our understanding of syphilis pathogenesis and human host immune response to Treponema pallidum, we will provide important data that will help in development of new point-of-care tests that could better identify active infection, leading to improved syphilis diagnosis and management. Findings could also contribute to vaccine development efforts.

High Lifetime Prevalence of Syphilis in Men Who Have Sex With Men and Transgender Women Versus Low Lifetime Prevalence in Female Sex Workers in Lima, Peru.

BACKGROUND: The syphilis epidemic continues to cause substantial morbidity worldwide and is worsening despite ongoing control efforts. Syphilis remains an important public health problem among 3 key populations: men who have sex with men (MSM), transgender women, and female sex workers. METHODS: We conducted a retrospective chart review of patients that received rapid point-of-care treponemal antibody tests from January 2019 to July 2019 in 4 sexually transmitted infection (STI) clinics in Lima, Peru. We assessed patient medical records for human immunodeficiency virus (HIV) infection, history of STIs, as well as sociodemographic and behavioral characteristics. Cross-sectional descriptive analyses were used to determine factors associated with treponemal positivity. RESULTS: We included 401 patient records in our analyses: 252 MSM, 31 transgender women, and 118 female sex workers. The overall median age of patients was 29.0 years (interquartile range, 24.0-36.0 years). Positivity on the treponemal test was 28.9% (95% confidence interval [CI], 24.3%-33.3%) overall, 37.7% (95% CI, 31.7%-44.0%) for MSM, 54.8% (95% CI, 36.0%-72.7%) for transgender women, and 3.4% (95% CI, 0.9%-8.5%) for female sex workers. In the bivariate analysis, treponemal positivity was also associated with receptive anal sex in the last 6 months in MSM (P < 0.01). Additionally, treponemal positivity increased with age (P = 0.0212) and varied by socioeconomic status (P < 0.01). Multivariate Least Absolute Shrinkage and Selection Operator logistic regression showed that treponemal positivity was highly associated with HIV coinfection (adjusted odds ratio, 5.42) and previous STI other than HIV or syphilis (adjusted odds ratio, 1.54). CONCLUSIONS: A review of the medical records of members of 3 key populations who had recently received a rapid point-of-care treponemal test in Lima, Peru, revealed that lifetime prevalence of syphilis was high among MSM and transgender women, but low among female sex workers. Those results may indicate a need for more frequent, regular testing among MSM and transgender women-possibly in conjunction with HIV testing, and appropriate treatment of those shown to be positive.

The Application of Cytokine Expression Assays to Differentiate Active From Previously Treated Syphilis.

To investigate the role of serum cytokine assays to distinguish between active from treated syphilis among serofast patients, we recruited individuals into a prospective cohort study. Participants underwent routine syphilis screening. We selected specimens from a majority cohort of serofast participants with treated and active syphilis. We analyzed specimens with a 62-cytokine multiplex bead-based enzyme-linked immunosorbent assay. Cytokines, brain-derived neurotrophic factor and tumor necrosis factor ß, were most predictive. We built a decision tree that was 82.4% accurate, 100% (95% confidence interval, 82%-100%) sensitive, and 45% (18%-75%) specific. Our decision tree differentiated between serum specimens from serofast participants with treated syphilis versus active syphilis.

Venue-Based HIV-Testing: An Effective Screening Strategy for High-Risk Populations in Lima, Peru.

Venue-based testing may improve screening efforts for HIV and syphilis, thereby reducing transmission. We offered onsite rapid dual HIV and syphilis testing at venues popular among MSM and/or transgender women in Lima, Peru. We used Poisson regression to calculate adjusted prevalence ratios (aPRs) for factors associated with each infection. Most (90.4%) of the 303 participants would test more frequently if testing was available at alternative venues. New cases of HIV (69) and syphilis infection (84) were identified. HIV was associated with recent sex work (aPR 1.11; 95% CI 1.02-1.22), sex with a partner of unknown serostatus (aPR 1.18; 95% CI 1.09-1.27), exclusively receptive anal sex role (aPR 1.16; 95% CI 1.03-1.30) or versatile sex role (aPR 1.17; 95% CI 1.06-1.30) compared to insertive. Syphilis was associated with reporting role versatility (aPR = 2.69; 95% CI 1.52-5.74). Sex work venues had higher syphilis prevalence 47% versus 28% in other venues, p value = 0.012. Venue-based testing may improve case finding.