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.

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.

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.

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.

The diagnosis of dengue disease, caused by the dengue virus (DENV) (a flavivirus), often requires serologic testing during acute and early convalescent phases of the disease. Some symptoms of DENV infection, such as nonspecific fever, are similar to those caused by infection with SARS-CoV-2, the virus that causes COVID-19. In studies with few COVID-19 cases, positive DENV immunoglobulin M (IgM) results were reported with various serologic tests, indicating possible cross-reactivity in these tests for DENV and SARS-CoV-2 infections (1,2). DENV antibodies can cross-react with other flaviviruses, including Zika virus. To assess the potential cross-reactivity of SARS-CoV-2, DENV, and Zika virus IgM antibodies, serum specimens from 97 patients from Puerto Rico and 12 U.S.-based patients with confirmed COVID-19 were tested using the DENV Detect IgM Capture enzyme-linked immunosorbent assay (ELISA) (InBios International).* In addition, 122 serum specimens from patients with confirmed dengue and 121 from patients with confirmed Zika virus disease (all from Puerto Rico) were tested using the SARS-CoV-2 pan-Ig Spike Protein ELISA (CDC).(†) Results obtained for DENV, Zika virus IgM, and SARS-CoV-2 antibodies indicated 98% test specificity and minimal levels of cross-reactivity between the two flaviviruses and SARS-CoV-2. These findings indicate that diagnoses of dengue or Zika virus diseases with the serological assays described in this report are not affected by COVID-19, nor do dengue or Zika virus diseases interfere with the diagnosis of COVID-19.

SARS-CoV-2 transmission from contaminated surfaces, or fomites, has been a concern during the COVID-19 pandemic. Households have been important sites of transmission throughout the COVID-19 pandemic, but there is limited information on SARS-CoV-2 contamination of surfaces in these settings. We describe environmental detection of SARS-CoV-2 in households of persons with COVID-19 to better characterize the potential risks of fomite transmission. Ten households with ≥1 person with laboratory-confirmed COVID-19 and with ≥2 members total were enrolled in Utah, U.S.A. Nasopharyngeal and anterior nasal swabs were collected from members and tested for the presence of SARS-CoV-2 by RT-PCR. Fifteen surfaces were sampled in each household and tested for presence and viability of SARS-CoV-2. SARS-CoV-2 RNA was detected in 23 (15%) of 150 environmental swab samples, most frequently on nightstands (4/6; 67%), pillows (4/23; 17%), and light switches (3/21; 14%). Viable SARS-CoV-2 was cultured from one sample. All households with SARS-CoV-2-positive surfaces had ≥1 person who first tested positive for SARS-CoV-2 ≤ 6 days prior to environmental sampling. SARS-CoV-2 surface contamination occurred early in the course of infection when respiratory transmission is most likely, notably on surfaces in close, prolonged contact with persons with COVID-19. While fomite transmission might be possible, risk is low.

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

BACKGROUND: Although many viral respiratory illnesses are transmitted within households, the evidence base for SARS-CoV-2 is nascent. We sought to characterize SARS-CoV-2 transmission within US households and estimate the household secondary infection rate (SIR) to inform strategies to reduce transmission. METHODS: We recruited laboratory-confirmed COVID-19 patients and their household contacts in Utah and Wisconsin during March 22-April 25, 2020. We interviewed patients and all household contacts to obtain demographics and medical histories. At the initial household visit, 14 days later, and when a household contact became newly symptomatic, we collected respiratory swabs from patients and household contacts for testing by SARS-CoV-2 rRT-PCR and sera for SARS-CoV-2 antibodies testing by enzyme-linked immunosorbent assay (ELISA). We estimated SIR and odds ratios (OR) to assess risk factors for secondary infection, defined by a positive rRT-PCR or ELISA test. RESULTS: Thirty-two (55%) of 58 households had evidence of secondary infection among household contacts. The SIR was 29% (n = 55/188; 95% confidence interval [CI]: 23-36%) overall, 42% among children (<18 years) of the COVID-19 patient and 33% among spouses/partners. Household contacts to COVID-19 patients with immunocompromised conditions had increased odds of infection (OR: 15.9, 95% CI: 2.4-106.9). Household contacts who themselves had diabetes mellitus had increased odds of infection (OR: 7.1, 95% CI: 1.2-42.5). CONCLUSIONS: We found substantial evidence of secondary infections among household contacts. People with COVID-19, particularly those with immunocompromising conditions or those with household contacts with diabetes, should take care to promptly self-isolate to prevent household transmission.

BACKGROUND: Detection of SARS-CoV-2 infection has principally been performed through the use of real-time reverse-transcription PCR (rRT-PCR) testing. Results of such tests can be reported as cycle threshold (Ct) values, which may provide semi-quantitative or indirect measurements of viral load. Previous reports have examined temporal trends in Ct values over the course of a SARS-CoV-2 infection. METHODS: Using testing data collected during a prospective household transmission investigation of outpatient and mild COVID-19 cases, we examined the relationship between Ct values of the viral RNA N1 target and demographic, clinical, and epidemiological characteristics collected through participant interviews and daily symptom diaries. RESULTS: We found Ct values are lowest (corresponding to higher viral RNA concentration) soon after symptom onset and are significantly correlated with time elapsed since onset (p<0.001); within 7 days after symptom onset, the median Ct value was 26.5 compared with a median Ct value of 35.0 occurring 21 days after onset. Ct values were significantly lower among participants under 18 years of age (p=0.01) and those reporting upper respiratory symptoms at the time of sample collection (p=0.001) and were higher among participants reporting no symptoms (p=0.05). CONCLUSIONS: These results emphasize the importance of early testing for SARS-CoV-2 among individuals with symptoms of respiratory illness and allows cases to be identified and isolated when their viral shedding may be highest.

On December 12, 2018, an immunocompromised man with non-Hodgkin’s lymphoma, aged 73 years, was evaluated by an ophthalmologist for left eyelid redness, swelling, and eye discharge and received a diagnosis of ligneous (pseudomembranous) conjunctivitis. The pseudomembrane was debrided and sent for culture, and the patient was prescribed oral amoxicillin clavulanate and moxifloxacin eye drops, with topical loteprednol and cyclosporine to decrease the robust inflammatory response. Corynebacterium ulcerans, one of three species of Corynebacterium (in addition to C. diphtheriae and C. psuedotuberculosis) that can harbor the diphtheria toxin–producing gene was initially identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry performed on an isolate obtained from culture of the pseudomembrane at a Missouri hospital on December 13. The Missouri Department of Health and Senior Services (MDHSS) laboratory-confirmed C. ulcerans by culture and forwarded the isolate to CDC for toxin testing. On December 28, CDC confirmed toxin-producing C. ulcerans. The patient had no systemic symptoms, was not hospitalized, and did not receive diphtheria antitoxin. On January 11, 2019, following multiple membrane removals and no residual membrane; cultures of conjunctival swabs tested by the hospital were negative for C. ulcerans. The patient was not up-to-date for tetanus-diphtheria (Td) vaccine and had postponed vaccination because of his ongoing cancer treatment.

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.

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.

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.

BACKGROUND: Pertussis serodiagnosis is increasingly being used in the United States despite the lack of a US Food and Drug Administration-approved, commercially available assay. To better understand the utility of these assays in diagnosing pertussis, serology assays were evaluated for analytical parameters and clinical accuracy. METHODS: Forty-three antigen-antibody combinations were evaluated for single-point diagnosis of pertussis. Serum panels included sera from laboratory-confirmed cases, an international reference standard, and healthy donors. Phase I panel (n = 20) of sera was used to assess precision, linearity, and accuracy; Phase II panel (n = 226) followed with positive percent agreement (PPA) and negative percent agreement (NPA) estimates. Analytical analyses included coefficients of variation (CV) and concordance correlation coefficients (rc). RESULTS: Intra-analyst variability was found to be relatively low among samples per assay, with only 6% (78 of 1240) having CV >20%, primarily with the highly concentrated immunoglobulin (Ig)G anti-pertussis toxin (PT) specimens and IgM assays. The rc measurements to assess linearity ranged between 0.282 and 0.994, 0.332 and 0.999, and -0.056 and 0.482 for IgA, IgG, and IgM, respectively. Analytical accuracy for calibrated IgG anti-PT assays was 86%-115%. The PPA and NPA varied greatly for all assays; PPA/NPA ranges for IgA, IgG, and IgM assays, with culture and/or polymerase chain reaction positivity as control, were 29-90/13-100, 26-96/27-100, and 0-73/42-100, respectively. In IgG assays, mixing filamentous hemagglutinin antigen with PT increased PPA but decreased NPA. CONCLUSIONS: Seroassays varied substantially under both analytical and clinical parameters; however, those that were calibrated to a reference standard were highly accurate. Our findings support incorporation of calibrated pertussis seroassays to the pertussis case definition for improved diagnosis and surveillance.

BACKGROUND: A recent increase in B. pertussis without the pertactin protein, an acellular vaccine immunogen, has been reported in the U.S. Determining whether pertactin-deficient (PRN-) B. pertussis is evading vaccine-induced immunity or altering the severity of illness is needed. METHODS: We retrospectively assessed for associations between pertactin production and both clinical presentation and vaccine history. Cases with isolates collected between May 2011 and February 2013 from 8 states were included. We calculated unadjusted and adjusted odds ratios (ORs) using multivariable logistic regression analysis. RESULTS: Among 753 isolates, 640 (85%) were PRN-. The age distribution differed between cases caused by PRN- B. pertussis and cases caused by B. pertussis producing pertactin (PRN+) (p-value= 0.01). The proportion reporting individual pertussis symptoms was similar between the two groups, except a higher proportion of PRN+ case-patients reported apnea (p-value=0.005). 22 case-patients were hospitalized; 6% in the PRN+ group compared to 3% in the PRN- group (p-value=0.11). Cases having received at least one pertussis vaccine dose had a higher odds of having PRN- B. pertussis as compared to unvaccinated cases (adjusted OR=2.2; 95% CI= 1.3-4.0). When restricted to cases-patients at least 1 year of age and those age-appropriately vaccinated the adjusted OR increased to 2.7 (95% CI=1.2-6.1). CONCLUSIONS: The significant association between vaccination and isolate pertactin produciton suggests the likelihood of having reported disease caused by PRN- compared to PRN+ strains is greater in vaccinated persons. Additional studies are needed to assess whether vaccine effectiveness is diminished against PRN- strains.

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 remains an important public health problem in many countries despite extensive immunization. Cultures and real-time PCR (RT-PCR) assays are the recommended pertussis diagnostic tests, but they lack sensitivity at the later stage of the disease. This study introduces the IgG anti-pertussis toxin enzyme-linked immunosorbent assay (PT ELISA) in our routine diagnosis to improve disease burden estimation. Serum samples and nasopharyngeal swabs (n = 503) were collected at the same time from patients presenting with cough illness suspected of being pertussis and tested by the PT ELISA and culture and/or RT-PCR, respectively. Patients were separated into three age groups: group 1, <1 year (n = 260; mean age, 3 months), group 2, 1 to 6 years (n = 81; mean age, 3 years), and group 3, ≥7 years (n = 162; mean age, 26 years). The times (means) from cough onset to specimen collection were 16, 24, and 26 days, respectively. In group 1, 83 (82.2%) of 101 positive cases were positive for pertussis by culture/RT-PCR, while 40 (39.6%) tested positive by PT ELISA. In group 2, 6 (19.4%) of 31 positive cases were culture/RT-PCR positive, and 29 (93.6%) were seropositive. In group 3, 13 (13.8%) of 94 positive cases were positive by culture/RT-PCR and 91 (96.8%) were positive by serology. Culture/RT-PCR detected more cases of pertussis in infants (P < 0.0001), whereas the PT ELISA detected more cases in adolescents and adults (P < 0.0001). The timing between cough onset and specimen collection or recent vaccination may have partially affected our results. Serology is a suitable, cost-effective, and complementary pertussis diagnostic tool, especially among older children, adolescents, and adults during the later disease phase.

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.

BACKGROUND: During May 9 2010-May 7 2011, an outbreak of pertussis-like illness (incidence 80 cases/100,000 persons) occurred in Franklin County, Ohio. The majority of cases were identified by IS481-directed polymerase chain reaction (PCR), which does not differentiate among Bordetella species. We sought to determine outbreak etiology and epidemiologic characteristics. METHODS: We obtained demographic, clinical, and vaccination-related data from the Ohio Disease Reporting System and Impact Statewide Immunization Information System. We tested sera from 14 patients for anti-pertussis toxin (PT) antibodies and used species-specific PCR on 298 nasopharyngeal specimens. RESULTS: Reported cases totaled 918. IS481 results were available for 10 serologically tested patients; 5/10 had discordant anti-PT antibody and IS481 results, suggestive of Bordetella holmesii, which lacks PT and harbors IS481. We identified specific Bordetella species in 164 of 298 specimens tested with multitarget PCR; B. holmesii and Bordetella pertussis were exclusively detected among 48 (29%) and 112 (68%), respectively; both were detected in 4 (2%). Among 48 patients with B. holmesii infections, 63% were aged 11-18 years, compared with 35% of 112 patients with B. pertussis infections (P = .001). Symptoms were similar among B. holmesii and B. pertussis-infected patients. Adolescent pertussis ("Tdap") booster vaccinations were more effective against B. pertussis than B. holmesii (effectiveness: 67% and 36%, respectively; 95% confidence intervals: 38%-82% and -33%-69%, respectively). CONCLUSIONS: We report the first documented mixed outbreak of B. pertussis and B. holmesii infections. B. holmesii particularly affected adolescents. Although laboratory capacity limitations might inhibit routine use of multitarget PCRs for clinical diagnosis; focused testing and enhanced surveillance might improve understanding the burden of B. holmesii infection.

BACKGROUND: An IgG anti-pertussis toxin (PT) enzyme-linked immunosorbent assay (ELISA) was analytically validated for diagnosis of pertussis, using the cut-off of 94 ELISA Units (EU)/mL. Little was known about the performance of this ELISA for diagnosis in adults recently vaccinated with tetanus-diphtheria-acellular pertussis (Tdap), which contains PT. The goal of this study was to determine when the assay can be used following Tdap vaccination. METHODS: A cohort of 102 asymptomatic health care personnel (HCP) vaccinated with Tdap (Adacel, Sanofi Pasteur) were aged 19 to 79 years (median 47 years) at vaccination. For each HCP, specimens were available for evaluation at 2-10 time points (pre-vaccination to 24 months post-vaccination), and geometric mean concentrations (GMC) for the cohort were calculated at each time point. Among 97 HCP who responded to vaccination, a mixed model analysis with prediction and tolerance intervals was performed to estimate the time at which serodiagnosis can be used following vaccination. RESULTS: The GMC was 8, 21, and 9 EU/mL at pre-vaccination, four, and 12 months post-vaccination, respectively. Eight of 102 (8%) HCP reached antibody titers ≥ 94 EU/mL during their peak response but none had these titers by six months post-vaccination. Calculated prediction and tolerance intervals were < 94 EU/mL by 45 and 75 days post-vaccination, respectively. CONCLUSIONS: Tdap vaccination six months prior to testing did not confound result interpretation. This seroassay remains a valuable diagnostic tool for adult pertussis.

BACKGROUND AND OBJECTIVES: We investigated a pertussis outbreak characterized by atypical cases, confirmed by polymerase chain reaction (PCR) alone at a single laboratory, which persisted despite high vaccine coverage and routine control measures. We aimed to determine whether Bordetella pertussis was the causative agent and advise on control interventions. METHODS: We conducted case ascertainment, confirmatory testing for pertussis and other pathogens, and an assessment for possible sources of specimen contamination, including a survey of clinic practices, sampling clinics for B pertussis DNA, and review of laboratory quality indicators. RESULTS: Between November 28, 2008, and September 4, 2009, 125 cases were reported, of which 92 (74%) were PCR positive. Cases occurring after April 2009 (n = 79; 63%) had fewer classic pertussis symptoms (63% vs 98%; P < .01), smaller amounts of B pertussis DNA (mean PCR cycle threshold value: 40.9 vs 33.1; P < .01), and a greater proportion of PCR-positive results (34% vs 6%; P < .01). Cultures and serology for B pertussis were negative. Other common respiratory pathogens were detected. We identified factors that likely resulted in specimen contamination at the point of collection: environmentally present B pertussis DNA in clinics from vaccine, clinic standard specimen collection practices, use of liquid transport medium, and lack of clinically relevant PCR cutoffs. CONCLUSIONS: A summer pertussis pseudo-outbreak, multifactorial in cause, likely occurred. Recommendations beyond standard practice were made to providers on specimen collection and environmental cleaning, and to laboratories on standardizing PCR protocols and reporting results, to minimize false-positive results from contaminated clinical specimens.

In an effort to improve the reliability and reproducibility of serological assays for Bordetella pertussis, a collaborative study was conducted to compare four different sources of pertussis toxin (PT) as coating antigens in the immunoglobulin G (IgG) anti-PT enzyme-linked immunosorbent assay (ELISA). Four sources of PT were used as coating antigens in the IgG anti-PT ELISA in four different testing laboratories (Labs A-D) to determine if the different antigen preparations and different laboratories influenced assay results. A panel of 60 sera consisting of de-identified human specimens from previous vaccination trials of normal healthy adults and infants and clinical specimens from outbreak settings was tested. In the four laboratories, each sample was tested three times with the four PT antigens according to the standard coating optimization and IgG anti-PT ELISA testing procedures used in that laboratory. Differences among the antigens, as well as intra- and inter-laboratory variability, were evaluated. Excellent agreement was observed with the test panel results among the four antigens within each laboratory. Concordance correlation coefficient (r(c)) measurements among the different antigens ranged from 0.99, 0.99-1.00, 1.00, and 0.97-1.00 for Labs A-D, respectively. The comparisons between pairs of laboratories also indicated a high degree of concordance for each PT preparation, with r(c) measurements between 0.90-0.98, 0.93-0.99, 0.92-0.98 and 0.93-0.99 for antigens 1-4, respectively. Relatively minor differences in results were observed among laboratories or among antigens, suggesting that the four PT antigens are quite similar and could be considered for acceptance in harmonized immunoassays used for serodiagnosis or vaccine evaluation.

BACKGROUND: Pertussis is among the most poorly controlled bacterial vaccine-preventable diseases in the United States. In 2006, a tetanus, reduced-dose diphtheria, and acellular pertussis (Tdap) booster was recommended for adolescents and adults. Tdap vaccines were licensed on the basis of antibody response without vaccine effectiveness data. METHODS: From 30 September 2007 through 19 December 2007, a pertussis outbreak occurred at a nursery through twelfth grade school on St. Croix, US Virgin Islands. We screened all students for cough and collected clinical history, including Tdap receipt. Coughing students were offered diagnostic testing. We defined clinical case patients as students with cough 14 days in duration plus either whoop, paroxysms, or post-tussive vomiting, and we defined confirmed case patients as students with any cough with isolation of Bordetella pertussis or those with clinical cases and polymerase chain reaction or serological evidence of pertussis; other clinical cases were classified as probable. RESULTS: There were 51 confirmed or probable cases among 499 students (attack rate, 10%). Disease clustered in grades 6-12, with a peak attack rate of 38% among 10th graders. Of 266 students aged 11 years with complete data, 31 (12%) had received Tdap. Forty-one unvaccinated students (18%) had confirmed or probable pertussis, compared with 2 (6%) of the vaccinated students (relative risk, 2.9); vaccine effectiveness was 65.6% (95% confidence interval, -35.8% to 91.3%; [Formula: see text]). CONCLUSIONS: This first evaluation of Tdap vaccine effectiveness in the outbreak setting suggests that Tdap provides protection against pertussis. Increased coverage is needed to realize the full benefit of the vaccine program. Serological testing was an important tool for case identification and should be considered for inclusion in the Council of State and Territorial Epidemiologists case definition.

Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae.

Adequately sensitive and specific methods to diagnose pertussis in adolescents and adults are not widely available. Currently, no Food and Drug Administration approved diagnostic assays are available for the serodiagnosis of Bordetella pertussis. Since concentrations of B. pertussis-specific antibodies tend to be high during the later-phases of disease, a simple, rapid, easily transferable serodiagnostic test was developed. This paper describes test development, initial evaluation of a prototype kit enzyme-linked immunosorbent assay (ELISA) in an inter-laboratory collaborative study, and the analytical validation. The data presented here demonstrate that the kit met all pre-specified criteria for precision, linearity, and accuracy for samples with anti-pertussis toxin (PT) immunoglobulin G (IgG) antibody concentrations in the range of 50 to 150 ELISA units (EU)/mL, the range believed to be most relevant for serodiagnosis. The assay met the precision and linearity criteria for a wider range, namely from 50 to 200 EU/mL, however, the accuracy criterion was not met at 200 EU/mL. When the newly adopted World Health Organization International Standard for pertussis antiserum (human) reference reagent was used to evaluate accuracy, the accuracy criteria were met from 50 to 200 international units (IU)/mL. In conclusion, the IgG anti-PT ELISA assay met all assay validation parameters within the range considered most relevant for serodiagnosis. This ELISA was developed and analytically validated to be a user-friendly kit that can be used in both qualitative and quantitative formats. The technology for producing the kit is transferrable to public health laboratories.