Ocular syphilis is a serious complication of Treponema pallidum infection that can occur at any stage of syphilis and affect any eye structure. It remains unknown if certain T. pallidum strains are associated with ocular infections; therefore, we performed genotyping and whole genome sequencing (WGS) to characterize strains from patients with ocular syphilis. Seventy-five ocular or non-ocular specimens from 55 ocular syphilis patients in 14 states within the United States were collected between February 2016 and November 2020. Sufficient T. pallidum DNA was available from nine patients for genotyping and three for WGS. Genotyping was done using the augmented Centers for Disease Control and Prevention typing scheme, and WGS was performed on Illumina platforms. Multilocus sequence typing allelic profiles were predicted from whole genome sequence data. T. pallidum DNA was detected in various specimens from 17 (30.9%) of the 55 patients, and typing was done on samples from 9 patients. Four complete strain types (14d10/g, 14b9/g, 14d9/g, and 14e9/f) and five partial types were identified. WGS was successful on samples from three patients and all three strains belonged to the SS14 clade of T. pallidum. Our data reveal that multiple strain types are associated with ocular manifestations of syphilis. While genotyping and WGS were challenging due to low amounts of T. pallidum DNA in specimens, we successfully performed WGS on cerebrospinal fluid, vitreous fluid, and whole blood.IMPORTANCESyphilis is caused by the spirochete Treponema pallidum. Total syphilis rates have increased significantly over the past two decades in the United States, and the disease remains a public health concern. In addition, ocular syphilis cases has also been on the rise, coinciding with the overall increase in syphilis rates. We conducted a molecular investigation utilizing traditional genotyping and whole genome sequencing over a 5-year period to ascertain if specific T. pallidum strains are associated with ocular syphilis. Genotyping and phylogenetic analysis show that multiple T. pallidum strain types are associated with ocular syphilis in the United States.

Downstream next generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA using the NEBNext Microbiome DNA Enrichment Kit followed by MDA with the REPLI-g Single Cell Kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/ul of sample for SWGA and >129 genomic copies/ul for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Downstream next-generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole-genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome and the capture and removal of 5'-C-phosphate-G-3' (CpG) methylated host DNA using the NEBNext Microbiome DNA enrichment kit followed by MDA with the REPLI-g single cell kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93 to 98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit-propagated isolates, containing >14 T. pallidum genomic copies/μL of sample for SWGA and >129 genomic copies/μL for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole-genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which has been challenging until now. IMPORTANCE Syphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions and, if left untreated, can progress through multiple stages with various clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past 2 decades, WGS data are needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of an SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

OBJECTIVE: To evaluate the field performance of a multiplex PCR (M-PCR) assay for detection of herpes simplex virus (HSV)-1 and HSV-2, Treponema pallidum (T. pallidum) and Haemophilus ducreyi (H. ducreyi) in genital ulcer disease (GUD) specimens. METHODS: GUD M-PCR was performed on 186 remnant specimens, previously collected for HSV testing, by four public health laboratories (PHLs) and the Laboratory Reference and Research Branch (LRRB) at the Centers for Disease Control and Prevention. The results from the PHLs were compared with those of LRRB, which served as the reference testing method, and percentage agreement was calculated. RESULTS: HSV was detected in 31 of 52 (59.6%), 20 of 40 (50%), 43 of 44 (97.7%) and 19 of 50 (38.0%) specimens from PHL1, PHL2, PHL3 and PHL4, respectively. There were seven discrepant results for HSV, and the overall percent agreement between the PHLs and the LRRB was 94%-100%, with a kappa value of 0.922, which demonstrates high agreement. T. pallidum was identified in 7 of 51 (13.7%) specimens from PHL1 with 94.1% agreement and in 2 of 40 (5.0%) specimens from PHL2 with 100% agreement. The LRRB identified three additional T. pallidum-positive specimens from PHL1. The kappa value (0.849) for T. pallidum testing suggests good agreement. Consistent with the LRRB results, no T. pallidum was detected in specimens from PHL3 and PHL4, and H. ducreyi was not detected at any of the study sites. CONCLUSIONS: The GUD M-PCR assay performed well in four independent PHLs and 12 suspected syphilis cases were identified in this study. The M-PCR assay could provide improved diagnostic options for GUD infections in state and local PHLs.

Lymphogranuloma venereum (LGV) can be differentiated from non-LGV chlamydial infection using Sanger sequencing or molecular assays, including those that are commercially-available internationally. Here, we describe the performance of a rapid real-time PCR (RT-PCR)-based strategy in differentiating Chlamydia trachomatis infections associated with LGV or non-LGV serovars. One hundred three rectal swabs, previously genotyped using Sanger sequencing of the ompA gene as a reference method, were tested in the RT-PCR assays. All non-LGV specimens were correctly identified, but the RT-PCR failed to detect 1 LGV specimen, resulting in a sensitivity of 87.5% for the non-LGV/LGV RT-PCR assay. Additional performance characteristics (e.g., specificity, accuracy, and reproducibility) were all between 93% and 100% with a limit of detection ≤100 copies/reaction. Thus, this rapid RT-PCR method for LGV detection in clinical specimens is comparable to the reference method.

The frequency of lymphogranuloma venereum (LGV) or invasive Chlamydia trachomatis (CT) infection with serovar L1, L2 or L3 is unknown in the United States. While no diagnostic test is commercially available, we used a laboratory-developed test and detected LGV-associated serovar L2 in 14% of 132 remnant CT-positive rectal swabs.

WHO and its partners aim to interrupt yaws transmission in endemic countries and to certify others as being yaws-free. Transmission can be assessed using rapid plasma reagin (RPR) tests, reflecting current or recent infection, but RPR is operationally impractical.We evaluated changes in antibody levels against two recombinant treponemal antigens, rp17 (also known as Tp17) and TmpA, after antibiotic treatment given as part of a randomized controlled trial for yaws in Ghana and Papua New Guinea. Paired serum samples from children aged 6-15 years with confirmed yaws, collected before and after treatment, were tested for antibodies to rp17 and TmpA using a semi-quantitative bead-based immunoassay.Of 344 baseline samples, 342 tested positive for anti-rp17 antibodies and 337 tested positive for anti-TmpA antibodies. Six months after treatment, the median decrease in anti-rp17 signal was 3.2%, whereas the median decrease in anti-TmpA was 53.8%. The magnitude of change in the anti-TmpA response increased with increasing RPR titer fold-change. These data demonstrate that responses to TmpA decrease markedly within 6 months of treatment whereas (as expected) those to rp17 do not.Incorporating responses to TmpA as a marker of recent infection within an integrated sero-surveillance platform could provide a way to prioritize areas for yaws mapping.

Clinical isolates of Treponema pallidum subspecies pallidum (T. pallidum) would facilitate study of prevalent strains. We describe the first successful rabbit propagation of T. pallidum from cryopreserved ulcer specimens. Fresh ulcer exudates were collected and cryopreserved with consent from syphilis-diagnosed patients (N = 8). Each of eight age-matched adult male rabbits were later inoculated with a thawed specimen, with two rabbits receiving 1.3 ml intratesticularly (IT), and six receiving 0.6 ml intravenously (IV) and IT. Monitoring of serology, blood PCR and orchitis showed that T. pallidum grew in 2/8 rabbits that were inoculated IV and IT with either a penile primary lesion specimen (CDC-SF003) or a perianal secondary lesion specimen (CDC-SF007). Rabbit CDC-SF003 was seroreactive by T. pallidum Particle Agglutination (TP-PA) and Rapid Plasma Reagin (RPR) testing, PCR+, and showed orchitis by week 6. Euthanasia was performed in week 7, with treponemal growth in the testes confirmed and quantified by qPCR and darkfield microscopy (DF). Serial passage of the extract in a second age-matched rabbit also yielded treponemes. Similarly, rabbit CDC-SF007 showed negligible orchitis, but was seroreactive and PCR+ by week 4 and euthanized in week 6 to yield T. pallidum, which was further propagated by second passage. Using the 4-component molecular typing system for syphilis, 3 propagated strains (CDC-SF003, CDC-SF007, CDC-SF008) were typed as 14d9f, 14d9g, and 14d10c, respectively. All 3 isolates including strain CDC-SF011, which was not successfully propagated, had the A2058G mutation associated with azithromycin resistance. Our results show that immediate cryopreservation of syphilitic ulcer exudate can maintain T. pallidum viability for rabbit propagation.

A guanine mononucleotide repeat in the rpsA (tp0279) gene was evaluated for improved strain discrimination using 72 Treponema pallidum-positive specimens. The tandem repeat combined with the enhanced Centers for Disease Control and Prevention typing system resulted in increased discrimination and should be useful for molecular epidemiologic studies on syphilis especially in outbreaks and among men who have sex with men.

We explored the association between violence victimization and increased risk for acquiring sexually transmitted infections (STIs) in women by measuring cellular immune barrier properties from the female reproductive tract. STI-negative participants reporting repeated prior victimization occurrences through the lifetime trauma and victimization history (LTVH) instrument were more likely to exhibit alterations in barrier homeostasis and the composition of critical immune mediators irrespective of demographic parameters or presence of bacterial vaginosis. By combining cellular data with mixed-effect linear modeling, we uncovered differences in local T cells, MHCII+ antigen-presenting cells, and epithelial cells indicative of altered trafficking behavior, increased immunosuppressive function, and decreased barrier integrity at sites of STI exposure that correlate most strongly with LTVH score. These data evidence a biological link between a history of violence victimization and risk of STI acquisition through immune dysregulation in the female reproductive tract.

Lymphogranuloma venereum is a sexually transmitted infection caused by serovars L1, L2, and L3 of Chlamydia trachomatis. We here report a case of Lymphogranuloma venereum, confirmed by PCR testing, which mimicked malignancy on CT imaging.

Yaws is a neglected tropical disease caused by the bacterium Treponema pallidum subspecies pertenue. The disease primarily affects children under 15 years of age living in low socioeconomic conditions in tropical areas. As a result of a renewed focus on the disease owing to a recent eradication effort initiated by the World Health Organization, we have evaluated a typing method, adapted from and based on the enhanced Centers for Disease Control and Prevention typing method for T. pallidum subsp. pallidum, for possible use in epidemiological studies. Thirty DNA samples from yaws cases in Vanuatu and Ghana, 11 DNA samples extracted from laboratory strains, and 3 published genomic sequences were fully typed by PCR/RFLP analysis of the tpr E, G, and J genes and by determining the number of 60-bp repeats within the arp gene. Subtyping was performed by sequencing a homonucleotide "G" tandem repeat immediately upstream of the rpsA gene and an 84-bp region of tp0548. A total of 22 complete strain types were identified; two strain types in clinical samples from Vanuatu (5q11/ak and 5q12/ak), nine strain types in clinical samples from Ghana (3q12/ah, 4r12/ah, 4q10/j, 4q11/ah, 4q12/ah, 4q12/v, 4q13/ah, 6q10/aj, and 9q10/ai), and twelve strain types in laboratory strains and published genomes (2q11/ae, 3r12/ad, 4q11/ad, 4q12/ad, 4q12/ag, 4q12/v, 5r12/ad, 6r12/x, 6q11/af, 10q9/r, 10q12/r, and 12r12/w). The tpr RFLP patterns and arp repeat sizes were subsequently verified by sequencing analysis of the respective PCR amplicons. This study demonstrates that the typing method for subsp. pallidum can be applied to subsp. pertenue strains and should prove useful for molecular epidemiological studies on yaws.

INTRODUCTION: The WHO yaws eradication strategy consists of one round of total community treatment (TCT) of single-dose azithromycin with coverage of > 90%.The efficacy of the strategy to reduce the levels on infection has been demonstrated previously in isolated island communities in the Pacific region. We aimed to determine the efficacy of a single round of TCT with azithromycin to achieve a decrease in yaws prevalence in communities that are endemic for yaws and surrounded by other yaws-endemic areas. METHODS: Surveys for yaws seroprevalence and prevalence of skin lesions were conducted among schoolchildren aged 5-15 years before and one year after the TCT intervention in the Abamkrom sub-district of Ghana. We used a cluster design with the schools as the primary sampling unit. Among 20 eligible primary schools in the sub district, 10 were assigned to the baseline survey and 10 to the post-TCT survey. The field teams conducted a physical examination for skin lesions and a dual point-of-care immunoassay for non-treponemal and treponemal antibodies of all children present at the time of the visit. We also undertook surveys with non-probabilistic sampling to collect lesion swabs for etiology and macrolide resistance assessment. RESULTS: At baseline 14,548 (89%) of 16,287 population in the sub-district received treatment during TCT. Following one round of TCT, the prevalence of dual seropositivity among all children decreased from 10.9% (103/943) pre-TCT to 2.2% (27/1211) post-TCT (OR 0.19; 95%CI 0.09-0.37). The prevalence of serologically confirmed skin lesions consistent with active yaws was reduced from 5.7% (54/943) pre-TCT to 0.6% (7/1211) post-TCT (OR 0.10; 95% CI 0.25-0.35). No evidence of resistance to macrolides against Treponema pallidum subsp. pertenue was seen. DISCUSSION: A single round of high coverage TCT with azithromycin in a yaws affected sub-district adjoining other endemic areas is effective in reducing the prevalence of seropositive children and the prevalence of early skin lesions consistent with yaws one year following the intervention. These results suggest that national yaws eradication programmes may plan the gradual expansion of mass treatment interventions without high short-term risk of reintroduction of infection from contiguous untreated endemic areas.

BACKGROUND: A dose of 30 mg/kg of azithromycin is recommended for treatment of yaws, a disease targeted for global eradication. Treatment with 20 mg/kg of azithromycin is recommended for the elimination of trachoma as a public health problem. In some settings, these diseases are co-endemic. We aimed to determine the efficacy of 20 mg/kg of azithromycin compared with 30 mg/kg azithromycin for the treatment of active and latent yaws. METHODS: We did a non-inferiority, open-label, randomised controlled trial in children aged 6-15 years who were recruited from schools in Ghana and schools and the community in Papua New Guinea. Participants were enrolled based on the presence of a clinical lesion that was consistent with infectious primary or secondary yaws and a positive rapid diagnostic test for treponemal and non-treponemal antibodies. Participants were randomly assigned (1:1) to receive either standard-dose (30 mg/kg) or low-dose (20 mg/kg) azithromycin by a computer-generated random number sequence. Health-care workers assessing clinical outcomes in the field were not blinded to the patient's treatment, but investigators involved in statistical or laboratory analyses and the participants were blinded to treatment group. We followed up participants at 4 weeks and 6 months. The primary outcome was cure at 6 months, defined as lesion healing at 4 weeks in patients with active yaws and at least a four-fold decrease in rapid plasma reagin titre from baseline to 6 months in patients with active and latent yaws. Active yaws was defined as a skin lesion that was positive for Treponema pallidum ssp pertenue in PCR testing. We used a non-inferiority margin of 10%. This trial was registered with ClinicalTrials.gov, number NCT02344628. FINDINGS: Between June 12, 2015, and July 2, 2016, 583 (65.1%) of 895 children screened were enrolled; 292 patients were assigned a low dose of azithromycin and 291 patients were assigned a standard dose of azithromycin. 191 participants had active yaws and 392 had presumed latent yaws. Complete follow-up to 6 months was available for 157 (82.2%) of 191 patients with active yaws. In cases of active yaws, cure was achieved in 61 (80.3%) of 76 patients in the low-dose group and in 68 (84.0%) of 81 patients in the standard-dose group (difference 3.7%; 95% CI -8.4 to 15.7%; this result did not meet the non-inferiority criterion). There were no serious adverse events reported in response to treatment in either group. The most commonly reported adverse event at 4 weeks was gastrointestinal upset, with eight (2.7%) participants in each group reporting this symptom. INTERPRETATION: In this study, low-dose azithromycin did not meet the prespecified non-inferiority margin compared with standard-dose azithromycin in achieving clinical and serological cure in PCR-confirmed active yaws. Only a single participant (with presumed latent yaws) had definitive serological failure. This work suggests that 20 mg/kg of azithromycin is probably effective against yaws, but further data are needed. FUNDING: Coalition for Operational Research on Neglected Tropical Diseases.

BACKGROUND: Community mass treatment with 30mg/kg azithromycin is central to the new WHO strategy for eradicating yaws. Both yaws and trachoma- which is earmarked for elimination by 2020 using a strategy that includes mass treatment with 20mg/kg azithromycin-are endemic in the Pacific, raising the possibility of an integrated approach to disease control. Community mass treatment with azithromycin for trachoma elimination was conducted in the Solomon Islands in 2014. METHODS: We conducted a study to assess the impact of mass treatment with 20mg/kg azithromycin on yaws. We examined children aged 5-14 years and took blood and lesion samples for yaws diagnosis. RESULTS: We recruited 897 children, 6 months after mass treatment. There were no cases of active yaws. Serological evidence of current infection was found in 3.6% (95% CI= 2.5-5.0%). This differed significantly between individuals who had and had not received azithromycin (2.8% vs 6.5%, p=0.015); the prevalence of positive serology in 5-14 year-olds had been 21.7% (95% CI=14.6%-30.9%) 6 months prior to mass treatment. Not receiving azithromycin was associated with an odds of 3.9 for infection (p=0.001). National figures showed a 57% reduction in reported cases of yaws following mass treatment. DISCUSSION: Following a single round of treatment we did not identify any cases of active yaws in a previously endemic population. We found a significant reduction in latent infection. Our data support expansion of the WHO eradication strategy and suggest an integrated approach to the control of yaws and trachoma in the Pacific may be viable.

Yaws, caused by Treponema pallidum ssp. pertenue, is a neglected tropical disease closely related to venereal syphilis and is targeted for eradication by 2020. Latent yaws represents a diagnostic challenge, and current tools cannot adequately distinguish between individuals with true latent infection and individuals who are serofast following successful treatment. PCR on blood has previously been shown to detect T. pallidum DNA in patients with syphilis, suggesting that this approach may be of value in yaws. We performed real-time PCR for Treponema pallidum ssp. pertenue on blood samples from 140 children with positive T. pallidum Particle Agglutination (TPPA) and Rapid Plasma Reagin (RPR) tests and 7 controls (negative serology), all collected as part of a prospective study of yaws in the Solomon Islands. All samples were also tested by a nested PCR for T. pallidum. 12 patients had clinical evidence of active yaws whilst 128 were considered to have latent yaws. 43 children had high titre rapid plasma reagins (RPRs) of ≥1:32. PCR testing with both assays gave negative results in all cases. It is possible that the failure to detect T. pallidum ssp. pertenue in blood reflects lower loads of organism in latent yaws compared to those in latent infection with T. pallidum ssp. pertenue, and/or a lower propensity for haematogenous dissemination in yaws than in syphilis. As the goal of the yaws control programme is eradication, a tool that can differentiate true latent infection from individuals who are serofast would be of value; however, PCR of blood is not that tool.

Yaws, caused by Treponema pallidum ssp. pertenue, is reportedly endemic in Ghana. Mass distribution of azithromycin is now the cornerstone of the WHO yaws eradication campaign. Mass distribution of azithromycin at a lower target dose was previously undertaken in two regions of Ghana for the control of trachoma. Ongoing reporting of yaws raises the possibility that resistance may have emerged in T. pallidum pertenue, or that alternative infections may be responsible for some of the reported cases. We conducted a cross-sectional survey in thirty communities in two districts of Ghana where MDA for trachoma had previously been conducted. Children aged 5-17 years with ulcerative lesions compatible with yaws were enrolled. Samples for treponemal serology and lesion PCR were collected from all children. 90 children with 98 lesions were enrolled. Syphilis serology was negative in all of them. PCR for T. pallidum ssp pertenue was negative in all children, but Haemophilus ducreyi DNA was detected in 9 lesions. In these communities, previously treated for trachoma, we found no evidence of ongoing transmission of yaws. H. ducreyi was associated with a proportion of skin lesions, but the majority of lesions remain unexplained. Integration of diagnostic testing into both pre and post-MDA surveillance systems is required to better inform yaws control programmes.

We developed a TaqMan-based real-time quadriplex polymerase chain reaction (PCR) to simultaneously detect Treponema pallidum subspecies pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum, the causative agents of venereal syphilis, yaws, and bejel, respectively. The PCR assay was applied to samples from skin ulcerations of clinically presumptive yaws cases among children on Tanna Island, Vanuatu. Another real-time triplex PCR was used to screen for the point mutations in the 23S rRNA genes that have previously been associated with azithromycin resistance in T. pallidum subsp. pallidum strains. Seropositivity by the classical syphilis serological tests was 35.5% among children with skin ulcerations clinically suspected with yaws, whereas the presence of T. pallidum subsp. pertenue DNA was only found in lesions from 15.5% of children. No evidence of T. pallidum subsp. pertenue infection, by either PCR or serology was found in approximately 59% of cases indicating alternative causes of yaws-like lesions in this endemic area.

During a survey of yaws prevalence in the Solomon Islands, we collected samples from skin ulcers of 41 children. Using PCR, we identified Haemophilus ducreyi infection in 13 (32%) children. PCR-positive and PCR-negative ulcers were phenotypically indistinguishable. Emergence of H. ducreyi as a cause of nongenital ulcers may affect the World Health Organization's yaws eradication program.

Syphilis is a systemic disease caused by the spirochete Treponema pallidum that is usually acquired through sexual exposure.

Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.

BACKGROUND: The 23S rRNA A2058G point mutation in Treponema pallidum is associated with macrolide antibiotic treatment failure. Its prevalence and potential association with a molecular subtype within the United States are unknown. METHODS: During 2007 to 2009, 11 clinics across the United States sent samples from genital ulcers to the Centers for Disease Control and Prevention. Molecular techniques were used to identify T. pallidum DNA sequences, the A2058G mutation, and subtype of T. pallidum. Accompanying epidemiologic information was abstracted from medical records. RESULTS: A total of 141 samples with T. pallidum were collected from individuals whose median age was 33 years (range, 13-68 years): 118 were male (69% reported as men having sex with men [MSM]). The A2058G mutation was carried in 75 samples (53%) with T. pallidum, with samples from MSM (versus women and other men) more likely carrying the A2058G mutation (65/82 samples versus 8/57 samples; prevalence ratio, 5.7; 95% confidence interval, 2.9-10.8). Of 98 strain-typed samples, 61 (62%) were the 14d9 subtype of T. pallidum, which was also associated with samples with T. pallidum from MSM (prevalence ratio, 3.5; 95% confidence interval, 1.9-6.5). However, among T. pallidum from MSM, the A2058G mutation was not associated with the 14d9 subtype. CONCLUSIONS: The A2058G mutation and 14d9 subtype of T. pallidum were present throughout the United States. Both were more commonly found in T. pallidum from MSM compared with women or other men but were not associated with each other. Treating syphilis with azithromycin should be done cautiously and only when treatment with penicillin or doxycycline is not feasible. (Copyright 2012 American Sexually Transmitted Diseases Association All rights reserved.)

BACKGROUND: In Russia, the microscopy- and culture-based diagnostics of trichomoniasis is mainly suboptimal. Recent years, domestically produced diagnostic PCR assays have been implemented; however, any evaluation of these PCRs has never been internationally reported. OBJECTIVE: To assess the performance characteristics of PCR assays developed and currently used in Russia to detect Trichomonas vaginalis. MATERIALS AND METHODS: Five PCR assays were assessed on 448 samples (317 vaginal and 131 male urethral) collected from symptomatic attendees of youth centres (n = 415) and patients of a dermatovenereological dispensary that were previously diagnosed with trichomoniasis (n = 33). As reference assay, a sensitive and specific real-time multiplex PCR was used. RESULTS: T. vaginalis DNA was detected in five (all females) of the 415 patients of youth centres (1.2%). All 33 patients previously diagnosed at the venereological dispensary proved to be true positive. For 445 (99.3%) of these 448 samples identical results were obtained by all PCRs, 35 positive and 410 negative. The three discordant samples were positive in all PCRs except one conventional PCR assay. The sensitivities of the PCRs were 94.3-100% and 66.7-100% for vaginal and urethral swabs, respectively. All evaluated assays were 100% specific. The detection limits of the different PCRs ranged from 0.1 to 5 genome equivalents per reaction. CONCLUSION: The PCR assays currently used in Russia for the detection of T. vaginalis have in general high sensitivities and excellent specificities for both vaginal samples and urethral samples from males.