BACKGROUND:Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola virus disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen. METHODS:A cohort of 131 male EVD survivors in Liberia were enrolled in a case-case study. "Early clearers" were defined as those with 2 consecutive negative EBOV semen test results by real-time reverse-transcription polymerase chain reaction (rRT-PCR) ≥2 weeks apart within 1 year after discharge from the Ebola treatment unit or acute EVD. "Late clearers" had detectable EBOV RNA by rRT-PCR >1 year after discharge from the Ebola treatment unit or acute EVD. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical examinations and blood work. RESULTS:Compared with early clearers, late clearers were older (median, 42.5 years; P < .001) and experienced fewer severe clinical symptoms (median 2, P = .006). Late clearers had more lens opacifications (odds ratio, 3.9 [95% confidence interval, 1.1-13.3]; P = .03), after accounting for age, higher total serum immunoglobulin G3 (IgG3) titers (P = .005), and increased expression of the HLA-C*03:04 allele (0.14 [.02-.70]; P = .007). CONCLUSIONS:Older age, decreased illness severity, elevated total serum IgG3 and HLA-C*03:04 allele expression may be risk factors for the persistence of EBOV in the semen of EVD survivors. EBOV persistence in semen may also be associated with its persistence in other immunologically protected sites, such as the eye.

The frequency of lymphogranuloma venereum or invasive Chlamydia trachomatis 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 lymphogranuloma venereum-associated serovar L2 in 14% of 132 remnant C. trachomatis-positive rectal swabs.

ABSTRACT:During the 2017-2018 National Health and Nutrition Examination Survey, urine samples from participants aged 14 to 59 years were tested for Mycoplasma genitalium infection. Overall prevalence was 1.7% (95% confidence interval [CI], 1.1%-2.7%). Prevalence was similar between males (1.8% [95% CI, 0.9%-3.1%]) and females (1.7% [95% CI, 0.8%-3.0%]).

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.

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.

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.

Background: The Centers for Disease Control and Prevention (CDC) recommends specific regimens for chlamydia and dual therapy for gonorrhea to mitigate antimicrobial-resistant gonorrhea in the CDC 2015 sexually transmitted disease treatment guidelines. Only limited studies examining adherence to these recommendations have been conducted at private practices in the United States. Methods: We used the OptumLabs Data Warehouse, a comprehensive, longitudinal data asset with deidentified persons with linked commercial insurance claims and clinical information, to identify persons aged 15 to 60 years who had valid nucleic acid amplification testing results demonstrating urogenital or extragenital gonorrhea or chlamydia in 2016 to 2018. We defined valid laboratory results as positive or negative. We then assessed the time of their first positive test result and the type of treatment within 30 days to determine if there was evidence in the claims record that the CDC-recommended treatment was provided. We defined presumed treatment if the date of treatment was before the date of the positive test result within 30 days. Results: Among 6476 patients with positive gonorrhea test results and 26,847 patients with positive chlamydia test results only, 34.8% and 64.2% had evidence of receiving the CDC-recommended therapy, respectively. Approximately 11.6% of patients with positive gonorrhea test results with recommended dual treatment and 7.1% of patients with positive chlamydia test results only with recommended chlamydia treatment were presumptively treated. Conclusion: Analysis of treatment claims and medical records from private settings indicated low rates of recommended gonorrhea and chlamydia treatment. Validation of treatment claims is needed to support further quality of care interventions based on these data.

Multilocus sequence typing (MLST) provides allele-based characterization of bacterial pathogens in a standardized framework. However, classical MLST schemes for Bordetella pertussis, the causative agent of whooping cough, seldom reveal diversity among the small number of gene targets and thereby fail to delineate population structure. To improve the discriminatory power of allele-based molecular typing of B. pertussis, we have developed a whole-genome MLST (wgMLST) scheme from 225 reference-quality genome assemblies. Iterative refinement and allele curation resulted in a scheme of 3,506 coding sequences and covering 81.4% of the B. pertussis genome. This wgMLST scheme was further evaluated with data from a convenience sample of 2,389 B. pertussis isolates sequenced on Illumina instruments, including isolates from known outbreaks and epidemics previously characterized by existing molecular assays, as well as replicates collected from individual patients. wgMLST demonstrated concordance with whole-genome single nucleotide polymorphism (SNP) profiles, accurately resolved outbreak and sporadic cases in a retrospective comparison, and clustered replicate isolates collected from individual patients during diagnostic confirmation. Additionally, a reanalysis of isolates from two statewide epidemics using wgMLST reconstructed the population structures of circulating strains with increased resolution, revealing new clusters of related cases. Comparison with an existing core genome (cgMLST) scheme highlights the stable gene content of this bacterium and forms the initial foundation for necessary standardization. These results demonstrate the utility of wgMLST for improving B. pertussis characterization and genomic surveillance during the current pertussis disease resurgence.

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:Antimicrobial resistance in Mycoplasma genitalium (MG), a cause of urethritis, is a growing concern. Yet little is known about the geographic distribution of MG resistance in the United States or about its associated clinical outcomes. We evaluated the frequency of MG among men with urethritis, resistance mutations, and posttreatment symptom persistence. METHODS:We enrolled men presenting with urethritis symptoms to 6 US sexually transmitted disease (STD) clinics during June 2017-July 2018; men with urethritis were eligible for follow-up contact and, if they had persistent symptoms or MG, a chart review. Urethral specimens were tested for MG and other bacterial STDs. Mutations in 23S ribosomal ribonucleic acid (rRNA) loci (macrolide resistance-associated mutations [MRMs]) and in parC and gyrA (quinolone-associated mutations) were detected by targeted amplification/Sanger sequencing. RESULTS:Among 914 evaluable participants, 28.7% (95% confidence interval [CI], 23.8-33.6) had MG. Men with MG were more often Black (79.8% vs 66%, respectively), <30 years (72.9% vs 56.1%, respectively), and reported only female partners (83.7% vs 74.2%, respectively) than men without MG. Among MG-positive participants, 64.4% (95% CI, 58.2-70.3%) had MRM, 11.5% (95% CI, 7.9-16.0%) had parC mutations, and 0% had gyrA mutations. Among participants treated with azithromycin-based therapy at enrollment and who completed the follow-up survey, persistent symptoms were reported by 25.8% of MG-positive/MRM-positive men, 13% of MG-positive/MRM-negative men, and 17.2% of MG-negative men. CONCLUSIONS:MG infection was common among men with urethritis; the MRM prevalence was high among men with MG. Persistent symptoms following treatment were frequent among men both with and without MG.

ABSTRACT Multi-Locus Sequence Typing (MLST) provides allele-based characterization of bacterial pathogens in a standardized framework. However, current MLST schemes for Bordetella pertussis , the causative agent of whooping cough, seldom reveal diversity among the small number of gene targets and thereby fail to delineate population structure. To improve discriminatory power of allele-based molecular typing of B. pertussis , we have developed a whole-genome MLST (wgMLST) scheme from 214 reference-quality genome assemblies. Iterative refinement and allele curation resulted in a scheme of 3,506 coding sequences and covering 81.4% of the B. pertussis genome. This wgMLST scheme was further evaluated with data from a convenience sample of 2,389 B. pertussis isolates sequenced on Illumina instruments, including isolates from known outbreaks and epidemics previously characterized by existing molecular assays, as well as replicates collected from individual patients. wgMLST demonstrated concordance with whole-genome single nucleotide polymorphisms (SNP) profiles, accurately resolved outbreak and sporadic cases in a retrospective comparison, and clustered replicate isolates collected from individual patients during diagnostic confirmation. Additionally, a re-analysis of isolates from two statewide epidemics using wgMLST reconstructed the population structures of circulating strains with increased resolution, revealing new clusters of related cases. Comparison with an existing core-genome (cgMLST) scheme highlights the genomic stability of this bacterium and forms the initial foundation for necessary standardization. These results demonstrate the utility of wgMLST for improving B. pertussis characterization and genomic surveillance during the current pertussis disease resurgence.

Abstract Background Chlamydia trachomatis (CT) causes pelvic inflammatory disease (PID) and other sequelae; however, these associations are not fully characterized. CT serologic assays including Pgp3 ELISA may detect prior CT infection and may better elucidate these associations. We used a serologic Pgp3 multiplex bead array assay (Pgp3MBA) to measure CT seroprevalence in reproductive-age US women and assess the association with PID. Methods We performed CT Pgp3MBA on sera collected from women 18–39 years old during the 2013–2016 cycles of the National Health and Nutrition Examination Survey (NHANES) who had available urine CT nucleic acid amplification test results. Weighted Pgp3MBA CT seroprevalence and 95% confidence intervals (95% CI) were calculated. We also determined weighted prevalence ratios (PRs) and 95% CIs of self-reported lifetime PID among women with and without detectable Pgp3MBA and other characteristics to estimate these US national statistics. Results Among 2,339 women, 1,725 (73.7%) had available sera. Of these women, 1,425 (or 93.4% of those with data) were sexually experienced and had a CT seroprevalence of 35.9% (95% CI 33.4–38.4). When weighted for US women, CT seroprevalence was 30.5% (95% CI 26.6–34.4%), ranging from 16.9% (95% CI 11.0–22.8%) among non-Hispanic Asian women to 70.2% (95% CI 62.4–78.0%) among non-Hispanic black women. PID was reported by 4.2% (95% CI 3.1–5.2) of 1,413 sexually-experienced women with PID data or an estimated 3.8% (95% CI 2.6–5.0) of US women. Among US women, estimated PID varied by Pgp3MBA status; 7.3% (95% CI 4.3–10.2) of Pgp3MBA-positive women were estimated to report PID versus 2.3% (95% CI 1.3–3.4) of Pgp3MBA-negative women (PR 3.1; 95% CI 1.7–5.9). PID prevalence did not vary by age, nor self-reported recent sexually transmitted disease among US women, but was higher among non-Hispanic black women compared to non-Hispanic white women (PR 2.2; 95% CI 1.4–3.5). Conclusion Nearly one-third of US women have had CT by Pgp3MBA, with differences by race/ethnicity. Women with prior CT had three times the reported PID prevalence of women without CT. Further serologic research may refine the population-level impact of CT prevention activities, such as recommended annual CT screening, on PID incidence, particularly among non-Hispanic black women. Disclosures All Authors: No reported disclosures

Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae , with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis . While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene ( tox ).

Background The Centers for Disease Control and Prevention (CDC) recommends specific regimens for treatment of chlamydia and gonorrhea. Dual therapy is recommended for gonococcal infection to mitigate antimicrobial resistant gonorrhea (250 mg ceftriaxone plus 1g azithromycin). Previous studies examining adherence to these recommendations have had limited information on medical claims, prescription claims, and laboratory data in private practices in the United States. Methods We used the OptumLabs® Data Warehouse (OLDW) a comprehensive, longitudinal, real-world data asset with de-identified lives with linked private insurance claims and clinical information to identify persons aged 15–60 years who had valid nucleic acid amplified testing results demonstrating gonorrhea or chlamydia in 2016–2017. We defined valid lab results as positive or negative, but did not include lab records with indeterminate or missing results in our analysis. We then assessed the time of their first positive test and the type of treatment within 30 days to determine if there was evidence in the claims record that the CDC recommended treatment was provided. Results 4,972 patients were identified as having gonorrhea only. Of this group, 77% were male, and 70% were 15–35 years of age. Additionally among this group, 35% had evidence of receiving the CDC recommended combination therapy for gonorrhea, 26% had evidence of receiving 250 mg ceftriaxone without evidence of receiving 1g azithromycin, and 16% had evidence of receiving 1g azithromycin without ceftriaxone. A separate group of 24,044 patients were identified as having chlamydia only. Among this group, 40% were male, and 88% were 15–35 years of age. Additionally among this group, 65% had evidence of receiving a CDC-recommended chlamydia treatment, and 11% also had evidence of receiving 250 mg ceftriaxone. Conclusion There is variation in claims data regarding the treatment regimens administered for gonorrhea and chlamydia treatment. Further studies are needed to evaluate treatment claims data against medical record reviews. Disclosure No significant relationships.

Background Mycoplasma genitalium (MG) is an emerging cause of urethritis. Although an FDA-approved MG diagnostic test is now available in the U.S., syndromic management of urethritis remains widespread. Little is known about the geographic distribution of MG resistance in the U.S. and associated clinical outcomes. We evaluated the frequency of MG among men with urethritis, antimicrobial susceptibility of MG, and post-treatment symptom persistence. Methods We enrolled men presenting with urethritis symptoms to 6 U.S. STD clinics during June 2017–July 2018. Participants with urethritis confirmed on stained urethral smear were eligible for a follow-up phone call 14–17 days post-enrollment and chart review. Urethral specimens were tested locally for N. gonorrhoeae and C. trachomatis. CDC tested specimens for MG and T. vaginalis. MG resistance mutations were detected by targeted amplification/Sanger sequencing of 23S rRNA loci (macrolide resistance mutations [MRM]) and parC and gyrA (quinolone resistance mutations). Results Among 914 participants with evaluable MG results, MG was detected in 28.7% (95% CI 23.8–33.6). Men with MG were more often black (79.8% vs 66%), <30 years (72.9% vs 56.2%), and reported only female partners (83.7% vs 74.2%) than men without MG. Among MG+ participants, MRM was detected in 62.2% (range 53%–72.3%), parC mutations in 11.5% (range 6.6–18.4%), and gyrA in 0%. Among 763 participants with follow-up, 19.8% reported symptom persistence, without clinically meaningful difference by MG status. Among MG participants treated with azithromycin, those with MRM more often reported persistent symptoms (35.1%) and were more likely to return to clinic within 45 days than those without MRM. Conclusion MG was common among men with urethritis and MRM prevalence high. Persistent symptoms were frequent among men with and without MG. Many participants with macrolide-resistant MG experienced symptom persistence and returned to clinic for evaluation. Disclosure No significant relationships.

Orolabial lymphogranuloma venereum was diagnosed for a man in Michigan, USA, who had sex with men, some infected with HIV. High index of suspicion for lymphogranuloma venereum led to accurate diagnosis, successful therapy, and description of an L2b variant with a unique genetic mutation.

We characterized 170 complete genome assemblies from clinical Bordetella pertussis isolates representing geographic and temporal diversity in the United States. These data capture genotypic shifts, including increased pertactin deficiency, occurring amid the current pertussis disease resurgence and provide a foundation for needed research to direct future public health control strategies.

ABSTRACT Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genomic structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine the potential evolution of the chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. The observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS 481 , a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigating disease resurgence and molecular epidemiology. IMPORTANCE Whooping cough, primarily caused by Bordetella pertussis , has resurged in the United States even though the coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen's evolution. The chromosome of B. pertussis includes a large number of repetitive mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-encoding genes, which otherwise exhibit little nucleotide sequence diversity. By comparing the complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology.

ABSTRACT RNase PH, encoded by the rph gene, is a 3′→5′ exoribonuclease that in E. coli participates primarily in the 3′ maturation of pre-tRNAs and the degradation of rRNA in stationary-phase cells. Interestingly, the routinely used laboratory strains of MG1655 and W3110 have naturally acquired the rph-1 allele, encoding a truncated catalytically inactive RNase PH protein which is widely assumed to be benign. Contrary to this assumption, we show that the rph-1 -encoded Rph-1 protein inhibits RNase P-mediated 5′-end maturation of primary pre-tRNAs with leaders of <5 nucleotides in the absence of RppH, an RNA pyrophosphohydrolase. In contrast, RppH is not required for 5′-end maturation of endonucleolytically generated pre-tRNAs in the rph-1 strain and for any tRNAs in Δ rph mutant or rph + strains. We propose that the Rph-1 protein bound to the 3′ end of the substrate creates a steric hindrance that in the presence of a triphosphate at the 5′ end reduces the ability of RNase P to bind to the pre-tRNA. IMPORTANCE In this paper, we demonstrate that the rph-1 mutation found in commonly used E. coli strains leads to the synthesis of a truncated functionally inactive RNase PH protein that interferes with the 5′-end maturation of specific tRNAs with short 5′ leaders by RNase P in the absence of RppH, an RNA pyrophosphohydrolase that converts primary 5′ triphosphates into 5′ monophosphates. The data presented indicate that the presence of the triphosphate interferes with RNase P binding to the pre-tRNA.

During 2010 and 2012, California and Vermont, respectively, experienced statewide epidemics of pertussis with differences seen in the demographic affected, case clinical presentation, and molecular epidemiology of the circulating strains. To overcome limitations of the current molecular typing methods for pertussis, we utilized whole-genome sequencing to gain a broader understanding of how current circulating strains are causing large epidemics. Through the use of combined next-generation sequencing technologies, this study compared de novo, singlecontig genome assemblies from 31 out of 33 Bordetella pertussis isolates collected during two separate pertussis statewide epidemics and 2 resequenced vaccine strains. Final genome architecture assemblies were verified with whole-genome optical mapping. Sixteen distinct genome rearrangement profiles were observed in epidemic isolate genomes, all of which were distinct from the genome structures of the two resequenced vaccine strains. These rearrangements appear to be mediated by repetitive sequence elements, such as high-copy-number mobile genetic elements and rRNA operons. Additionally, novel and previously identified single nucleotide polymorphisms were detected in 10 virulence-related genes in the epidemic isolates. Whole-genome variation analysis identified state-specific variants, and coding regions bearing nonsynonymous mutations were classified into functional annotated orthologous groups. Comprehensive studies on whole genomes are needed to understand the resurgence of pertussis and develop novel tools to better characterize the molecular epidemiology of evolving B. pertussis populations.IMPORTANCE Pertussis, or whooping cough, is the most poorly controlled vaccine-preventable bacterial disease in the United States, which has experienced a resurgence for more than a decade. Once viewed as a monomorphic pathogen, B. pertussis strains circulating during epidemics exhibit diversity visible on a genome structural level, previously undetectable by traditional sequence analysis using shortread technologies. For the first time, we combine short-and long-read sequencing platforms with restriction optical mapping for single-contig, de novo assembly of 31 isolates to investigate two geographically and temporally independent U. S. pertussis epidemics. These complete genomes reshape our understanding of B. pertussis evolution and strengthen molecular epidemiology toward one day understanding the resurgence of pertussis.