Chlamydia trachomatis is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. U.S. cases have been steadily increasing for more than a decade in both the urogenital tract and rectum. C. trachomatis is an obligate intracellular bacterium that is not easily cultured, limiting the capacity for genome studies to understand strain diversity and emergence among various patient 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, efficiencies are only 60-80% for ≥95-100% genome coverage. We therefore re-designed and expanded the RNA bait library to augment enrichment of the organism from clinical samples to improve efficiency. We describe the expanded library, the limit of detection for C. trachomatis genome copy input, and the 100% efficiency and high-resolution of generated genomes where genomic recombination among paired vaginal and rectal specimens from four patients was identified. 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, among geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men.Importance Chlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, and there is limited information on rectal C. trachomatis transmission and its impact on morbidity. To improve efficiency of previous studies involving whole genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we expanded the RNA bait library to augment enrichment of the organism from clinical samples. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis. We show the new system is sensitive for near identical genomes of C. trachomatis from two body sites in four women. Further, we provide 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.Competing Interest StatementThe authors have declared no competing interest.

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.

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 two consecutive negative EBOV semen tests by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) at least two weeks apart within 1 year after discharge from the Ebola Treatment Unit (ETU) or acute EVD. "Late clearers" had detectable EBOV RNA by rRT-PCR over one year following ETU discharge or acute EVD. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical exams and blood work. RESULTS: Compared to early clearers, late clearers were older (median 42.5 years, p = 0.0001) and experienced fewer severe clinical symptoms (median 2, p = 0.006). Late clearers had more lens opacifications (OR 3.9, 95%CI 1.1-13.3, p = 0.03), after accounting for age, higher total serum IgG3 titers (p = 0.007) and increased expression of the HLA-C*03:04 allele (OR 0.14, 95% CI 0.02-0.70, p = 0.007). 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.

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 (ESI) 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%) in comparison to 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.

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.

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

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 SureSelect(XT) 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.IMPORTANCE Chlamydia 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.

Multi-Locus 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 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 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 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.

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). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for tox, Diph_rpoB, and CUP_rpoB targets, respectively. Twenty-nine NTTB Corynebacterium isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in tox or the promoter region. This new Corynebacterium RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation.

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 STD 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 (OLDW), a comprehensive, longitudinal data asset with de-identified persons with linked commercial insurance claims and clinical information, to identify persons aged 15-60 years who had valid nucleic acid amplification testing results demonstrating urogenital or extragenital gonorrhea or chlamydia in 2016-2018. We defined valid lab results as positive or negative. 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. We defined presumed treatment if the date of treatment was before the date of the positive test within 30 days. RESULTS: Among 6,476 patients with positive gonorrhea tests and 26,847 patients with positive chlamydia tests only, 34.8% and 64.2% had evidence of receiving the CDC-recommended therapy, respectively. About 11.6% of patients with positive gonorrhea tests with recommended dual treatment and 7.1% of patients with positive chlamydia tests 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.

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 U.S. or associated clinical outcomes. We evaluated the frequency of MG among men with urethritis, resistance mutations, and post-treatment symptom persistence. METHODS: We enrolled men presenting with urethritis symptoms to 6 U.S. sexually transmitted disease (STD) clinics during June 2017-July 2018; men with urethritis were eligible for follow-up contact and (if persistent symptoms or MG) chart review. Urethral specimens were tested for MG and other bacterial STDs. Mutations in 23S rRNA loci (macrolide-associated mutations [MRMs]) and parC and gyrA (quinolone-associated mutations [QRMs]) were detected by targeted amplification/Sanger sequencing. RESULTS: Among 914 evaluable participants, 28.7% (95% CI 23.8-33.6) had MG. Men with MG were more often black (79.8% vs 66%), <30 years (72.9% vs 56.1%), and reported only female partners (83.7% vs 74.2%) 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; MRM prevalence was high among men with MG. Persistent symptoms following treatment were frequent among men with and without MG.

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.

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 genome 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 potential evolution of 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. Observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, 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 investigation of disease resurgence and molecular epidemiology. IMPORTANCE: Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though 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 high 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-coding genes which otherwise exhibit little nucleotide sequence diversity. By comparing 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.

Species of the genus Bordetella associate with various animal hosts, frequently causing respiratory disease. Bordetella pertussis is the primary agent of whooping cough and other Bordetella species can cause similar cough illness. Here, we report four complete genome sequences from isolates of different Bordetella species recovered from human respiratory infections.

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, single-contig 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 short-read 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.

While PCR is the most common method used for detecting Bordetella pertussis in the US, most laboratories use insertion sequence 481 (IS481), which is not specific for B. pertussis; therefore, the relative contribution of other Bordetella species is not understood. The objectives of this study were to evaluate the proportion of other Bordetella spp. misidentified as B. pertussis during a period of increased pertussis incidence, determine the level of agreement in Bordetella species detection between US commercial laboratories and CDC, and assess the relative diagnostic sensitivity of CDC's PCR assay when using a different PCR master mix. Specimens collected between May 2012-2013 were tested at two US commercial laboratories for B. pertussis and B. parapertussis detection. Every fifth specimen positive for IS481 and/or IS1001 with Ct values ≤35 was sent to CDC for PCR testing that identifies Bordetella species. Specimens with CDC PCR indeterminate or negative results were tested using an alternate PCR master mix. Of 755 specimens, there was agreement in species identification for 83.4% (n=630). Of those with different identifications (n=125), 79.2% (n=99) were identified as indeterminate B. pertussis at CDC. Overall, 0.66% (n=5) of the specimens were identified as B. holmesii or B. bronchiseptica at CDC. Of 115 specimens with indeterminate or negative results, 46.1% (n=53) were B. pertussis positive when tested by an alternate master mix, suggesting possible increase in assay sensitivity. This study demonstrates good agreement between the two US commercial laboratories and CDC and little misidentification of Bordetella species during the 2012 US epidemic.

Although pertussis disease is vaccine-preventable, Washington State experienced a substantial rise in pertussis incidence beginning in 2011. By June 2012, the reported cases reached 2,520 (37.5 cases per 100,000 residents), a 1,300% increase compared with the same period in 2011. We assessed the molecular epidemiology of this statewide epidemic using 240 isolates collected from case-patients reported from 19 of 39 Washington counties during 2012-2013. Typing methods included pulsed-field gel electrophoresis (PFGE), multilocus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), and pertactin gene (prn) mutational analysis. Using the scheme PFGE-MLVA-MLST-prn mutations-Prn deficiency, the 240 isolates comprised 65 distinct typing profiles. Thirty-one PFGE types were found, with the most common types, CDC013 (n=51), CDC237 (n=44), and CDC002 (n=42), accounting for 57% of them. Eleven MLVA types were observed, mainly comprising type 27 (n=183; 76%). Seven MLST types were identified, with the majority of the isolates typing as prn2-ptxP3-ptxA1-fim3-1 (n=157; 65%). Four different prn mutations accounted for the 76% of isolates exhibiting pertactin-deficiency. PFGE provided the highest discriminatory power (D=0.87) and was found to be a more powerful typing method than MLVA and MLST combined (D=0.67). This study provides evidence for the continued predominance of MLVA 27 and prn2-ptxP3-ptxA1, along with the reemergence of the fim3-1 allele. Our results indicate that the B. pertussis population causing this epidemic was diverse, with a few molecular types predominating. PFGE, MLVA, and MLST profiles were consistent with predominate types circulating in the US and other countries. For prn, several mutations were present in multiple molecular types.

Pertussis has made a striking resurgence in the US, returning to record numbers of reported cases last observed in the 1950s. Bordetella pertussis isolates lacking pertactin, a key antigen component of the acellular pertussis vaccine, have been observed, suggesting that B. pertussis is losing pertactin in response to vaccine immunity. Screening of 1300 isolates from outbreak and surveillance studies (historical isolates collected from 1935 up to 2009, the 2010 California pertussis outbreak, US isolates from routine surveillance between 2010-2012, and the 2012 Washington pertussis outbreak) by conventional PCR and later by Western blot and prn sequencing analyses ultimately identified 306 pertactin-deficient isolates. Of these pertactin-deficient strains, 276 were identified as having an IS481 in the prn gene (prnIS481-positive). The first prnIS481-positive isolate was found in 1994, with the next prnIS481-positive isolates not detected until 2010. Pertactin-deficient isolates increased substantially to over 50% in 2012. Sequence analysis of pertactin-deficient isolates revealed various types of mutations in the prn gene, including two deletions, single nucleotide substitutions resulting in a stop codon, an inversion in the promoter, and a single nucleotide insertion resulting in a frame shift mutation. All but one mutation type were found in prn2 alleles. CDC013 was a predominant PFGE profile in the pertactin-positive isolates (203/994), but was found in only 5% (16/306) of the pertactin-deficient isolates. Interestingly, PFGE profiles CDC002 and CDC237 represented 55% (167/306) of the identified pertactin-deficient isolates. These results indicate that there has been a recent, dramatic increase in pertactin-deficient B. pertussis isolates throughout the US.