Pandemic Demand for SARS-CoV-2 Testing Led to Critical Supply and Workforce Shortages in U.S. Clinical and Public Health Laboratories.

COVID-19 has brought unprecedented challenges to clinical and public health laboratories. While U.S. laboratories have continued striving to provide quality test results during the pandemic, the uncertainty and lack of supplies became a significant hurdle, hindering day-to-day laboratory operations and the ability to increase testing capacity for both SARS-CoV-2 and non-COVID-19 testing. In addition, long-standing laboratory workforce shortages became apparent, hindering the ability of clinical and public health laboratories to rapidly increase testing. The American Society for Microbiology, the College of American Pathologists, the National Coalition of STD Directors, and the Emerging Infections Network independently conducted surveys in 2020 and early 2021 to assess the capacity of the nation's clinical laboratories to respond to the increase in demand for testing during the COVID-19 pandemic. The results of these surveys highlighted the shortages of crucial supplies for SARS-CoV-2 testing and supplies for other routine laboratory diagnostics, as well as a shortage of trained personnel to perform testing. The conclusions are based on communications, observations, and the survey results of the clinical laboratory, public health, and professional organizations represented here. While the results of each survey considered separately may not be representative of the entire community, when considered together they provide remarkably similar results, further validating the findings and highlighting the importance of laboratory supply chains and the personnel capable of performing these tests for any response to a large-scale public health emergency.

In Vitro Testing of Trichomonas vaginalis Drug Susceptibility: Evaluation of Minimal Lethal Concentrations for Metronidazole and Tinidazole That Correspond With Treatment Failure.

BACKGROUND: The only drugs approved by the US Food and Drug Administration for oral treatment of trichomoniasis belong to the 5-nitroimidazole group. Most individuals infected with Trichomonas vaginalis can be cured with a standard treatment of metronidazole or tinidazole, but it is estimated that more than 159,000 people fail treatment each year. Although a minimal lethal concentration (MLC) corresponding to treatment failure has been reported for metronidazole, the MLC for tinidazole associated with treatment failure has not been determined. We conducted a study using T. vaginalis isolates from women with reported treatment success or failure to determine these values. METHODS: We measured MLCs of 47 isolates obtained from women who had failed metronidazole treatment, 33 isolates from women who had failed tinidazole treatment, and 48 isolates from women successfully cured with metronidazole. The cutoff was calculated as the 95th percentile of MLCs of susceptible isolates for each drug. RESULTS: Our data confirmed that the MLC previously associated with metronidazole treatment failure is ≥50 µg/mL and identified the MLC associated with tinidazole treatment failure as ≥6.3 µg/mL. For metronidazole, the agreement between laboratory result and treatment outcome was 93.7%; for tinidazole, this agreement was 88.9%. CONCLUSIONS: The T. vaginalis susceptibility assay is useful for determining whether 5-nitroimidazole treatment failure in persons with trichomoniasis can be attributed to drug resistance. These results are useful for establishing interpretive guidance of test results, and MLC levels can help guide appropriate patient treatment.

Phylogenomic Comparison of Neisseria gonorrhoeae Causing Disseminated Gonococcal Infections and Uncomplicated Gonorrhea in Georgia, United States.

Disseminated gonococcal infection (DGI) is a rare complication caused by the systemic dissemination of Neisseria gonorrhoeae to normally sterile anatomical sites. Little is known about the genetic diversity of DGI gonococcal strains and how they relate to other gonococcal strains causing uncomplicated mucosal infections. We used whole genome sequencing to characterize DGI isolates (n = 30) collected from a surveillance system in Georgia, United States, during 2017-2020 to understand phylogenetic clustering among DGI as well as uncomplicated uro- and extragenital gonococcal infection (UGI) isolates (n = 110) collected in Fulton County, Georgia, during 2017-2019. We also investigated the presence or absence of genetic markers related to antimicrobial resistance (AMR) as well as surveyed the genomes for putative virulence genetic factors associated with normal human-serum (NHS) resistance that might facilitate DGI. We found that DGI strains demonstrated significant genetic variability similar to the population structure of isolates causing UGI, with sporadic incidences of geographically clustered DGI strains. DGI isolates contained various AMR markers and genetic mechanisms associated with NHS resistance. DGI isolates had a higher frequency of the porB1A allele compared with UGI (67% vs 9%, P < .0001); however, no single NHS resistance marker was found in all DGI isolates. Continued DGI surveillance with genome-based characterization of DGI isolates is necessary to better understand specific factors that promote systemic dissemination.

Characterization of a Neisseria gonorrhoeae Ciprofloxacin panel for an antimicrobial resistant Isolate Bank.

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 µg/mL), 8 isolates with susceptible phenotypes (MIC < = 0.06 µg/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.

Chlamydia trachomatis Variants Escaping Detection in the Aptima Combo 2 Assay in the United States.

BACKGROUND: The Aptima Combo 2 (AC2) assay manufactured by Hologic, Inc., detects Neisseria gonorrhoeae and/or Chlamydia trachomatis (CT) in urogenital and extragenital specimens by targeting either a 16S rRNA (N. gonorrhoeae) 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 5-week period in late 2019/early 2020 were evaluated using an updated AC2 assay. RESULTS: Although the FI-nvCT variant was not detected within this specimen panel, 2 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 and 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.

Surveillance for Disseminated Gonococcal Infections, Active Bacterial Core Surveillance (ABCs)-United States, 2015-2019.

BACKGROUND: Disseminated gonococcal infections (DGIs) are thought to be uncommon; surveillance is limited, and case reports are analyzed retrospectively or in case clusters. We describe the population-level burden of culture-confirmed DGIs through the Active Bacterial Core surveillance (ABCs) system. METHODS: During 2015-2016, retrospective surveillance was conducted among residents in 2 ABCs areas and prospectively in 3 ABCs areas during 2017-2019. A DGI case was defined as isolation of Neisseria gonorrhoeae from a normally sterile site. A case report form was completed for each case and antimicrobial susceptibility testing (AST) was performed on available isolates. RESULTS: During 2015-2019, 77 DGI cases were identified (a rate of 0.13 cases per 100 000 population) and accounted for 0.06% of all reported gonorrhea cases in the 3 surveillance areas. Most DGI cases were male (64%), non-Hispanic Black (68%), and ranged from 16 to 67 years of age; blood (55%) and joint (40%) were the most common sterile sites. Among 29 isolates with AST results during 2017-2019, all were susceptible to ceftriaxone. CONCLUSIONS: DGI is an infrequent complication of N gonorrhoeae; because it can quickly develop antimicrobial resistance, continued DGI surveillance, including monitoring trends in antimicrobial susceptibility, could help inform DGI treatment recommendations.

Global Emergence and Dissemination of Neisseria gonorrhoeae ST-9363 Isolates with Reduced Susceptibility to Azithromycin.

Neisseria gonorrhoeae multilocus sequence type (ST) 9363 core-genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the United States. 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 United States. 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 United States 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.

Use of real-time PCR as an alternative to conventional genotyping methods for the laboratory detection of lymphogranuloma venereum (LGV).

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.

Implementation and Evaluation of Gradient Strip Antimicrobial Susceptibility Testing in US Public Health Laboratories to Respond to Resistant Gonorrhea.

BACKGROUND: Gradient strip antimicrobial susceptibility testing using Etest is conducted by local public health jurisdictions participating in the Strengthening the US 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/nonalert) agreement was calculated for MICs determined using Etest and agar dilution methods. RESULTS: Strengthening the US Response to Resistant Gonorrhea laboratories had high proficiency testing scores (≥98%) and low levels of interlaboratory 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% and 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.

Assessment and utility of 2 Chlamydia trachomatis Pgp3 serological assays for seroprevalence studies among women in the United States.

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.

Exploring and Comparing the Structure of Sexual Networks Affected by Neisseria gonorrhoeae Using Sexual Partner Services Investigation and Genomic Data.

BACKGROUND: Sexual networks are difficult to construct because of 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 8 jurisdictions participating in Centers for Disease Control and Prevention's Strengthening the US 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 4627 diagnoses of NG infection (81% sequenced), 2455 people received a PSI, 393 people were negative contacts of cases, and 495 were contacts with an 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.

At-Home Specimen Self-Collection and Self-Testing for Sexually Transmitted Infection Screening Demand Accelerated by the COVID-19 Pandemic: a Review of Laboratory Implementation Issues.

The coronavirus disease 2019 (COVID-19) pandemic reduced the sexually transmitted infection (STI) testing volume due to social-distancing and stay-at-home orders, among other reasons. These events highlighted previously known benefits of at-home STI self-testing or specimen self-collection and accelerated testing demand via telemedicine. We review testing outside traditional clinical settings. We focus on three curable bacterial STIs among the top 10 U.S. nationally notifiable conditions with screening recommendations: syphilis, gonorrhea (Neisseria gonorrhoeae, also known as the gonococcus [GC]), and chlamydia (Chlamydia trachomatis). At least 19 million GC/C. trachomatis (GC/CT) screening or diagnostic tests are performed annually, presenting a considerable challenge during the pandemic. Unlike for HIV, STI at-home tests are currently not commercially available. However, innovative telemedicine providers currently offer services where specimen self-collection kits are mailed to patients at home who then ship them to laboratories for processing. We discuss technical and regulatory aspects of modifications for home-based specimen self-collection. The telemedicine provider typically manages and communicates results, provides linkage to care, and is responsible for billing and case reporting. We also describe rapid testing devices in development that may present an opportunity for future self-testing. In summary, COVID-19 has accelerated the evaluation and development of STI self-tests and specimen self-collection. The remaining obstacles are high price, regulatory approval, support for laboratories offering the service, and uncertainty regarding whether target populations with the greatest need are reached effectively. However, increased testing, convenience, and privacy are potential benefits that may enhance uptake and outlast the pandemic.

Phylogenomic analysis reveals persistence of gonococcal strains with reduced-susceptibility to extended-spectrum cephalosporins and mosaic penA-34.

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.

Genomic Analysis of the Predominant Strains and Antimicrobial Resistance Determinants Within 1479 Neisseria gonorrhoeae Isolates From the US Gonococcal Isolate Surveillance Project in 2018.

BACKGROUND: The prevalence of Neisseria gonorrhoeae (GC) isolates with elevated minimum inhibitory concentrations to various antibiotics continues to rise in the United States and globally. Genomic analysis provides a powerful tool for surveillance of circulating strains, antimicrobial resistance determinants, and understanding of transmission through a population. METHODS: Neisseria gonorrhoeae isolates collected from the US Gonococcal Isolate Surveillance Project in 2018 (n = 1479) were sequenced and characterized. Whole-genome sequencing was used to identify sequence types, antimicrobial resistance profiles, and phylogenetic relationships across demographic and geographic populations. RESULTS: Genetic characterization identified that (1) 80% of the GC isolates were represented in 33 multilocus sequence types, (2) isolates clustered in 23 major phylogenetic clusters with select phenotypic and demographic prevalence, and (3) common antimicrobial resistance determinants associated with low-level or high-level decreased susceptibility or resistance to relevant antibiotics. CONCLUSIONS: Characterization of this 2018 Gonococcal Isolate Surveillance Project genomic data set, which is the largest US whole-genome sequence data set to date, sets the basis for future prospective studies, and establishes a genomic baseline of GC populations for local and national monitoring.

High Plasmid Gene Protein 3 (Pgp3) Chlamydia trachomatis Seropositivity, Pelvic Inflammatory Disease, and Infertility Among Women, National Health and Nutrition Examination Survey, United States, 2013-2016.

BACKGROUND: Chlamydia trachomatis causes pelvic inflammatory disease (PID) and tubal infertility. Plasmid gene protein 3 antibody (Pgp3Ab) detects prior chlamydial infections. We evaluated for an association of high chlamydial seropositivity with sequelae using a Pgp3Ab multiplex bead array (Pgp3AbMBA). METHODS: We performed chlamydia Pgp3AbMBA on sera from women 18-39 years old participating in the 2013-2016 National Health and Nutrition Examination Survey (NHANES) with urine chlamydia nucleic acid amplification test results. High chlamydial seropositivity was defined as a median fluorescence intensity (MFI ≥ 50 000; low-positive was MFI > 551-<50 000. Weighted US population high-positive, low-positive, and negative Pgp3Ab chlamydia seroprevalence and 95% confidence intervals (CI) were compared for women with chlamydial infection, self-reported PID, and infertility. RESULTS: Of 2339 women aged 18-39 years, 1725 (73.7%) had sera, and 1425 were sexually experienced. Overall, 104 women had high positive Pgp3Ab (5.4% [95% CI 4.0-7.0] of US women); 407 had lowpositive Pgp3Ab (25.1% [95% CI 21.5-29.0]), and 914 had negative Pgp3Ab (69.5% [95% CI 65.5-73.4]). Among women with high Pgp3Ab, infertility prevalence was 2.0 (95% CI 1.1-3.7) times higher than among Pgp3Ab-negative women (19.6% [95% CI 10.5-31.7] versus 9.9% [95% CI 7.7-12.4]). For women with low Pgp3Ab, PID prevalence was 7.9% (95% CI 4.6-12.6) compared to 2.3% (95% CI 1.4-3.6) in negative Pgp3Ab. CONCLUSIONS: High chlamydial Pgp3Ab seropositivity was associated with infertility although small sample size limited evaluation of an association of high seropositivity with PID. In infertile women, Pgp3Ab may be a marker of prior chlamydial infection.

A community-driven resource for genomic epidemiology and antimicrobial resistance prediction of Neisseria gonorrhoeae at Pathogenwatch.

BACKGROUND: Antimicrobial-resistant (AMR) Neisseria gonorrhoeae is an urgent threat to public health, as strains resistant to at least one of the two last-line antibiotics used in empiric therapy of gonorrhoea, ceftriaxone and azithromycin, have spread internationally. Whole genome sequencing (WGS) data can be used to identify new AMR clones and transmission networks and inform the development of point-of-care tests for antimicrobial susceptibility, novel antimicrobials and vaccines. Community-driven tools that provide an easy access to and analysis of genomic and epidemiological data is the way forward for public health surveillance. METHODS: Here we present a public health-focussed scheme for genomic epidemiology of N. gonorrhoeae at Pathogenwatch ( https://pathogen.watch/ngonorrhoeae ). An international advisory group of experts in epidemiology, public health, genetics and genomics of N. gonorrhoeae was convened to inform on the utility of current and future analytics in the platform. We implement backwards compatibility with MLST, NG-MAST and NG-STAR typing schemes as well as an exhaustive library of genetic AMR determinants linked to a genotypic prediction of resistance to eight antibiotics. A collection of over 12,000 N. gonorrhoeae genome sequences from public archives has been quality-checked, assembled and made public together with available metadata for contextualization. RESULTS: AMR prediction from genome data revealed specificity values over 99% for azithromycin, ciprofloxacin and ceftriaxone and sensitivity values around 99% for benzylpenicillin and tetracycline. A case study using the Pathogenwatch collection of N. gonorrhoeae public genomes showed the global expansion of an azithromycin-resistant lineage carrying a mosaic mtr over at least the last 10 years, emphasising the power of Pathogenwatch to explore and evaluate genomic epidemiology questions of public health concern. CONCLUSIONS: The N. gonorrhoeae scheme in Pathogenwatch provides customised bioinformatic pipelines guided by expert opinion that can be adapted to public health agencies and departments with little expertise in bioinformatics and lower-resourced settings with internet connection but limited computational infrastructure. The advisory group will assess and identify ongoing public health needs in the field of gonorrhoea, particularly regarding gonococcal AMR, in order to further enhance utility with modified or new analytic methods.

Whole-Genome Enrichment and Sequencing of Chlamydia trachomatis Directly from Patient Clinical Vaginal and Rectal Swabs.

Chlamydia trachomatis, an obligately intracellular bacterium, is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. Numbers of U.S. infections of the urogenital tract and rectum have increased annually. Because C. trachomatis is not easily cultured, comparative genomic studies are limited, restricting our understanding of strain diversity and emergence among populations globally. While Agilent SureSelectXT target enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal samples, public access to these libraries is not available. We therefore designed an RNA bait library (34,795 120-mer probes based on 85 genomes, versus 33,619 probes using 74 genomes in a previous one) to augment organism sequencing from clinical samples that can be shared with the scientific community, enabling comparison studies. We describe the library and limit of detection for genome copy input, and we present results of 100% efficiency and high-resolution determination of recombination and identical genomes within vaginal-rectal specimen pairs in women. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporary C. trachomatis STIs across sample types, geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men.IMPORTANCEChlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, which limits our understanding of urogenital and rectal C. trachomatis transmission and impact on morbidity. To provide a publicly available workflow for whole-genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we developed and report on an RNA bait library to enrich the organism from clinical samples for sequencing. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis and identified recombinant and identical C. trachomatis genomes in paired vaginal-rectal samples from women. Our workflow provides a robust genomic epidemiologic approach to advance our understanding of C. trachomatis strains causing ocular, urogenital, and rectal infections and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.

Atypical Mutation in Neisseria gonorrhoeae 23S rRNA Associated with High-Level Azithromycin Resistance.

A2059G mutation in the 23S rRNA gene is the only reported mechanism conferring high-level azithromycin resistance (HL-AZMR) in Neisseria gonorrhoeae Through U.S. gonococcal antimicrobial resistance surveillance projects, we identified four HL-AZMR gonococcal isolates lacking this mutational genotype. Genetic analysis revealed an A2058G mutation of 23S rRNA alleles in all four isolates. In vitro selected gonococcal strains with homozygous A2058G recapitulated the HL-AZMR phenotype. Taken together, we postulate that the A2058G mutation confers HL-AZMR in N. gonorrhoeae.