This report describes the complete genome sequence assemblies from four representative isolates of the human pathogen Corynebacterium belfantii. These data provide necessary references to aid accurate sequence-based species discrimination among closely related Corynebacterium spp. pathogens.

In the United States, the general laboratory method for diagnosing pertussis, caused by Bordetella pertussis, is real-time PCR (rt-PCR) targeting insertion sequence 481 (IS481). Other Bordetella species (parapertussis, holmesii, and bronchiseptica) can also cause a pertussis-like syndrome, and some commercial laboratory assays include the insertion sequence 1001 (pIS1001) that can detect B. parapertussis/B. bronchiseptica (BppBb). Because IS481 exists in B. pertussis and B. holmesii, current commercial assays cannot differentiate these two species. We used a multiplex rt-PCR assay containing species-specific targets to Bordetella to evaluate clinical specimens detected as B. pertussis/B. holmesii (BpBh) or BppBb by commercial laboratories. A sample of 3,984 clinical specimens positive for IS481 or pIS1001 from two commercial laboratories during 2012-2019 were re-tested at CDC. Agreement of Bordetella species between the CDC and commercial laboratory assays, and the proportion of commercial laboratory specimens that were non-B. pertussis by CDC's assay was assessed. Overall agreement in Bordetella species detection and identification between the CDC and commercial lab assays was 85%. Agreement for identifying B. pertussis was 87% for 3,663 BpBh specimens and 98% for identifying B. parapertussis in 310 BppBb specimens. CDC's assay detected B. holmesii in 55/3,984 (1.4%) specimens. Most discrepant results (410/490, 82%) were BpBh specimens interpreted as indeterminate B. pertussis at CDC. We found a small portion of B. holmesii in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays, suggesting that commercial PCR assays are a reliable diagnostic tool for correctly identifying Bordetella species in most patients with suspected pertussis. IMPORTANCE: When testing specimens collected from patients with suspected pertussis, large-scale commercial laboratories in the United States employ an IS481-based assay that cannot differentiate between Bordetella pertussis and Bordetella holmseii. The level of B. holmesii causing pertussis-like illness in the United States is not well-understood given that only B. pertussis is nationally notifiable. After re-testing with a multiplex, species-specific rt-PCR assay, our data show low levels of B. holmesii identified in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays. These results reinforce the validity of large-scale commercial rt-PCR testing as a reliable diagnostic tool for pertussis in the United States.

BACKGROUND: In 2020, the Council of State and Territorial Epidemiologists (CSTE) pertussis case definition was modified; the main change was classifying PCR-positive cases as confirmed, regardless of cough duration. Pertussis data reported through Enhanced Pertussis Surveillance (EPS) in seven sites and the National Notifiable Diseases Surveillance System (NNDSS) were used to evaluate the impact of the new case definition. METHODS: We compared the number of EPS cases with cough onset in 2020 to the number that would have been reported based on the prior (2014) CSTE case definition. To assess the impact of the change nationally, the proportion of EPS cases newly reportable under the 2020 CSTE case definition was applied to 2020 NNDSS data to estimate how many additional cases were captured nationally. RESULTS: Among 442 confirmed and probable cases reported to EPS states in 2020, 42 (9.5%) were newly reportable according to the 2020 case definition. Applying this proportion to the 6,124 confirmed and probable cases reported nationally in 2020, we estimated that the new definition added 582 cases. Had the case definition not changed, reported cases in 2020 would have decreased by 70% from 2019; the observed decrease was 67%. CONCLUSIONS: Despite a substantial decrease in reported pertussis cases in the setting of COVID-19, our data show that the 2020 pertussis case definition change resulted in additional case reporting compared with the previous case definition, providing greater opportunities for public health interventions such as prophylaxis of close contacts.

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis, the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA "baits" targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.

Volume 3, no. 4, e00965-15, 2015. Page 1: The byline and affiliation line should read as given above.

Pertussis remains a public health concern in South Africa, with an increase in reported cases and outbreaks in recent years. Whole genome sequencing was performed on 32 Bordetella pertussis isolates sourced from three different surveillance programmes in South Africa between 2015 and 2019. Genome sequences were characterized using multilocus sequence typing, vaccine antigen genes (ptxP, ptxA, ptxB, prn and fimH) and overall genome structure. All isolates were sequence type 2 and harboured the pertussis toxin promoter allele ptxP3. The dominant genotype was ptxP3-ptxA1-ptxB2-prn2-fimH2 (31/32, 96.9 %), with no pertactin-deficient or other mutations in vaccine antigen genes identified. Amongst 21 isolates yielding closed genome assemblies, eight distinct genome structures were detected, with 61.9 % (13/21) of the isolates exhibiting three predominant structures. Increases in case numbers are probably not due to evolutionary changes in the genome but possibly due to other factors such as the cyclical nature of B. pertussis disease, waning immunity due to the use of acellular vaccines and/or population immunity gaps.

Pertussis, caused by Bordetella pertussis, can cause debilitating respiratory symptoms, so whole-cell pertussis vaccines (wPVs) were introduced in the 1940s. However, reactogenicity of wPV necessitated the development of acellular pertussis vaccines (aPVs) that were introduced in the 1990s. Since then, until the COVID-19 pandemic began, reported pertussis incidence was increasing, suggesting that aPVs do not induce long-lasting immunity and may not effectively prevent transmission. Additionally, aPVs do not provide protection against other Bordetella species that are observed during outbreaks. The significance of this work is in determining potential new vaccine antigens for multiple Bordetella species that are predicted to elicit long-term immune responses. Genome-based approaches have aided the development of novel vaccines; here, these methods identified Bordetella vaccine candidates that may be cross-protective and predicted to induce strong memory responses. These targets can lead to an improved vaccine with a strong safety profile while also strengthening the longevity of the immune response.

Few data exist on asymptomatic carriage of Bordetella species among populations receiving acellular pertussis vaccine. We conducted a cross-sectional study among acellular-vaccinated children presenting to an emergency department. B. pertussis carriage prevalence was <1% in this population, a lower prevalence than that found in recent studies among whole-cell pertussis-vaccinated participants.

Respiratory diphtheria is a serious infection caused by toxigenic Corynebacterium diphtheriae, and disease transmission mainly occurs through respiratory droplets. Between 2017 and 2019, a large diphtheria outbreak among forcibly displaced Myanmar nationals densely settled in Bangladesh was investigated. Here we utilized whole-genome sequencing (WGS) to characterize recovered isolates of C. diphtheriae and two co-circulating non-diphtheritic Corynebacterium (NDC) species - C. pseudodiphtheriticum and C. propinquum. C. diphtheriae isolates recovered from all 53 positive cases in this study were identified as toxigenic biovar mitis, exhibiting intermediate resistance to penicillin, and formed four phylogenetic clusters circulating among multiple refugee camps. Additional sequenced isolates collected from two patients showed co-colonization with non-toxigenic C. diphtheriae biovar gravis, one of which exhibited decreased susceptibility to the first-line antibiotics and harboured a novel 23-kb multidrug resistance plasmid. Results of phylogenetic reconstruction and virulence-related gene contents of the recovered NDC isolates indicated they were likely commensal organisms, though 80.4 %(45/56) were not susceptible to erythromycin, and most showed high minimum inhibition concentrations against azithromycin. These results demonstrate the high resolution with which WGS can aid molecular investigation of diphtheria outbreaks, through the quantification of bacterial genetic relatedness, as well as the detection of virulence factors and antibiotic resistance markers among case isolates.

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: Post-exposure prophylaxis (PEP) for pertussis is recommended for household contacts of pertussis cases in the United States within 21 days of exposure, but data on PEP effectiveness for prevention of secondary cases in the setting of widespread pertussis vaccination are limited. We implemented a multi-state evaluation of azithromycin PEP use and effectiveness among household contacts. METHODS: Culture- or PCR-confirmed pertussis cases were identified through surveillance. Household contacts were interviewed within 7 days of case report and again 14-21 days later. Interviewers collected information on exposure, demographics, vaccine history, prior pertussis diagnosis, underlying conditions, PEP receipt, pertussis symptoms, and pertussis testing. A subset of household contacts provided nasopharyngeal and blood specimens during interviews. RESULTS: Of 299 household contacts who completed both interviews, 12 (4%) reported not receiving PEP. There was no evidence of higher prevalence of cough or pertussis symptoms among contacts who did not receive PEP. Of 168 household contacts who provided at least one nasopharyngeal specimen, four (2.4%) were culture or PCR positive for B. pertussis; three of these received PEP prior to their positive test result. Of 156 contacts with serologic results, 14 (9%) had blood specimens that were positive for IgG anti-pertussis toxin (PT) antibodies; all had received PEP. CONCLUSIONS: Very high PEP uptake was observed among household contacts of pertussis patients. Although the number of contacts who did not receive PEP was small, there was no difference in prevalence of pertussis symptoms or positive laboratory results among these contacts compared with those who did receive PEP.

This report describes the complete genome sequences of four isolates of the nondiphtheritic Corynebacterium (NDC) species Corynebacterium pseudodiphtheriticum and Corynebacterium propinquum, recovered during investigation of a large diphtheria outbreak in Bangladesh. These data will assist in better delineating the boundary between these related species and understanding their virulence potential.

Bacterial genetic diversity is often described solely using base-pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplication/amplifications are thought to be widespread among bacteria but due to their often-intractable size and instability, comprehensive studies of these mutations are rare. We define a methodology to investigate amplifications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for amplifications to occur. Analysis of data for 2430 B. pertussis isolates identified 272 putative amplifications, of which 94 % were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of amplifications, suggesting unstable and sporadic characteristics. Genome instability was further described in vitro using long-read sequencing via the Nanopore platform, which revealed that clonally derived laboratory cultures produced heterogenous populations rapidly. We extended this research to analyse a population of 1000 isolates of another important pathogen, Mycobacterium tuberculosis. We found 590 amplifications in M. tuberculosis, and like B. pertussis, these occurred primarily at hotspots. Genes amplified in B. pertussis include those involved in motility and respiration, whilst in M. tuberuclosis, functions included intracellular growth and regulation of virulence. Using publicly available short-read data we predicted previously unrecognized, large amplifications in B. pertussis and M. tuberculosis. This reveals the unrecognized and dynamic genetic diversity of B. pertussis and M. tuberculosis, highlighting the need for a more holistic understanding of bacterial genetics.

Many countries have reported antigenic divergence among circulating Bordetella pertussis strains, mainly in those countries which introduced the acellular pertussis (aP) vaccine. This phenomenon can be seen, for example, with the recent rise of pertactin (Prn)-deficient B. pertussis strains, one of the antigens included in aP vaccine formulas. The whole cell pertussis (wP) vaccine has been used in Brazil since 1977 for the primary pertussis, diphtheria and tetanus immunization series. In 2014, the aP vaccine was recommended for women during pregnancy to protect infants in the first months of life. Our objective was to determine the prevalence of Prn-deficiency in 511 isolates of B. pertussis collected in Brazil during 2010–2016. All isolates were characterized, through PFGE and serotyping, and screened for the loss of Prn by ELISA. Prn-deficiency was confirmed by immunoblotting, and identification of the possible genetic markers was performed with PCR and Sanger sequencing. Results indicate that 110 PFGE profiles are currently circulating, with five profiles representing the majority, and the predominant serotype 3, has been gradually replaced by serotype 2 and serotype 2,3. ELISA screening and immunoblotting identified three Prn-deficient isolates. Genotypic characterization by PCR and sequencing indicated that one isolate had a promoter mutation in prn, while the other two did not have an obvious genetic explanation for their deficiency. While the lack of Prn was identified in a few isolates, this study did not detect a relevant occurrence of Prn-deficiency, until 2016, confirming previous observations that Prn-deficiency is likely aP vaccine-driven. © 2021

Background: Maternal Tetanus, diphtheria, and acellular pertussis (Tdap) vaccination provides antibody transfer to newborn infants and may affect their antibody response to the primary vaccination series. This study aimed to assess the effect of Tdap vaccination during pregnancy on infant antibody response to the whole cell pertussis (DTwP) primary series. Methods: Plasma from 318 pregnant women (243 Tdap-vaccinated and 75 unvaccinated) and their infants (cord blood) was collected at delivery; infant blood was again collected at 2 and 7 months, before and after their primary DTwP series. Anti-pertussis toxin (PT), pertactin (PRN), filamentous hemagglutinin (FHA), fimbriae 2/3 (FIM) and adenylate cyclase toxin (ACT) IgG antibodies were quantified by a microsphere-based multiplex antibody capture assay and anti-PT neutralizing antibodies by the Real Time Cell analysis system. Results: Infant geometric mean concentrations (GMCs) of IgG anti-Tdap antigens were significantly higher (p < 0.001) among the Tdap-vaccinated (PT: 57.22 IU/mL; PRN: 464.86 IU/mL; FHA: 424.0 IU/mL), versus the unvaccinated group (4 IU/mL, 15.43 IU/mL, 31.99 IU/mL, respectively) at delivery. Anti-FIM and ACT GMCs were similar between the two groups. At 2 months of age, anti-PT, PRN, and FHA GMCs remained higher (p < 0.001) in the Tdap-vaccinated group (12.64 IU/mL; 108.76 IU/mL; 87.41 IU/mL, respectively) than the unvaccinated group (1.02 IU/mL; 4.46 IU/mL; 6.89 IU/mL). However, at 7 months, after receiving the third DTwP dose, the anti-PT GMC was higher (p = 0.016) in the unvaccinated group (7.91 IU/mL) compared to the vaccinated group (2.27 IU/mL), but without differences for anti-PRN, FHA, FIM and ACT GMCs. Conclusion: Elevated antibody levels suggest that maternal Tdap vaccination might protect infants until 2 months of age. Reduced anti-PT levels at 7 months indicate potential blunting of immune response in infants. Surveillance would help determine if blunting alters vaccine immunity and impacts pertussis prevention in infants. © 2021 The Authors

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.

BACKGROUND: Diphtheria, a life-threatening respiratory disease, is caused mainly by toxin-producing strains of Corynebacterium diphtheriae, while nontoxigenic Corynebacteria, such as C. pseudodiphtheriticum rarely causes diphtheria-like illness. Recently several global diphtheria outbreaks have resulted from the breakdown of healthcare infrastructures particularly in countries experiencing political conflict. This report summarizes a laboratory and epidemiological investigation of a diphtheria outbreak among Forcibly Displaced Myanmar Nationals in Bangladesh. METHODS: Specimens and clinical information were collected from patients presenting at Diphtheria Treatment Centers. Swabs were tested for toxin-gene (tox) bearing C. diphtheriae by real-time (RT) PCR and culture. The isolation of another Corynebacterium species prompted further laboratory investigation. RESULTS: Among 382 patients; 153 (40%) tested tox-positive for C. diphtheriae by RT-PCR; 31 (20%) PCR-positive swabs were culture-confirmed. RT-PCR revealed 78% (298/382) of patients tested positive for C. pseudodiphtheriticum. Of patients positive for only C. diphtheriae, 63% (17/27) had severe disease compared to 55% (69/126) positive for both Corynebacterium species, and 38% (66/172) for only C. pseudodiphtheriticum. CONCLUSIONS: We report the confirmation of a diphtheria outbreak and identification of a co-circulating Corynebacterium species. The high proportion of C. pseudodiphtheriticum co-detection may explain why many suspected patients testing negative for C. diphtheriae presented with diphtheria-like symptoms.

Between July 2018 and May 2019, Corynebacterium diphtheriae was isolated from eight patients with non-respiratory infections, seven of whom experienced homelessness and had stayed at shelters in King County, WA, USA. All isolates were microbiologically identified as nontoxigenic C. diphtheriae biovar mitis. Whole-genome sequencing confirmed that all case isolates were genetically related, associated with sequence type 445 and differing by fewer than 24 single-nucleotide polymorphisms (SNPs). Compared to publicly available C. diphtheriae genomic data, these WA isolates formed a discrete cluster with SNP variation consistent with previously reported outbreaks. Virulence-related gene content variation within the highly related WA cluster isolates was also observed. These results indicated that genome characterization can readily support epidemiology of nontoxigenic C. diphtheriae.

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.

Whooping cough (pertussis), primarily caused by Bordetella pertussis, has resurged in the United States, and circulating strains exhibit considerable chromosome structural fluidity in the form of rearrangement and deletion. The genus Bordetella includes additional pathogenic species infecting various animals, some even causing pertussis-like respiratory disease in humans; however, investigation of their genome evolution has been limited. We studied chromosome structure in complete genome sequences from 167 Bordetella species isolates, as well as 469 B. pertussis isolates, to gain a generalized understanding of rearrangement patterns among these related pathogens. Observed changes in gene order primarily resulted from large inversions and were only detected in species with genomes harboring multicopy insertion sequence (IS) elements, most notably B. holmesii and B. parapertussis While genomes of B. pertussis contain >240 copies of IS481, IS elements appear less numerous in other species and yield less chromosome structural diversity through rearrangement. These data were further used to predict all possible rearrangements between IS element copies present in Bordetella genomes, revealing that only a subset is observed among circulating strains. Therefore, while it appears that rearrangement occurs less frequently in other species than in B. pertussis, these clinically relevant respiratory pathogens likely experience similar mutation of gene order. The resulting chromosome structural fluidity presents both challenges and opportunity for the study of Bordetella respiratory pathogens.IMPORTANCE Bordetella pertussis is the primary agent of whooping cough (pertussis). The Bordetella genus includes additional pathogens of animals and humans, including some that cause pertussis-like respiratory illness. The chromosome of B. pertussis has previously been shown to exhibit considerable structural rearrangement, but insufficient data have prevented comparable investigation in related species. In this study, we analyze chromosome structure variation in several Bordetella species to gain a generalized understanding of rearrangement patterns in this genus. Just as in B. pertussis, we observed inversions in other species that likely result from common mutational processes. We used these data to further predict additional, unobserved inversions, suggesting that specific genome structures may be preferred in each species.

BACKGROUND: Pertussis remains an important global public health concern, despite the presence of extensive immunization programs. Incidence and severity of pertussis are typically higher in neonates and young infants. As a strategy to protect these young infants, maternal vaccination with Tdap (tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis) has been recommended in Brazil. The objective of this study was to evaluate the effects of Tdap vaccination during pregnancy on the anti-pertussis toxin (PT) IgG response in mothers and their infants at birth. MATERIAL AND METHODS: Maternal and cord blood samples were collected from vaccinated (n=243) and unvaccinated (n=75) pregnant women, at the time of delivery, from July 2015 to August 2016 in Sao Paulo, Brazil. Anti-PT IgG antibodies were quantified by Enzyme-Linked Immunosorbent Assay (ELISA) and geometric mean concentrations (GMC) were calculated. Relationship between timing of vaccination and antibody concentrations were evaluated. RESULTS: Maternal and cord blood GMCs among the vaccinated group were 5.4 and 5.6 fold higher [66.5 International Units (IU)/mL and 89.8IU/mL] compared to the unvaccinated group (12.4IU/mL and 16.1IU/mL), respectively (p<0.001). Higher anti-PT IgG GMCs were observed when vaccination occurred >/=60days before delivery compared to <60days, suggesting that vaccination early in the third trimester may be more effective than later in pregnancy. CONCLUSION: Tdap maternal vaccination results in significantly higher anti-PT IgG in newborn infants and supports the current recommendation of the Brazilian Immunization Program.

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.

INTRODUCTION: The appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition. An additional objective was to describe the optimal timing of specimen collection for the various tests. METHODS: Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit. Patients who had been coughing for </= 2 weeks were asked to return in 2-4 weeks for collection of a second, convalescent blood specimen. Sensitivity and specificity of each diagnostic test were estimated using three methods-pertussis culture as the "gold standard," composite reference standard analysis (CRS), and latent class analysis (LCA). RESULTS: Overall, 868 patients were enrolled and 13.6% were B. pertussis positive by at least one diagnostic test. In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA. CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates. Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough. CONCLUSIONS: Timing of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis.

The Latin American Pertussis Project (LAPP), established in 2009, is a collaboration between the Centers for Disease Control and Prevention, Pan American Health Organization, Sabin Vaccine Institute, and the ministries of health of 6 countries in Latin America. The project goal is to expand understanding of pertussis epidemiology in Latin America to inform strategies for control and prevention. Here we describe LAPP structure and activities. After an initial surveillance evaluation, LAPP activities are tailored to individual country needs. LAPP activities align with Global Health Security Agenda priorities and have focused on expanding laboratory diagnostic capacity, implementing a laboratory quality control and quality assurance program, and providing epidemiologic support to strengthen reporting of pertussis surveillance data. Lessons learned include that ongoing mentoring is key to the successful adoption of new technologies and that sustainability of laboratory diagnostics requires a regional commitment to procure reagents and related supplies.

Despite high vaccine coverage, pertussis cases in the United States (US) have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The US exclusively employs acellular vaccines and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the US retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 US surveillance isolates collected from 2010-2016 identified two that were both Prn- and Fha-deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutation to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure.

Clinical isolates of the respiratory pathogen Bordetella pertussis in the United States have become predominantly deficient for the acellular vaccine immunogen pertactin through various independent mutations. Here, we report the complete genome sequences for four B. pertussis isolates that harbor novel deletions responsible for pertactin deficiency.

Bordetella pertussis infection has been increasing in the US, with reported cases reaching over 50,000 in 2012, a number last observed in the 1950s. Concurrently, B. pertussis lacking the pertactin protein, one of the immunogens included in the acellular vaccine formulations, has rapidly emerged since 2010, and has become the predominant circulating phenotype. Monitoring the production of the remaining acellular vaccine immunogens, such as pertussis toxin (Pt), is a critical next step. To date, methods for screening Pt have been either through genomic sequencing means or by conventional ELISAs. However, sequencing limits detection to the DNA level, missing potential disruptions in transcription or translation. Conventional ELISAs are beneficial for detecting the protein; however, they can often suffer from poor sensitivity and specificity. Here we describe a rapid, highly sensitive and specific electrochemiluminescent capture ELISA that can detect Pt production in prepared inactivated bacterial suspensions. Over 340 isolates were analyzed and analytical validation parameters, such as precision, reproducibility, and stability, were rigorously tested. Intra-plate and inter-plate variability measured at 9.8% and 11.5%, respectively. Refrigerated samples remained stable for two months and variability was unaffected (coefficient of variation was 12%). Interestingly, despite the intention of being a qualitative method, the assay was sensitive enough to detect a small, but statistically significant, difference in protein production between different pertussis promoter allelic groups of strains, ptxP1 and ptxP3. This technology has the ability to perform screening of multiple antigens at one time, thus, improving testing characteristics while minimizing costs, specimen volume, and testing time.

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.

Serum Institute of India is among the world's largest vaccine producers. Here, we report the complete genome sequences for four Bordetella pertussis strains used by Serum Institute of India in the production of whole-cell pertussis vaccines.

BACKGROUND: In the United States, infants have the highest reported pertussis incidence and death rates. Improved understanding of infant risk factors is needed to optimize prevention strategies. METHODS: We prospectively enrolled infants ≤4 months of age with incident-confirmed pertussis from 4 sites during 2002-2005 (preceding pertussis-antigen-containing vaccination recommendations for adolescents/adults); each case-patient was age- and site-matched with 2 control subjects. Caregivers completed structured interviews. Infants and their contacts ≥11 years of age were offered serologic testing for IgG; being seropositive was defined as ≥94 anti-pertussis toxin IgG enzyme-linked immunosorbent assay units/mL. RESULTS: Enrolled subjects (115 case-patients; 230 control subjects) had 4,396 contacts during incubation periods; 83 (72%) case-patients had ≥1 contact with prolonged (≥5 days) new cough in primary or secondary households. In multivariable analysis, the odds for pertussis were higher for infants with primary/secondary household contacts who had a prolonged new cough, compared with infants who did not. These contacts included mother (adjusted matched odds ratio [aMOR] 43.8; 95% confidence interval [CI], 6.45-298.0) and ≥1 nonmother contact (aMOR, 20.1; 95% CI, 6.48-62.7). Infants receiving breast milk with 0-1 formula feedings daily had decreased pertussis odds (aMOR, 0.27; 95% CI, 0.08-0.89), compared with those receiving more formula. Of 41 tested case-patients, 37 (90%) were seropositive. CONCLUSIONS: Pertussis in infants was associated with prolonged new cough (≥5 days) in infants' household contacts. Findings suggest breastfeeding protects against pertussis, and warrants recommendation with pertussis prevention strategies, which currently include pertussis vaccination of pregnant mothers and infants' close contacts.