INTRODUCTION: Neisseria gonorrhoeae (Ng) has successively developed resistance to all previously recommended antimicrobial therapies, with ceftriaxone being the last option for monotherapy of gonorrhea. Global emergence and international spread of the FC428 clone derived mosaic penA-60 allele, associated with highlevel ceftriaxone minimum inhibitory concentrations (MICs) in non FC428 clone Ng lineages, has become an increasing concern. The penA-60 allele carrying Ng was first identified in the U.S. in Las Vegas, Nevada (2019; GCWGS-102723), with a multi-locus sequence type (MLST)-1901 strain, in a non FC428 clone Ng lineage, which is associated with a historically ceftriaxone susceptible core genogroup. Later in 2022, an allele genetically similar to penA-60, mosaic penA-237, was identified in the UK (H22-722) and France (F92) with high-level ceftriaxone MICs and both belonged to MLST-1901. METHODS: In this study, we assessed phylogenomic relatedness and antimicrobial resistance (AMR) determinant profiles of these three isolates with high-level ceftriaxone MICs among a global collection of 2,104 genomes belonging to the MLST-1901 core genome cluster group 31, which includes strains separated by a locus threshold of 200 or fewer differences (Ng_cgc_200). Recombination events in and around the penA coding region were catalogued and potential sources of inter species recombinant DNA were also inferred. RESULTS: The global population structure of MLST-1901 core genogroup falls into 4 major lineages. Isolates GCWGS-10723, F92, and H22-722 clustered within Lineage 1, which was dominated by non-mosaic penA-5 alleles. These three isolates formed a clade within Lineage 1 that consisted of isolates from North America and southeast Asia. Neisseria subflava and Neisseria sicca were identified as likely progenitors of two independent recombination events that may have led to the generation of mosaic penA-60 and penA-237, within a possible non-mosaic penA-5 background. DISCUSSIONS: Our study suggests that there are multiple evolutionary pathways that could generate concerning mosaic penA alleles via homologous recombination of historically susceptible Ng lineages with Neisseria commensals. Enhanced surveillance of gonococcal strains and Neisseria commensals is crucial for understanding of the evolution of AMR, particularly in less-studied regions (e.g., Asia), where high-level ceftriaxone MICs and multi-drug resistance are more prevalent.

Neisseria meningitidis (Nm) and Neisseria gonorrhoeae (Ng) are human pathogens that sometimes occupy the same anatomical niche. Ng, the causative agent of gonorrhea, infects 87 million individuals annually worldwide and is an urgent threat due to increasing drug resistance. Ng is a pathogen of the urogenital tract and may infect the oropharyngeal or rectal site, often asymptomatically. Conversely, Nm is an opportunistic pathogen. While often a commensal in the oropharyngeal tract, it is also the leading cause of bacterial meningitis with 1.2 million cases globally, causing significant morbidity and mortality. Horizontal gene transfer (HGT) is likely to occur between Ng and Nm due to their shared anatomical niches and genetic similarity, which poses challenges for accurate detection and treatment. Routine surveillance through the Gonococcal Isolate Surveillance Project and Strengthening the U.S. Response to Resistant Gonorrhea detected six concerning urogenital Neisseria isolates with contradicting species identification in Milwaukee (MIL). While all six isolates were positive for Ng using nucleic acid amplification testing (NAAT) and matrix-assisted laser desorption/ionization time of flight identified the isolates as Ng, two biochemical tests, Gonochek-II and API NH, classified them as Nm. To address this discrepancy, we performed whole-genome sequencing (WGS) using Illumina MiSeq on all isolates and employed various bioinformatics tools. Species detection analysis using BMScan, which uses WGS data, identified all isolates as Ng. Furthermore, Kraken revealed over 98% of WGS reads mapped to the Ng genome and <1% to Nm. Recombination analysis identified putative HGT in all MIL isolates within the γ-glutamyl transpeptidase (ggt) gene, a key component in the biochemical tests used to differentiate between Nm and Ng. Further analysis identified Nm as the source of HGT event. Specifically, the active Nm ggt gene replaced the Ng pseudogenes, ggt1 and ggt2. Together, this study demonstrates that closely related Neisseria species sharing a niche underwent HGT, which led to the misidentification of species following biochemical testing. Importantly, NAAT accurately detected Ng. The misidentification highlights the importance of using WGS to continually evaluate diagnostic or bacterial identification tests.

INTRODUCTION: Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) infections are often asymptomatic; screening increases early detection and prevents disease, sequelae and further spread. To increase Ct and Ng testing, several countries have implemented specimen self-collection outside a clinical setting. While specimen self-collection at home is highly acceptable to patients and as accurate as specimens collected by healthcare providers, this strategy is new or not being used in some countries. To understand how offering at home specimen self-collection will affect testing uptake, test results, diagnosis and linkage to care, when compared with collection in clinical settings, we conducted a systematic literature review and meta-analysis of peer-reviewed studies. METHODS: We searched Medline, Embase, Global Health, Cochrane Library, CINAHL (EBSCOHost), Scopus and Clinical Trials. Studies were included if they directly compared specimens self-collected at home or in other non-clinical settings to specimen collection at a healthcare facility (self or clinician) for Ct and/or Ng testing and evaluated the following outcomes: uptake in testing, linkage to care, and concordance (agreement) between the two settings for the same individuals. Risk of bias (RoB) was assessed using Cochrane Risk of Bias (RoB2) tool for randomised control trials (RCTs). RESULTS: 19 studies, from 1998 to 2024, comprising 15 RCTs with a total of 62 369 participants and four concordance studies with 906 participants were included. Uptake of Ct or Ng testing was 2.61 times higher at home compared with clinical settings. There was a high concordance between specimens collected at home and in clinical settings, and linkage to care was not significantly different between the two settings (prevalence ratio 0.96 (95% CI 0.91-1.01)). CONCLUSION: Our meta-analysis and systematic literature review show that offering self-collection of specimens at home or in other non-clinical settings could be used as an additional strategy to increase sexually transmitted infection testing in countries that have not yet widely adopted this collection method.

Neisseria gonorrhoeae is a global threat to public health due to the accumulation of antimicrobial resistance mechanisms. ST-1901 is an internationally important sequence type (ST) because of its high incidence and the usual occurrence of chromosomally determined resistance. In this study, we describe the evolution of the ST-1901 and its single locus variants in Rio de Janeiro from 2006 to 2022. We analyzed 82 N. gonorrhoeae isolates according to antimicrobial susceptibility profile, resistance mechanisms, molecular typing, and phylogenetics. Six different single locus variants were detected. Phylogenetic analysis identified five clades, which share similar characteristics. Resistance rates for penicillin and tetracycline decreased due to the lower occurrence of resistance plasmids, but intermediary resistance to penicillin rose. Resistance to ciprofloxacin remained high throughout all clades and the years of the study. Regarding resistance to azithromycin, alterations in mtrR promoter and gene, and 23S rRNA encoding gene rrl were detected, with a notable rise in the incidence of C2611T mutations in more recent years occurring in 4 out of 5 clades. In contrast, beta-lactam resistance associated penA 34 mosaic was found only in one persisting clade (Clade D), as well as unique G45D and A39T mutations in mtrR gene and its promoter (Nm-Like) were found in only Clade B. Taken together, these data suggest that ST-1901, a persistently circulating lineage of N. gonorrhoeae in Rio de Janeiro, has undergone changes over the years and may evolve to develop resistance to the current recommended dual therapy adopted in Brazil, ceftriaxone and azithromycin.

To determine antimicrobial susceptibility of Neisseria gonorrhoeae, we analyzed phenotypes and genomes of 72 isolates collected in Cambodia in 2023. Of those, 9/72 (12.5%) were extensively drug resistant, a 3-fold increase from 2022. Genomic analysis confirmed expansion of newly emerging resistant clones and ongoing resistance emergence across new phylogenetic backbones.

Genital herpes is caused by infection with herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and currently has no cure. The disease is the second-most common sexually transmitted infection in the United States, with an estimated 18.6 million prevalent genital infections caused by HSV-2 alone. Genital herpes diagnostics and treatments are not optimal, and no vaccine is currently available. The Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases convened a workshop entitled "CDC/NIAID Joint Workshop on Genital Herpes." This report summarizes 8 sessions on the epidemiology of genital herpes, neonatal HSV, HSV diagnostics, vaccines, treatments, cures, prevention, and patient advocacy perspective intended to identify opportunities in herpes research and foster the development of strategies to diagnose, treat, cure, and prevent genital herpes.

This Viewpoint discusses the US Food and Drug Administration’s authorization of marketing an at-home testing system for chlamydia and gonorrhea as a good first step in boosting access to screening and treatment and in reducing infection rates. | eng

Unsuccessful treatment of gonorrhoea has not yet occurred in the USA, and cases of gonorrhoea that are non-susceptible to cephalosporins have been rare. In 2019, non-susceptibility to ceftriaxone conferred by the mosaic penA 60.001 allele was found in a Neisseria gonorrhoeae multilocus sequence type (MLST) 1901 isolate from Nevada.1 In this Correspondence, we present two additional US cases of the penA 60.001 allele identified in MLST 8123, an emerging international multidrug non-susceptible N gonorrhoeae lineage. Although these cases responded to ceftriaxone treatment, N gonorrhoeae isolates from the first known patient (case 1) demonstrated in-vitro non-susceptibility to ceftriaxone as well as non-susceptibility or resistance to drugs previously recommended for front-line treatment. | | In August, 2022, N gonorrhoeae grown from urine culture from a patient with urethritis in primary care in Massachusetts displayed non-susceptibility to cephalosporins (the minimum inhibitory concentrations were 1·0 μg/mL for ceftriaxone and >1·0 μg/mL for cefixime by agar dilution; the minimum inhibitory concentration for cefixime was 1·5 μg/mL by gradient strip) and azithromycin and resistance to ciprofloxacin, penicillin, and tetracycline (appendix pp 6–7). Antimicrobial susceptibility testing was done with gradient strips at the state public health laboratory Massachusetts and then confirmed via agar dilution at the US Centers for Disease Control and Prevention (CDC). The patient (case 1) had already been successfully diagnosed on nucleic acid amplification test (NAAT) with gonorrhoea and was given 500 mg ceftriaxone intramuscularly and asked to return to primary care where, 9 days after treatment, he was asymptomatic, had normal results during examination, and tested negative by urine culture and pharyngeal and rectal NAAT recommended by the Massachusetts sexually transmitted diseases programme to document N gonorrhoeae clearance from any site of infection. The patient reported that he had not travelled outside USA in the 60 days before onset of symptoms. He disclosed female sex worker contacts, but insufficient information was provided to trace the contacts.

INTRODUCTION: The reemergence of syphilis, especially congenital syphilis, presents a significant public health threat. Accurate diagnosis of syphilis depends on recognition of a constellation of symptoms, review of medical and sexual history, and multiple laboratory tests. While reliable, current tests for syphilis can be difficult to interpret, which can lead to delays in treatment. AREA COVERED: This review summarizes the major advantages and limitations of available diagnostic laboratory methods for syphilis, provides an update on recent advances in laboratory tools, and highlights the urgent need for coordinated efforts to create new tools to halt the resurgence of syphilis. EXPERT OPINION: In syphilis, the wide variety of short-lived signs and symptoms followed by periods of latency create diagnostics challenges. Currently available laboratory tests, when positive, require additional information to interpret (prior testing, treatment, and sexual history). Point-of-care tests that can rapidly and accurately detect both treponemal and non-treponemal antibodies would be a huge step toward reducing test turnaround time and time to treatment. Incorporating biological insights and technology innovations to advance the development of direct detection assays is urgently needed. A comprehensive coordinated effort is critical to stem the tide of rising syphilis in the United States and globally.

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.

Neisseria gonorrhoeae multi-locus sequence type (ST) 9363 genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the U.S. Here we analyze a global collection of ST-9363 genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009-2011 and again during the 2012-2013 time, facilitating the clonal expansion of this genogroup with AZMrs in the U.S. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the U.S. and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers.Competing Interest StatementThe authors have declared no competing interest.

Recent reports suggest that mosaic-like sequences within the mtr (multiple transferable resistance) efflux pump locus of Neisseria gonorrhoeae likely originating from commensal Neisseria sp. by transformation can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within the mtrR gene encoding the transcriptional repressor (MtrR) of the mtrCDE efflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (Wadsworth et al. 2018. MBio. doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates including Azi. We report that a strong cis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of the mtrCDE promoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-like mtr locus.IMPORTANCE Historically, after introduction of an antibiotic for treatment of gonorrhea, strains of N. gonorrhoeae emerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of the Neisseria genus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented herein provide a framework for understanding how mosaic-like DNA sequences from commensal Neisseria that recombine within the gonococcal mtr efflux pump locus function to decrease bacterial susceptibility to antimicrobials including antibiotics used in therapy of gonorrhea.

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.

Syphilis is a sexually transmitted disease (STD) caused by the bacterium Treponema pallidum, subspecies pallidum (T. pallidum).1 The resurgence of syphilis in the last 2 decades is a major public health concern in the United States. In 2020, a total of 133 945 cases of all stages of syphilis were reported in the United States, an increase of more than 70% since 2015. The national rate of congenital syphilis has increased 254% since 2016. In 2020, a total of 2148 cases of congenital syphilis were reported, including 149 congenital syphilis–related stillbirths and infant deaths.2 However, syphilis diagnosis is still challenging, partially because of overlapping and ambiguous clinical presentation, particularly during early infection.3 For laboratory testing, darkfield microscopy examinations and direct molecular detection of T. pallidum from clinical specimens are definitive methods for diagnosing early syphilis, but both methods are currently not widely performed at local, reference, or public health laboratories and have challenges in identifying asymptomatic or early-stage infection without the presence of visible lesions for suitable specimen collection.4 Darkfield microscopy requires special equipment and experienced operators for reliable results.5 US Food and Drug Administration (FDA)–cleared molecular tests for syphilis are still not available, and laboratory-developed molecular tests are limited in availability. Therefore, serological tests that detect treponemal and nontreponemal antibodies remain the mainstay for routine laboratory diagnosis of active syphilis infection.6

Automated nontreponemal rapid plasma reagin (RPR) tests were recently introduced in the United States for syphilis testing and limited performance data are available. In collaboration with the Association of Public Health Laboratories, three public health laboratories (PHL) were chosen through a competitive selection process to evaluate the performance of three FDA-cleared automated RPR test systems: BioPlex 2200 Syphilis Total & RPR assay (Bio-Rad Laboratories), AIX 1000 (Gold Standard Diagnostics), and ASI Evolution (Arlington Scientific). Panels prepared at the CDC included: a qualitative panel comprised of 734 syphilis reactive/nonreactive sera; a quantitative panel of 50 syphilis reactive sera (RPR titer 1:64 to 1:1,024); and a reproducibility panel of 15 nonreactive and reactive sera (RPR titer 1:1 to 1:64). Panels were shipped frozen to the PHL and tested on the automated RPR systems following manufacturers' instructions. Prior test results were blinded to all laboratories. When compared to manual RPR (Arlington Scientific) performed at the CDC as a reference test, the qualitative panel results demonstrated an overall concordance of 95.9% for AIX 1000, 94.6% for ASI Evolution, and 92.6% for Bioplex RPR; quantitative panel showed within range titer of 2-fold for 94% of specimens for AIX 1000, 68% for ASI Evolution, and 64% for BioPlex RPR, and the reproducibility testing panel demonstrated point estimates ranging from 69 to 95%. Automated RPR instruments could reduce turnaround time and minimize interpretation errors. However, additional evaluations with more specimens could assist laboratories with implementing automated RPR tests and understanding their limitations.

This study characterized high-quality whole-genome sequences of a sentinel, surveillance-based collection of 1710 Neisseria gonorrhoeae (GC) isolates from 2019 collected in the USA as part of the Gonococcal Isolate Surveillance Project (GISP). It aims to provide a detailed report of strain diversity, phylogenetic relationships and resistance determinant profiles associated with reduced susceptibilities to antibiotics of concern. The 1710 isolates represented 164 multilocus sequence types and 21 predominant phylogenetic clades. Common genomic determinants defined most strains' phenotypic, reduced susceptibility to current and historic antibiotics (e.g. bla (TEM) plasmid for penicillin, tetM plasmid for tetracycline, gyrA for ciprofloxacin, 23S rRNA and/or mosaic mtr operon for azithromycin, and mosaic penA for cefixime and ceftriaxone). The most predominant phylogenetic clade accounted for 21 % of the isolates, included a majority of the isolates with low-level elevated MICs to azithromycin (2.0 µg ml(-1)), carried a mosaic mtr operon and variants in PorB, and showed expansion with respect to data previously reported from 2018. The second largest clade predominantly carried the GyrA S91F variant, was largely ciprofloxacin resistant (MIC ≥1.0 µg ml(-1)), and showed significant expansion with respect to 2018. Overall, a low proportion of isolates had medium- to high-level elevated MIC to azithromycin ((≥4.0 µg ml(-1)), based on C2611T or A2059G 23S rRNA variants). One isolate carried the penA 60.001 allele resulting in elevated MICs to cefixime and ceftriaxone of 1.0 µg ml(-1). This high-resolution snapshot of genetic profiles of 1710 GC sequences, through a comparison with 2018 data (1479 GC sequences) within the sentinel system, highlights change in proportions and expansion of select GC strains and the associated genetic mechanisms of resistance. The knowledge gained through molecular surveillance may support rapid identification of outbreaks of concern. Continued monitoring may inform public health responses to limit the development and spread of antibiotic-resistant gonorrhoea.

A commentary on recent successes and challenges for home STI specimen collection, for point-of-care test usage close to the home, and implications of COVID-19 self-tests for future STI self-test development.

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.

OBJECTIVES: Neisseria gonorrhoeae (gonococcus) infection is one of the most commonly reported nationally notifiable conditions in the United States. Gonococcus has developed antimicrobial resistance to each previously used antibiotic for gonorrhea therapy. However, some isolates may be still susceptible to no longer recommended, yet still effective antibiotics. This in turn suggests that targeted therapy could slow resistance development to currently recommended empirical treatments. We curated a gonococcal Ciprofloxacin Antibiotic Resistance Isolate Bank panel (Cipro-panel) as a tool for validating or developing new tests to determine ciprofloxacin susceptibility. METHOD: The Cipro-panel was selected using whole genome sequencing, bioinformatic tools, and antimicrobial susceptibility testing (AST) data. Isolates were further selected based on nucleotide variations in gyrA and parC genes. RESULTS: We selected 14 unique N. gonorrhoeae isolates from the 2006-2012 Gonococcal Isolate Surveillance Project (GISP) collection. They represented a wide range of antimicrobial susceptibility to ciprofloxacin and commonly observed nucleotide variations of gyrA and parC genes. This Cipro-panel consists of 5 isolates with resistant phenotypes (MIC > = 1 g/mL), 8 isolates with susceptible phenotypes (MIC < = 0.06 g/mL), and 1 isolate falling in the Clinical and Laboratory Standards Institute defined intermediate range. Among the gyrA variations we observed a total of 18 SNPs. Four positions had nonsynonymous changes (nucleotide positions 272, 284, 1093, and 1783). The first two positions (272 and 284) have been linked previously with resistance to ciprofloxacin (i.e. amino acid positions 91 and 95). For the parC gene, we observed a total of 21 possible SNPs. Eight of those SNPs resulted in non-synonymous amino acid changes. One location (amino acid 87) has been previously reported to be associated with ciprofloxacin resistance. CONCLUSIONS: This Cipro-Panel is useful for researchers interested in developing clinical tests related to ciprofloxacin. It could also provide additional choices for validation, quality assurance purposes and improve antibiotic usage.

GdhR is a transcriptional repressor of the virulence factor gene lctP, which encodes a unique l-lactate permease that has been linked to pathogenesis of Neisseria gonorrhoeae, and loss of gdhR can confer increased fitness of gonococci in a female mouse model of lower genital tract infection. In this work, we identified a single nucleotide polymorphism (SNP) in gdhR, which is often present in both recent and historical gonococcal clinical strains and results in a proline (P)-to-serine (S) change at amino acid position 6 (P6S) of GdhR. This mutation (gdhR6) was found to reduce GdhR transcriptional repression at lctP in gonococcal strains containing the mutant protein compared to wild-type GdhR. By using purified recombinant proteins and in vitro DNA-binding and cross-linking experiments, we found that gdhR6 impairs the DNA-binding activity of GdhR at lctP without an apparent effect on protein oligomerization. By analyzing a panel of U.S. (from 2017 to 2018) and Danish (1928 to 2013) clinical isolates, we observed a statistical association between gdhR6 and the previously described adenine deletion in the promoter of mtrR (mtrR-P A-del), encoding the repressor (MtrR) of the mtrCDE operon that encodes the MtrCDE multidrug efflux pump that can export antibiotics, host antimicrobials, and biocides. The frequent association of gdhR6 with the mtrR promoter mutation in these clinical isolates suggests that it has persisted in this genetic background to enhance lctP expression, thereby promoting virulence. IMPORTANCE We report the frequent appearance of a novel SNP in the gdhR gene (gdhR6) possessed by Neisseria gonorrhoeae. The resulting amino acid change in the GdhR protein resulted in enhanced expression of a virulence gene (lctP) that has been suggested to promote gonococcal survival during infection. The mutant GdhR protein expressed by gdhR6 had a reduced ability to bind to its target DNA sequence upstream of lctP. Interestingly, gdhR6 was found in clinical gonococcal strains isolated in the United States and Denmark at a high frequency and was frequently associated with a mutation in the promoter of the gene encoding a repressor (MtrR) of both the mtrCDE antimicrobial efflux pump operon and gdhR. Given this frequent association and the known impact of these regulatory mutations, we propose that virulence and antibiotic resistance properties are often phenotypically linked in contemporary gonococcal strains.

BACKGROUND: The Aptima Combo 2 (AC2) assay manufactured by Hologic, Inc. detects Neisseria gonorrhoeae (NG) and/or Chlamydia trachomatis (CT) in urogenital and extragenital specimens by targeting either a 16S rRNA (NG) or 23S rRNA (CT) region. In 2019, a mutation (C1515T) in the 23S rRNA region was reported to cause false negative/equivocal results in specimens collected in Finland. Specimens containing this variant (Fl-nvCT) were also discovered internationally. Working with specimens submitted to a large commercial laboratory, we sought to determine if this variant was also present in the United States. METHODS: A subset (N = 401) of specimens tested with the AC2 assay collected during a five-week period in late 2019/early 2020 were evaluated using an updated AC2 assay. RESULTS: While the FI-nvCT variant was not detected within this specimen panel, two CT variants containing 23S rRNA mutations (A1518G, G1526A) were identified. The updated AC2 assay targeting an additional region of the 23S rRNA detected both of these variants. A retrospective study of >18 million AC2 results tested between 2018-2019 did not display a decrease in CT positivity. CONCLUSIONS: Although we did not detect the Fl-nvCT variant among US specimens, we show evidence that the low occurrence of similar diagnostic escape mutants can be detected with an updated AC2 assay using multiple 23S rRNA targets.

The effect of extended refrigerated storage of 14 serum and plasma specimens on 5 syphilis serologic tests was evaluated for 16 weeks. Higher stability of nontreponemal and treponemal antibodies in serum was recorded compared to plasma. Described work may provide insights on refrigerated specimens' stability and suitability for syphilis tests.

Neisseria gonorrhoeae multi-locus sequence type (ST) 9363 core-genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the U.S. Here we analyze a global collection of ST-9363 core-genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 core-genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 core-genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009-2011 and again during the 2012-2013 time, facilitating the clonal expansion of this core-genogroup with AZMrs in the U.S. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the U.S. and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young core-genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers.

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.

BACKGROUND: Gradient strip antimicrobial susceptibility testing (AST) using Etest® is conducted by local public health jurisdictions participating in the Strengthening the U.S. Response to Resistant Gonorrhea (SURRG) program to inform public health responses to resistant gonorrhea. Proficiency testing results across the participating laboratories were analyzed and a comparison of Etest® with the agar dilution method was conducted. METHODS: Laboratories participating in SURRG performed Etest® for azithromycin (AZM), cefixime (CFX), and ceftriaxone (CRO). Concurrence between minimum inhibitory concentrations (MICs) obtained with Etest® versus the agar dilution method using corresponding isolates was defined as +/- 1 double dilution. Specific levels of reduced susceptibility were termed "alerts" and included isolates with the following MICs: ≥ 2.0 μg/ml (AZM), ≥ 0.25 μg/ml (CFX), and ≥ 0.125 μg/ml (CRO). Categorical (alert/non-alert) agreement was calculated for MICs determined using Etest® and agar dilution methods. RESULTS: SURRG laboratories had high proficiency testing scores (≥98%) and low levels of inter-laboratory variations in MICs. The overall concurrence of MICs (essential agreement) determined using agar dilution and Etest® was 96% (CRO), 96% (CFX), and 95% (AZM). Depending on the antibiotic tested, between 27-66% of isolates with alert MICs determined by Etest® also had alert MICs using the reference agar dilution methodology, however most of these alert MICs were detected at threshold levels. CONCLUSIONS: This study demonstrates that MICs produced by SURRG laboratories using Etest® have a high level of concurrence with agar dilution. Although confirmation of specific alert MICs varied, Etest® facilities rapid detection and response to emerging resistant gonorrhea.

Advancing the understanding of pelvic inflammatory disease (PID) requires access to advanced diagnostic approaches for evaluating reproductive sequelae of sexually transmitted infections (STIs). Current limitations of clinical criteria and advanced imaging technologies for diagnosing reproductive sequelae make diagnosis and surveillance of PID a challenge. We summarize and comment on major challenges in diagnostic evaluation of reproductive sequelae: limited point-of-care clinical diagnostic options for reproductive sequelae, economic and geographical obstacles to accessing state-of-the-art diagnostics, an expanding list of STIs that may cause reproductive sequelae and the complexities in evaluating them, and the need for coordinated research efforts to systematically evaluate biomarkers with gold-standard, well-defined specimens and associated clinical data. The future use of biomarkers in readily accessible mucosal or blood-derived specimens as a noninvasive approach to determining STI etiologies may be fruitful and requires more research. Biomarkers under consideration include cytokines, STI-specific antibody responses, and mRNA transcriptional profiles of inflammatory markers.

Two plasmid gene protein (Pgp3)-based serological assays, the Pgp3-ELISA and multiplex bead assay (Pgp3-MBA), were compared and used to estimate seropositivity of Chlamydia trachomatis (CT) among females 14 to 39 years old participating in the National Health and Nutrition Examination Survey between 2013-2016. Of the 2,201 specimens tested, 502 (29.5%, 95% CI 27.6-31.5) were positive using Pgp3-ELISA and 624 (28.4%, 95% CI 26.5-30.3) were positive using Pgp3-MBA. The overall agreement between the assays was 87.7%. Corresponding nucleic acid amplification test (NAAT) results were available for 1,725 specimens (from women 18-39 years old); of these, 42 (2.4%, 95% CI 1.8-3.3) were CT NAAT-positive. Most of the CT NAAT-positive specimens had corresponding positive serological assay results; 33 (78.6%, 95% CI 62.8-89.2) were Pgp3-ELISA-positive and 36 (85.7%, 95% CI 70.8-94.1) were Pgp3-MBA-positive. Although Pgp3-ELISA and Pgp3-MBA demonstrated equivalent performance in this study, an advantage of the Pgp3-MBA over Pgp3-ELISA is that it is well suited for high sample throughput applications.

BACKGROUND: Sexual networks are difficult to construct due to incomplete sexual partner data. The proximity of people within a network may be inferred from genetically similar infections. We explored genomic data combined with partner services investigation (PSI) data to extend our understanding of sexual networks affected by Neisseria gonorrhoeae (NG). METHODS: We used 2017-2019 PSI and whole-genome sequencing (WGS) data from eight jurisdictions participating in CDC's Strengthening the United States Response to Resistant Gonorrhea (SURRG) project. Clusters were identified from sexual contacts and through genetically similar NG isolates. Sexual mixing patterns were characterized by describing the clusters by the individual's gender and gender of their sex partners. RESULTS: Our study included 4,627 diagnoses of NG infection (81% sequenced), 2,455 people received a PSI, 393 people were negative contacts of cases, and 495 contacts with unknown NG status. We identified 823 distinct clusters using PSI data combined with WGS data. Of cases that were not linked to any other case using PSI data, 37% were linked when using WGS data. Overall, 40% of PSI cases were allocated to a larger cluster when PSI and WGS data were combined compared with PSI data alone. Mixed clusters containing women, men who report sex with women, and men who report sex with men were common when using the WGS data either alone or in combination with the PSI data. CONCLUSIONS: Combining PSI and WGS data improves our understanding of sexual network connectivity.

Congenital syphilis (CS) is on the rise in the United States and is a growing public health concern. CS is an infection with Treponema pallidum in an infant or fetus, acquired via transplacental transmission when a pregnant woman has untreated or inadequately treated syphilis. Pregnant women with untreated syphilis are more likely to experience pregnancies complicated by stillbirth, prematurity, low birth weight, and early infant death, while their children can develop clinical manifestations of CS such as hepatosplenomegaly, bone abnormalities, developmental delays, and hearing loss. One of the ways CS can be prevented is by identifying and treating infected women during pregnancy with a benzathine penicillin G regimen that is both appropriate for the maternal stage of syphilis and initiated at least 30 days prior to delivery. In this article we discuss many of the challenges faced by both public health and healthcare systems with regards to this preventable infection, summarize missed opportunities for CS prevention, and provide practical solutions for future CS prevention strategies.

The recent emergence of strains of Neisseria gonorrhoeae associated with treatment failures to ceftriaxone, the foundation of current treatment options, has raised concerns over a future of untreatable gonorrhea. Current global data on gonococcal strains suggest that several lineages, predominately characterized by mosaic penA alleles, are associated with elevated minimum inhibitory concentrations (MICs) to extended spectrum cephalosporins (ESCs). Here we report on whole genome sequences of 813 N. gonorrhoeae isolates collected through the Gonococcal Isolate Surveillance Project in the United States. Phylogenomic analysis revealed that one persisting lineage (Clade A, multi-locus sequence type [MLST] ST1901) with mosaic penA-34 alleles, contained the majority of isolates with elevated MICs to ESCs. We provide evidence that an ancestor to the globally circulating MLST ST1901 clones potentially emerged around the early to mid-20th century (1944, credibility intervals [CI]: 1935-1953), predating the introduction of cephalosporins, but coinciding with the use of penicillin. Such results indicate that drugs with novel mechanisms of action are needed as these strains continue to persist and disseminate globally.