Background: Transgender women (TGW) are disproportionately impacted by sexually transmitted infections (STIs). To date, few studies have been done on STIs in TGW from Latin America. We sought to estimate the prevalence of human immunodeficiency virus, human papillomavirus (HPV), hepatitis B and C, syphilis, gonorrhea, and chlamydia among TGW in Cali, Colombia. Methods: We conducted a cross-sectional study using respondent-driven sampling between May and November 2023. Eight TGW seeds initiated recruitment chains. Eligible participants completed a survey, underwent a physical examination, and were tested for STIs, including pooled swabs for HPV, chlamydia, and gonorrhea. Network size-adjusted prevalence estimates with their corresponding 95% confidence intervals (95% CI) were calculated using respondent-driven sampling-II weighting. Results: A total of 169 TGW were enrolled. The median age was 39 years, 69% reported engaging in transactional sex in the past year, and 68% reported condomless sex during their last encounter. Only 50% had been tested for any STIs in the past year. Adjusted prevalence estimates were 45.8% (95% CI: 31.9-59.6) for human immunodeficiency virus, 41.3% (95% CI: 27.0-55.7) for high-risk HPV, 16.9% (95% CI: 3.7-30.1) for gonorrhea, 15.5% (95% CI: 3.9-27.1) for chlamydia, 14.8% (95% CI: 6.9-22.6) for presumptive active syphilis, 4.6% (95% CI: 0-10.1) for hepatitis B, and 1.7% (95% CI: 0-3.4) for hepatitis C. Approximately 40% had multiple infections. Most cases of chlamydia and gonorrhea were asymptomatic. Conclusions: STIs are highly prevalent among TGW in Cali. These findings highlight the urgent need for expanded diagnostic capacity and community-centered interventions to improve sexual health among TGW.

BACKGROUND:Rapid dual treponemal/non-treponemal (lipoidal antigen) tests could improve syphilis detection strategies among people living with human immunodeficiency virus (PLHIV). The World Health Organization (WHO) recommends minimum performance targets for syphilis point-of-care tests: sensitivity >80% for the treponemal component, >95% for the non-treponemal component, and specificity >90% for both. We evaluated the diagnostic accuracy of a dual treponemal/non-treponemal test in PLHIV. METHODS:Cross-sectional study using stored serum samples donated by PLHIV who had been previously enrolled in a study conducted in Cali, Colombia. The index test was the Dual Path Platform® Syphilis Screen & Confirm Assay (DPP-SSCA, Chembio Diagnostic Systems, Inc.). A combination of Treponema pallidum hemagglutination assay and treponemal enzyme-linked immunosorbent assay (ELISA) served as the reference standard for the treponemal component, and the rapid plasma reagin (RPR) test for the non-treponemal component. We estimated the sensitivity and specificity of both components. RESULTS:We performed 280 DPP-SSCA on serum from 220 PLHIV (60 samples tested in duplicate). Median age: 35.2 years; 75.9% male; 53.0% with undetectable viral load; and 35.9% with previously treated syphilis. The treponemal component had 74.5% sensitivity (95% confidence interval [CI], 58.4-85.9) and 97.2% specificity (95% CI, 92.8-98.9). The non-treponemal component had 90.4% sensitivity (95% CI, 74.9-96.7) and 46.5% specificity (95% CI, 38.0-55.3). CONCLUSIONS:In PLHIV, the serum-based DPP-SSCA did not meet WHO performance targets. Given the low specificity of the non-treponemal component, the development of improved diagnostic tools and rigorous validation are needed to ensure reliable and rapid diagnosis of active syphilis infection in this population.

Knowledge of Treponema pallidum subspecies pallidum (TPA) outer membrane protein (OMP) sequence variability is essential for understanding spirochete proliferation within endemic populations as well as the design of a globally effective syphilis vaccine. Our group has identified extracellular loops (ECLs) of TPA BamA (TP0326) and members of the FadL family (TP0548, TP0856, TP0858, TP0859, and TP0865) as potential components of a multivalent vaccine cocktail. As part of a consortium to explore TPA strain diversity, we mapped the variability of BamA and FadL orthologs in 186 TPA strains from Malawi, China, and Colombia onto predicted 3D structures. The 186 genomes fell into eight subclades (five Nichols- and three SS14-lineage) with substantial geographic restriction. Single nucleotide variants accounted for the large majority of proteoforms, with variability notably higher within the Nichols-lineage strains. Most mutations were in regions of the proteins predicted to be extracellular and harboring B cell epitopes. We observed a striking difference in the degree of variability between the six OMPs, suggesting that these proteins are following divergent evolutionary paths. Concatenation of OMP sequences recapitulated the phylogenetic structure of the TPA strains, effectively segregating within clades and largely clustering by subclades. Finally, we noted that BamA and FadL candidate ECL vaccinogens, previously shown to elicit antibodies that kill treponemes during in vitro cultivation, are well conserved. Taken as a whole, our study establishes a structural-phylogenetic approach for analyzing the forces shaping the host-pathogen interface in syphilis within endemic populations while informing the selection of vaccine targets.IMPORTANCESyphilis remains a major global health concern, reinforcing the need for a safe and effective vaccine. Understanding the variability of TPA OMPs is essential for tracking pathogen evolution and informing vaccine design. Here, we analyzed the variability of six TPA OMPs in 186 strains from Malawi, China, and Colombia, identifying protein-specific evolutionary patterns. Most mutations were localized in extracellular regions and, notably, appeared to correlate with the phylogenetic structure of TPA. Despite OMP heterogeneity, several candidate vaccinogens remained highly conserved, reinforcing their potential as globally effective vaccine targets. Our study establishes a structural-phylogenetic framework for dissecting the forces shaping the host-spirochete interface within endemic populations and provides a foundation for designing a globally effective syphilis vaccine.

BACKGROUND:The increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We aimed to explore Treponema pallidum subspecies pallidum (TPA) molecular epidemiology essential for vaccine research by analysing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. METHODS:In this multicentre, cross-sectional, molecular epidemiology study, we enrolled patients with primary, secondary, or early latent syphilis from clinics in China, Colombia, Malawi, and the USA between Nov 28, 2019, and May 27, 2022. Participants aged 18 years or older with laboratory confirmation of syphilis by direct detection methods or serological testing, or both, were included. Patients were excluded from enrolment if they were unwilling or unable to give informed consent, did not understand the study purpose or nature of their participation, or received antibiotics active against syphilis in the past 30 days. TPA detection and WGS were conducted on lesion swabs, skin biopsies, skin scrapings, whole blood, or rabbit-passaged isolates. We compared our WGS data to publicly available genomes and analysed TPA populations to identify mutations associated with lineage and geography. FINDINGS:We screened 2802 patients and enrolled 233 participants, of whom 77 (33%) had primary syphilis, 154 (66%) had secondary syphilis, and two (1%) had early latent syphilis. The median age of participants was 28 years (IQR 22-35); 154 (66%) participants were cisgender men, 77 (33%) were cisgender women, and two (1%) were transgender women. Of the cisgender men, 66 (43%) identified as gay, bisexual, or other sexuality. Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants showed a predominance of SS14-lineage strains with geographical clustering. Phylogenomic analyses confirmed that Nichols-lineage strains were more genetically diverse than SS14-lineage strains and clustered into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models showed population-specific substitutions, some in outer membrane proteins (OMPs) of interest. INTERPRETATION:Our study substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains is vital for vaccine development and understanding syphilis pathogenesis on a population level. FUNDING:US National Institutes of Health National Institute for Allergy and Infectious Disease, the Bill & Melinda Gates Foundation, Connecticut Children's, and the Czech Republic National Institute of Virology and Bacteriology.

BACKGROUND:Venereal syphilis, caused by the spirochete Treponema pallidum subsp. pallidum (TPA), is surging worldwide, underscoring the need for a vaccine with global efficacy. Vaccine development requires an understanding of syphilis epidemiology and clinical presentation as well as genomic characterization of TPA strains circulating within at-risk populations. The aim of this study was to describe the clinical, demographic, and molecular features of early syphilis cases in Cali, Colombia. METHODS AND FINDINGS:We conducted a cross-sectional study to identify individuals with early syphilis (ES) in Cali, Colombia through a city-wide network of public health centers, private sector HIV clinics and laboratory databases from public health institutions. Whole blood (WB), skin biopsies (SB), and genital and oral lesion swabs were obtained for measurement of treponemal burdens by polA quantitative polymerase chain reaction (qPCR) and for whole-genome sequencing (WGS). Among 1,966 individuals screened, 128 participants met enrollment criteria: 112 (87%) with secondary (SS), 15 (12%) with primary (PS) and one with early latent syphilis; 66/128 (52%) self-reported as heterosexual, while 48 (38%) were men who have sex with men (MSM). Genital ulcer swabs had the highest polA copy numbers (67 copies/μl) by qPCR with a positivity rate (PR) of 73%, while SS lesions had 42 polA copies/μl with PR of 62%. WB polA positivity was more frequent in SS than PS (42% vs 7%, respectively; p = 0.009). Isolation of TPA from WB by rabbit infectivity testing (RIT) was achieved in 5 (56%) of 9 ES WB samples tested. WGS from 33 Cali patient samples, along with 10 other genomic sequences from South America (9 from Peru, 1 from Argentina) used as comparators, confirmed that SS14 was the predominant clade, and that half of all samples had mutations associated with macrolide (i.e., azithromycin) resistance. Variability in the outer membrane protein (OMP) and vaccine candidate BamA (TP0326) was mapped onto the protein's predicted structure from AlphaFold. Despite the presence of mutations in several extracellular loops (ECLs), ECL4, an immunodominant loop and proven opsonic target, was highly conserved in this group of Colombian and South American TPA isolates. CONCLUSIONS:This study offers new insights into the sociodemographic and clinical features of venereal syphilis in a highly endemic area of Colombia and illustrates how genomic sequencing of regionally prevalent TPA strains can inform vaccine development.

Background The continuing increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We conducted a multi-center, observational study to explore Treponema pallidum subsp. pallidum ( TPA ) molecular epidemiology essential for vaccine research by analyzing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. Methods We enrolled patients with primary (PS), secondary (SS) or early latent (ELS) syphilis from clinics in China, Colombia, Malawi and the United States between November 2019 - May 2022. Inclusion criteria included age ≥18 years, and syphilis confirmation by direct detection methods and/or serological testing. TPA detection and WGS were conducted on lesion swabs, skin biopsies/scrapings, whole blood, and/or rabbit-passaged isolates. We compared our WGS data to publicly available genomes, and analysed TPA populations to identify mutations associated with lineage and geography. Findings We screened 2,820 patients and enrolled 233 participants - 77 (33%) with PS, 154 (66%) with SS, and two (1%) with ELS. Median age of participants was 28; 66% were cis -gender male, of which 43% reported identifying as “gay”, “bisexual”, or “other sexuality”. Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants demonstrated a predominance of SS14-lineage strains with geographic clustering. Phylogenomic analysis confirmed that Nichols-lineage strains are more genetically diverse than SS14-lineage strains and cluster into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models demonstrated population-specific substitutions, some in outer membrane proteins (OMPs) of interest. Interpretation Our study involving participants from four countries substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains will be vital for vaccine development and improved understanding of syphilis pathogenesis on a population level. Funding National Institutes of Health, Bill and Melinda Gates Foundation

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 β-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.

ABSTRACT In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.

Syphilis is a multi-stage, sexually transmitted disease caused by the spirochete Treponema pallidum (Tp). Considered broadly, syphilis can be conceptualized as a dualistic process in which spirochete-driven inflammation, the cause of clinical manifestations, coexists to varying extents with bacterial persistence. Inflammation is elicited in the tissues, along with the persistence of spirochetes to keep driving a robust immune response while evading host defenses; this duality is best exemplified during the florid, disseminated stage called secondary syphilis (SS). SS lesions typically contain copious amounts of spirochetes along with a mixed cellular infiltrate consisting of CD4+ T cells, CD8+ T cells, NK cells, plasma cells, and macrophages. In the rabbit model, Tp are cleared by macrophages via antibody-mediated opsonophagocytosis. Previously, we demonstrated that human syphilitic serum (HSS) promotes efficient uptake of Tp by human monocytes and that opsonophagocytosis of Tp markedly enhances cytokine production. Herein, we used monocyte-derived macrophages to study Tp–macrophage interactions ex vivo. In the absence of HSS, monocyte-derived macrophages internalized low numbers of Tp and secreted little cytokine (e.g., TNF). By contrast, these same macrophages internalized large numbers of unopsonized Borrelia burgdorferi and secreted robust levels of cytokines. Maturation of macrophages with M-CSF and IFNγ resulted in a macrophage phenotype with increased expression of HLA-DR, CD14, inducible nitric oxide synthase, TLR2, TLR8, and the Fcγ receptors (FcγR) CD64 and CD16, even in the absence of LPS. Importantly, IFNγ-polarized macrophages resulted in a statistically significant increase in opsonophagocytosis of Tp accompanied by enhanced production of cytokines, macrophage activation markers (CD40, CD80), TLRs (TLR2, TLR7, TLR8), chemokines (CCL19, CXCL10, CXCL11), and TH1-promoting cytokines (IL-12, IL-15). Finally, the blockade of FcγRs, primarily CD64, significantly diminished spirochetal uptake and proinflammatory cytokine secretion by IFNγ-stimulated macrophages. Our ex vivo studies demonstrate the importance of CD64-potentiated uptake of opsonized Tp and suggest that IFNγ-activated macrophages have an important role in the context of early syphilis. Our study results also provide an ex vivo surrogate system for use in future syphilis vaccine studies.

Introduction Syphilis is a multi-stage, sexually transmitted disease caused by the spirochete Treponema pallidum (Tp). Clinical manifestations result from the treponeme’s ability to elicit a robust immune response while at the same time evading host defenses. Syphilitic lesions are comprised of a rich cellular infiltrate, which includes IFN-gamma (IFNg) producing T cells, NK cells and activated macrophages. We previously, we demonstrated that human syphilitic serum (HSS) promotes efficient uptake of Tp by human monocytes and that opsonophagocytosis of Tp markedly enhances cytokine production. The purpose of this study is to establish a potential role for macrophages and opsonic Ab in clearance of Tp and generation of tissue-based inflammation during human syphilis. Methods We used monocyte-derived macrophages to develop an ex vivo model for studying spirochete-macrophage interactions. We used macrophage-colony stimulating factor and IFNg for macrophage maturation and evaluated the immunophenotypic modulations by flow cytometry. We assessed Tp uptake, in the presence or absence of HSS by confocal microscopy. We also determined the cellular responses initiated by opsonophagocytosis of Tp using targeted transcriptional array analysis and cytokine bead array. Results IFNg polarisation of macrophages led to an increase in Fcg receptors (FcgRs) expression, phagocytosis of HSS opsonized Tp and cytokine production. Blockade of CD64 significantly diminished spirochetal uptake and pro-inflammatory cytokine secretion by the macrophages. Conclusion Our ex vivo studies provide a potential role for macrophages in clearance of Tp during human syphilis. These data are the first to demonstrate that CD64 in the primary FcR involved in opsonophagocytosis of Tp and IFNg plays a critical role in the macrophages responsiveness following uptake of the spirochete. Moreover, our study results also provide an ex vivo surrogate system for use in future syphilis vaccine studies.

Introduction Most women in Colombia receive antenatal care (ANC), but goals for the reduction of congenital syphilis (CS) have not been met. In 2015 Cali had an incidence of 3.0 cases of CS per 1000 births.Point of care testing (POC) has proven cost-effective, and since 2015 it is now recommended by the Colombian Ministry of Health Guidelines. Nevertheless, POC for gestational syphilis (GS) has not been implemented in ANC. We sought to test the feasibility of POC and identify barriers to its implementation in Floralia, a public, primary healthcare centre in Cali. Methods During a three-month period, we observed routine ANC in Floralia and offered women on-site rapid tests (RT) for syphilis. Process measures were compared with data from the clinical charts (CC) of women seen in 2015. Patients and administrators were interviewed and asked to describe ANC, syphilis screening procedures, and to identify opportunities for improvement. Results 55 women were offered POC testing, 42 agreed to participate and were screened. 55 CC from 2016, and 123 CC from 2015, were reviewed. The median gestational age at which women sought health care was eight weeks. The first trimester of pregnancy had the highest percentage of women screened for GS: 97% were screened in 2015, and 100% were screened in 2016. Changes to screening procedures were hindered by existing contracts with insurance companies (IC); they determine what tests can be ordered and who can order them. Administrative procedures lead to missed opportunities for screening and treatment.Two women (1.6%) from the 2015 group were diagnosed with GS, and three (5.4%) were diagnosed during the intervention. POC testing allowed us to treat GS on the same day of the positive result, while using routine testing methods took 6 to 24 days. Conclusion Diffusion of the national guidelines for GS has been insufficient to control GS and CS: There is still confusion among health care workers, and contracts with IC do not include RT. POC for syphilis can be a part of ANC, but barriers need to be addressed before implementation.

Introduction Outer membrane proteins (OMPs) play critical roles in disease pathogenesis and are vaccinogens. Topologic characterisation of surface-exposed β-barrels of Treponema pallidum (Tp) Nichols rare OMPs enabled a novel strategy to assess sequence diversity and evolution of Tp in geographically diverse locations. Methods Through early 2017, sequences encoding TprC (TP0117), TprD (TP0131), and BamA (TP0326) β-barrels were amplified from secondary syphilis patients from Cali (n=16) and swabs from patients in San Francisco (SF, n=6) and Czech Republic (CZ, n=9). Strains were assigned to the Nichols or SS14 clade based on tp0548 and/or tp0558 sequences. Results 23 assignable CZ and Cali strains belonged to either the SS14 or Nichols clade (SS14 predominant), while all 6 SF strains belong to the SS14 clade. Sequence diversity at the three OMP loci was greatest in Cali, with evidence of recombination within tprC and bamA alleles, as well as between strains and clades at all 3 genetic loci. SF strains contained nearly identical sequences at all 3 genetic loci. The SS14 and Mexico A reference strains, both belonging to the SS14 clade, have identical tprDs (tprD2) but different tprC and bamA alleles. Mexico A tprCs were common at all three geographic locations, including Nichols clade strains from Cali. Mexico A bamAs were prevalent in Cali and SF, while CZ SS14 clades contained only SS14 bamAs. OMP sequences were obtained from all three loci in 7 of 8 Nichols clade strains. Of these 7, only 1 matched the Nichols reference strain, while the other 6 contained Mexico A alleles in at least 1 OMP locus. Of the 21 SS14 clade strains, 10 contained Mexico A alleles at all 3 loci; 2 contained Mexico A trpCs and Nichols bamAs; and 9 contained Mexico A tprCs and SS14 bamAs. Conclusion OMP loci are evolving independently within Tp. Recombination of OMP sequences appears to be occurring between Tp strains and clades within patients. Mexico A OMP alleles are circulating widely among Tp strains. These findings have major ramifications for syphilis vaccine development.

Event Abstract Back to Event Human macrophage polarization enhances opsonophagocytosis and inflammatory response of the stealth pathogen, Treponema pallidum Kelly L. Hawley1*, Adriana R. Cruz2, Jorge Cervantes1, 3, Carson Karanian4, Morgan LeDoyt4, Rodolfo Trujillo1, Lady G. Ramirez2, Justin Radolf1, 4, 5 and Juan C. Salazar1, 3, 4 1 University of Connecticut Health Center, Department of Pediatrics, United States 2 Centro Internacional de Entrenamiento e Investigaciones Médicas CIDEIM, Colombia 3 Connecticut Children’s Medical Center, Division of Infectious Disease and Immunology, United States 4 University of Connecticut Health Center, Departments of Medicine, United States 5 University of Connecticut Health Center, Genetics and Developmental Biology, United States Venereal syphilis is a multi-stage, sexually transmitted disease caused by the spirochete Treponema pallidum (Tp). Congenital syphilis, a result of transplacental migration of Tp, is a major global threat to child health. Clinical manifestations result from the treponeme's ability to elicit a robust immune response while evading host defenses; this duality is best exemplified during the florid, disseminated stage called secondary syphilis (SS). SS lesions contain copious spirochetes along with a mixed cellular infiltrate consisting of CD4 T cells, CD8 T cells, NK cells, plasma cells and macrophages (MΦ). In the rabbit model, Tp are cleared by MΦs via antibody (Ab)-mediated opsonophagocytosis. Previously, we demonstrated that human syphilitic serum (HSS) promotes efficient uptake of Tp by human monocytes and that opsonophagocytosis of Tp markedly enhances cytokine production. The purpose of this study is to establish a potential role for MΦs and opsonic Ab in clearance of treponemes and generation of tissue-based inflammation during human syphilis. We used monocyte-derived MΦs to develop an ex vivo model for studying spirochete-MΦ interactions and cellular responses (e.g., phagosomal signaling) following uptake of Tp. We also examined SS skin biopsies for evidence of internalization of Tp by dermal MΦs. In the presence or absence of HSS, untreated (i.e., no M-CSF or IFNγ) MΦs internalized low numbers of Tp and secreted little cytokine (e.g., TNF and IL-6). In contrast, untreated MΦs internalized unopsonized Lyme disease spirochetes (Borrelia burgdorferi) and secreted robust levels of cytokines. Maturation of MΦs with M-CSF and IFNγ [MΦ(IFNγ)] resulted in increased TLR signaling and enhanced expression of the Fc receptors CD64 and CD16. Importantly, IFNγ polarization of MΦs led to a statistically significant, but modest, increase in opsonophagocytosis of Tp and cytokine production. Interestingly, immunofluorescence analysis of SS skin biopsies yielded only scant evidence for internalization of Tp by dermal MΦs. Our ex vivo studies demonstrate a potential role for MΦs in clearance of Tp during human syphilis. On the other hand, the in vivo data suggest that Tp, the stealth pathogen, is adept at evading MΦ-dependent clearance and suggest the possibility of MΦ-independent clearance mechanisms. Acknowledgements We gratefully acknowledge the patients who participated in this study. This work received support from NIAID grant A1090166, CCMC Arrison and Burr Curtis Research Funds and Fogarty/NIH R03TW009172. Keywords: Macrophages, Syphilis, FcγR, phagosomal signaling, Clearance Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Oral Presentation Topic: Innate Immunity Citation: Hawley KL, Cruz AR, Cervantes J, Karanian C, LeDoyt M, Trujillo R, Ramirez LG, Radolf J and Salazar JC (2015). Human macrophage polarization enhances opsonophagocytosis and inflammatory response of the stealth pathogen, Treponema pallidum. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00306 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 May 2015; Published Online: 15 Sep 2015. * Correspondence: PhD. Kelly L Hawley, University of Connecticut Health Center, Department of Pediatrics, Farmington, Connecticut, United States, Hawley@uchc.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kelly L Hawley Adriana R Cruz Jorge Cervantes Carson Karanian Morgan LeDoyt Rodolfo Trujillo Lady G Ramirez Justin Radolf Juan C Salazar Google Kelly L Hawley Adriana R Cruz Jorge Cervantes Carson Karanian Morgan LeDoyt Rodolfo Trujillo Lady G Ramirez Justin Radolf Juan C Salazar Google Scholar Kelly L Hawley Adriana R Cruz Jorge Cervantes Carson Karanian Morgan LeDoyt Rodolfo Trujillo Lady G Ramirez Justin Radolf Juan C Salazar PubMed Kelly L Hawley Adriana R Cruz Jorge Cervantes Carson Karanian Morgan LeDoyt Rodolfo Trujillo Lady G Ramirez Justin Radolf Juan C Salazar Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Background Cali is a syphilis-endemic area, with a very high prevalence of gestational syphilis (14.7/1000 live births) and congenital syphilis (7.7/1000 live births). Molecular typing techniques are useful for studying bacterial strain diversity, molecular epidemiologic patterns and antimicrobial resistance patterns. The study objective was to determine the T. pallidum (Tp) strain diversity and analyse for the mutations associated with macrolide-resistance in this syphilis-endemic region. Methods 19 secondary syphilis (SS) patients, 15 (RPR+, FTA-ABS+, HIV-) and 4 (RPR+, FTA-ABS+, HIV+) were enrolled in the study. Tp DNA was extracted from syphilis skin lesions and quantified by real-time-qPCR targeting polA gene. Molecular typing was performed using established typing and subtyping methods (arp, tpr, tp0279, tp0548) and strains were analysed for A2058G and A2059G mutations within the 23S rRNA gene. Strain diversity in Cali strains (arp and tpr) was compared with other published Tp molecular studies using the Shannon index. Results 14/19 SS patients were polA PCR positive (range 5.4 – 38.920; mean 3.227 copies/ug total DNA). Among 8/14 samples that were fully typed, 7 strain subtypes (21a11/d, 12d9/f, 10p9/f, 14j10f, 5p9/f, 14k9/f, 14d9/f) were identified with only 2 strains exhibiting identical patterns (21a11/d). The A2059G mutation was found in 1 specimen from an HIV-negative subject. Cali Tp strain types had a Shannon index of 2.2, higher than all 13 studies in other localities reported in two recent reviews. Conclusions There is a high T. pallidum strain diversity in Cali, Colombia, probably due to population mobilisation and close proximity to other syphilis-endemic regions (i.e. Buenaventura, Pacific Coast). Contact tracing and cluster identification is difficult to achieve in this setting. Although our sample size was small, the identification of the A2059G mutation suggests that macrolides should be used with caution for syphilis treatment in Cali and close monitoring for macrolide resistant strains should be initiated.

BACKGROUND:The clinical syndrome associated with secondary syphilis (SS) reflects the propensity of Treponema pallidum (Tp) to escape immune recognition while simultaneously inducing inflammation. METHODS:To better understand the duality of immune evasion and immune recognition in human syphilis, herein we used a combination of flow cytometry, immunohistochemistry (IHC), and transcriptional profiling to study the immune response in the blood and skin of 27 HIV(-) SS patients in relation to spirochetal burdens. Ex vivo opsonophagocytosis assays using human syphilitic sera (HSS) were performed to model spirochete-monocyte/macrophage interactions in vivo. RESULTS:Despite the presence of low-level spirochetemia, as well as immunophenotypic changes suggestive of monocyte activation, we did not detect systemic cytokine production. SS subjects had substantial decreases in circulating DCs and in IFNγ-producing and cytotoxic NK-cells, along with an emergent CD56-/CD16+ NK-cell subset in blood. Skin lesions, which had visible Tp by IHC and substantial amounts of Tp-DNA, had large numbers of macrophages (CD68+), a relative increase in CD8+ T-cells over CD4+ T-cells and were enriched for CD56+ NK-cells. Skin lesions contained transcripts for cytokines (IFN-γ, TNF-α), chemokines (CCL2, CXCL10), macrophage and DC activation markers (CD40, CD86), Fc-mediated phagocytosis receptors (FcγRI, FcγR3), IFN-β and effector molecules associated with CD8 and NK-cell cytotoxic responses. While HSS promoted uptake of Tp in conjunction with monocyte activation, most spirochetes were not internalized. CONCLUSIONS:Our findings support the importance of macrophage driven opsonophagocytosis and cell mediated immunity in treponemal clearance, while suggesting that the balance between phagocytic uptake and evasion is influenced by the relative burdens of bacteria in blood and skin and the presence of Tp subpopulations with differential capacities for binding opsonic antibodies. They also bring to light the extent of the systemic innate and adaptive immunologic abnormalities that define the secondary stage of the disease, which in the skin of patients trends towards a T-cell cytolytic response.

Background The inflammatory response is prominent in the pathogenesis of dermal leishmaniasis. We hypothesized that regulatory T cells (Tregs) may be diminished in chronic dermal leishmaniasis (CDL) and contribute to healing during treatment. Methodology/Principal Findings The frequency and functional capacity of Tregs were evaluated at diagnosis and following treatment of CDL patients having lesions of ≥6 months duration and asymptomatically infected residents of endemic foci. The frequency of CD4+CD25hi cells expressing Foxp3 or GITR or lacking expression of CD127 in peripheral blood was determined by flow cytometry. The capacity of CD4+CD25+ cells to inhibit Leishmania-specific responses was determined by co-culture with effector CD4+CD25− cells. The expression of FOXP3, IFNG, IL10 and IDO was determined in lesion and leishmanin skin test site biopsies by qRT-PCR. Although CDL patients presented higher frequency of CD4+CD25hiFoxp3+ cells in peripheral blood and higher expression of FOXP3 at leishmanin skin test sites, their CD4+CD25+ cells were significantly less capable of suppressing antigen specific-IFN-γ secretion by effector cells compared with asymptomatically infected individuals. At the end of treatment, both the frequency of CD4+CD25hiCD127− cells and their capacity to inhibit proliferation and IFN-γ secretion increased and coincided with healing of cutaneous lesions. IDO was downregulated during healing of lesions and its expression was positively correlated with IFNG but not FOXP3. Conclusions/Significance The disparity between CD25hiFoxp3+ CD4 T cell frequency in peripheral blood, Foxp3 expression at the site of cutaneous responses to leishmanin, and suppressive capacity provides evidence of impaired Treg function in the pathogenesis of CDL. Moreover, the concurrence of increased Leishmania-specific suppressive capacity with induction of a CD25hiCD127− subset of CD4 T cells during healing supports the participation of Tregs in the resolution of chronic dermal lesions. Treg subsets may therefore be relevant in designing immunotherapeutic strategies for recalcitrant dermal leishmaniasis caused by Leishmania (Viannia) species.

Venereal syphilis is a multi-stage, sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum (Tp). Herein we describe a cohort of 57 patients (age 18-68 years) with secondary syphilis (SS) identified through a network of public sector primary health care providers in Cali, Colombia. To be eligible for participation, study subjects were required to have cutaneous lesions consistent with SS, a reactive Rapid Plasma Reagin test (RPR-titer > or = 1 : 4), and a confirmatory treponemal test (Fluorescent Treponemal Antibody Absorption test- FTA-ABS). Most subjects enrolled were women (64.9%), predominantly Afro-Colombian (38.6%) or mestizo (56.1%), and all were of low socio-economic status. Three (5.3%) subjects were newly diagnosed with HIV infection at study entry. The duration of signs and symptoms in most patients (53.6%) was less than 30 days; however, some patients reported being symptomatic for several months (range 5-240 days). The typical palmar and plantar exanthem of SS was the most common dermal manifestation (63%), followed by diffuse hypo- or hyperpigmented macules and papules on the trunk, abdomen and extremities. Three patients had patchy alopecia. Whole blood (WB) samples and punch biopsy material from a subset of SS patients were assayed for the presence of Tp DNA polymerase I gene (polA) target by real-time qualitative and quantitative PCR methods. Twelve (46%) of the 26 WB samples studied had quantifiable Tp DNA (ranging between 194.9 and 1954.2 Tp polA copies/ml blood) and seven (64%) were positive when WB DNA was extracted within 24 hours of collection. Tp DNA was also present in 8/12 (66%) skin biopsies available for testing. Strain typing analysis was attempted in all skin and WB samples with detectable Tp DNA. Using arp repeat size analysis and tpr RFLP patterns four different strain types were identified (14d, 16d, 13d and 22a). None of the WB samples had sufficient DNA for typing. The clinical and microbiologic observations presented herein, together with recent Cali syphilis seroprevalence data, provide additional evidence that venereal syphilis is highly endemic in this region of Colombia, thus underscoring the need for health care providers in the region to be acutely aware of the clinical manifestations of SS. This study also provides, for the first time, quantitative evidence that a significant proportion of untreated SS patients have substantial numbers of circulating spirochetes. How Tp is able to persist in the blood and skin of SS patients, despite the known presence of circulating treponemal opsonizing antibodies and the robust pro-inflammatory cellular immune responses characteristic of this stage of the disease, is not fully understood and requires further study.