Understanding COVID-19 Vaccine Hesitancy Among K-12 Staff, Parents, and Students: District of Columbia, February to April, 2022.

OBJECTIVE: Despite widespread availability of COVID-19 vaccines, millions of Americans have not received the recommended vaccine doses. In the District of Columbia (DC), COVID-19 vaccination rates are lowest among residents who are Non-Hispanic (NH) Black and among school-aged children. We assessed COVID-19 vaccine hesitancy among staff and parents of students in DC K-12 public and public charter schools. METHODS: We conducted a telephone-based survey from February 6 to April 16, 2022 to staff, students, and parents of students who participated in school-based COVID-19 screening testing. COVID-19-related survey items included: vaccination status, reasons for not getting vaccinated, perceived vaccine access, and trusted COVID-19 information sources. Utilizing time-to-event analyses, we evaluated differences across demographic groups. RESULTS: The interview response rate was 25.8% (308/1193). Most unvaccinated participants were NH Black and ages 5 to 11 years. Median time from vaccine eligibility to uptake was 236 days for NH Black participants vs. 10 days for NH White participants. Vaccine safety was the top concern among unvaccinated participants. Government and healthcare providers were the most trusted COVID-19 information sources. CONCLUSIONS: Differences in timing of vaccine uptake among respondents and greater vaccine hesitancy among NH Black participants compared to other racial/ethnic groups highlight a need for continued tailored outreach and communication using trusted sources to convey the importance, benefits, and safety of COVID-19 vaccination.

Clinical evaluation and validation of laboratory methods for the diagnosis of Bordetella pertussis infection: Culture, polymerase chain reaction (PCR) and anti-pertussis toxin IgG serology (IgG-PT).

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.

Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the "out" position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections.IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts.

Conveyance Contact Investigation for Imported Middle East Respiratory Syndrome Cases, United States, May 2014.

In 2014, the Centers for Disease Control and Prevention conducted conveyance contact investigations for 2 Middle East respiratory syndrome cases imported into the United States, comprising all passengers and crew on 4 international and domestic flights and 1 bus. Of 655 contacts, 78% were interviewed; 33% had serologic testing. No secondary cases were identified.

Human cathelicidin, LL-37, inhibits respiratory syncytial virus infection in polarized airway epithelial cells.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract illness in young children worldwide. Treatment options for severe RSV disease remain limited and the development of therapeutic treatment strategies remains a priority. LL-37, a small cationic host defense peptide involved in anti-inflammatory and anti-bacterial responses, reduces replication of or infection by multiple viruses, including influenza virus, in vitro, and protects against lethal challenge with influenza virus in vivo. LL-37 also protects against RSV infection of HEp-2 cells in vitro; however, HEp-2 are not reflective of polarized airway epithelial cells and respond differently to RSV infection. An air-liquid interface (ALI) Calu-3 model that more closely mimics the human airway epithelium was established. Using this in vitro model, the effectiveness of LL-37 in preventing RSV infection and replication was examined. RESULTS: LL-37, when pre-incubated with virus prior to RSV infection (prophylactic), significantly reduced the level of viral genome detected in infected Calu-3 cells, and decreased chemokine expression associated with RSV infection in vitro. In contrast, therapeutic treatment of RSV-infected ALI Calu-3 at 24 h and 3 days post-infection had minimal impact on RSV infection. CONCLUSIONS: Differences in the efficacy of LL-37 at reducing RSV infection under prophylactic and therapeutic conditions may in part be ascribed to differences in the method of peptide exposure. However, the efficacy of LL-37 at reducing RSV infection under prophylactic conditions indicates that further studies examining the efficacy of LL-37 as a small peptide inhibitor of RSV are warranted.

Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators.

Multifacility Outbreak of Middle East Respiratory Syndrome in Taif, Saudi Arabia.

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is a novel respiratory pathogen first reported in 2012. During September 2014-January 2015, an outbreak of 38 cases of MERS was reported from 4 healthcare facilities in Taif, Saudi Arabia; 21 of the 38 case-patients died. Clinical and public health records showed that 13 patients were healthcare personnel (HCP). Fifteen patients, including 4 HCP, were associated with 1 dialysis unit. Three additional HCP in this dialysis unit had serologic evidence of MERS-CoV infection. Viral RNA was amplified from acute-phase serum specimens of 15 patients, and full spike gene-coding sequencing was obtained from 10 patients who formed a discrete cluster; sequences from specimens of 9 patients were closely related. Similar gene sequences among patients unlinked by time or location suggest unrecognized viral transmission. Circulation persisted in multiple healthcare settings over an extended period, underscoring the importance of strengthening MERS-CoV surveillance and infection-control practices.

Qualitative assessment of pertussis diagnostics in United States laboratories.

BACKGROUND: United States national surveillance data show that the use of culture for pertussis diagnostics has sharply declined, whereas polymerase chain reaction (PCR) is now the most common testing method. PCR testing for pertussis is rapid and sensitive, but the lack of standardization and variable specificity is concerning. METHODS: A web-based survey containing 12 questions was sent to public health, commercial and hospital-based US laboratories performing clinical diagnostics to determine the pertussis diagnostics used. An extensive real-time PCR (RT-PCR) questionnaire accompanied a proficiency panel assessing the types of extraction methods, RT-PCR methods and current quality control in place at the laboratories. The proficiency panel of 12 specimens containing Bordetella pertussis at various concentrations and negative controls was created to detect cross-contamination and assess the lower limit of detection. RESULTS: One hundred twenty-three (35%) of 355 respondents from the web-based survey performed diagnostic tests for the presence of B. pertussis. Eighty-three (71%) labs reported performing culture, whereas 67 (54%) labs used PCR. All 41 laboratories that consented to participate in the proficiency exercise used the IS481 RT-PCR target; however, a variety of extraction and RT-PCR methods were employed. The laboratories correctly identified 92% of the B. pertussis specimens, and 5% of the laboratories (1.8% of the panel specimens) reported at least 1 false-positive. CONCLUSIONS: The small percentage of false-positives suggests that adequate procedures are in place to prevent cross-contamination. Differing extraction and PCR methods as well as variable analytic sensitivity emphasize the necessity for an external well-defined quality control program and interlaboratory pertussis PCR harmonization.

Draft genome sequences of Bordetella holmesii strains from blood (F627) and nasopharynx (H558).

Bordetella holmesii, a human pathogen, can confound the diagnosis of respiratory illness caused by Bordetella pertussis. We present the draft genome sequences of two B. holmesii isolates, one from blood, F627, and one from the nasopharynx, H558. Interestingly, important virulence genes that are present in B. pertussis are not found in B. holmesii.

Utilization of multiple real-time PCR assays for the diagnosis of Bordetella spp. in clinical specimens.

Bordetella pertussis causes an upper respiratory infection in infants, adolescents, and adults. Diagnosis of pertussis, a vaccine-preventable disease, can be difficult, but recent implementation of real-time PCR assays in laboratories has hastened the ability of clinicians to make an accurate diagnosis. In this paper we describe the method of nasopharyngeal specimen collection, extraction of DNA, and real-time PCR assays that will allow the detection and identification of Bordetella spp. in clinical specimens.

Epidemiologic and laboratory features of a large outbreak of pertussis-like illnesses associated with cocirculating Bordetella holmesii and Bordetella pertussis--Ohio, 2010-2011.

BACKGROUND: During 9 May 2010-7 May 2011, an outbreak of pertussis-like illness (incidence, 80 cases per 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 of 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% to 69%, respectively). CONCLUSIONS: We report the first documented mixed outbreak of B. pertussis and B. holmesii infections. Bordetella holmesii particularly affected adolescents. Although laboratory capacity limitations might inhibit routine use of multitarget PCR for clinical diagnosis, focused testing and enhanced surveillance might improve understanding the burden of B. holmesii infection.

Sorotipos e perfis genéticos de cepas de Bordetella pertussis isoladas na cidade de São Paulo, 2006-2008 / Serotypes and genetic profiles of Bordetella pertussis strains isolated in the city of São Paulo, 2006-2008

OBJETIVO: O conhecimento de Bordetella pertussis circulante na América Latina é limitado. Portanto, o objetivo deste estudo foi usar a técnica da eletroforese em campo pulsado e a sorotipagem para caracterizar cepas de B. pertussis isoladas na cidade de São Paulo (SP). MÉTODOS: Este estudo, conduzido entre 2006 e 2008, analisou 652 swabs de nasofaringe coletados de casos suspeitos e comunicantes de coqueluche, provenientes de 37 hospitais sentinela de São Paulo. Foram realizadas as técnicas da eletroforese em campo pulsado e sorotipagem em 91 (70%) cepas de B. pertussis, escolhidas aleatoriamente. RESULTADOS: Noventa e sete por cento das cepas de São Paulo foram sorotipadas como Fim3. Foram identificados 14 perfis genéticos pela eletroforese em campo pulsado; o mais prevalente (57%) também é o mais prevalente nos EUA. CONCLUSÕES: Esses dados, em conjunto com ações da vigilância, podem ter um impacto nas estratégias de prevenção e controle de coqueluche na região, oferecendo informações úteis para a introdução de estratégias novas de vacinação e redução do risco de transmissão para bebês menores de 6 meses de idade.

OBJECTIVE: Knowledge of Bordetella pertussis circulating in Latin America is limited. Therefore, the goal of this study was to use pulsed-field gel electrophoresis and serotyping to characterize B. pertussis strains isolated in the city of São Paulo, Brazil. METHODS: This study, conducted between 2006 and 2008, analyzed 652 nasopharyngeal swabs from suspected pertussis cases and contacts, collected from 37 sentinel hospitals in São Paulo. Randomized samples of 91 (70%) strains of B. pertussis were subtyped by pulsed-field gel electrophoresis and serotyping. RESULTS: Ninety-seven percent of strains from São Paulo were serotyped as Fim3. Fourteen pulsed-field gel electrophoresis profiles were identified; the most prevalent (57%) is also the most prevalent in the USA. CONCLUSIONS: These data, in conjunction with surveillance activities, may impact strategies regarding prevention and control of pertussis in the region, providing useful information for introduction of new vaccination strategies and reduction of risk of transmission to infants less than 6 months of age.

Population diversity among Bordetella pertussis isolates, United States, 1935-2009.

Since the 1980s, pertussis notifications in the United States have been increasing. To determine the types of Bordetella pertussis responsible for these increases, we divided 661 B. pertussis isolates collected in the United States during 1935-2009 into 8 periods related to the introduction of novel vaccines or changes in vaccination schedule. B. pertussis diversity was highest from 1970-1990 (94%) but declined to ≈ 70% after 1991 and has remained constant. During 2006-2009, 81.6% of the strains encoded multilocus sequence type prn2-ptxP3-ptxS1A-fim3B, and 64% were multilocus variable number tandem repeat analysis type 27. US trends were consistent with those seen internationally; emergence and predominance of the fim3B allele was the only molecular characteristic associated with the increase in pertussis notifications. Changes in the vaccine composition and schedule were not the direct selection pressures that resulted in the allele changes present in the current B. pertussis population.

Serotypes and genetic profiles of Bordetella pertussis strains isolated in the city of São Paulo, 2006-2008.

OBJECTIVE: Knowledge of Bordetella pertussis circulating in Latin America is limited. Therefore, the goal of this study was to use pulsed-field gel electrophoresis and serotyping to characterize B. pertussis strains isolated in the city of São Paulo, Brazil. METHODS: This study, conducted between 2006 and 2008, analyzed 652 nasopharyngeal swabs from suspected pertussis cases and contacts, collected from 37 sentinel hospitals in São Paulo. Randomized samples of 91 (70%) strains of B. pertussis were subtyped by pulsed-field gel electrophoresis and serotyping. RESULTS: Ninety-seven percent of strains from São Paulo were serotyped as Fim3. Fourteen pulsed-field gel electrophoresis profiles were identified; the most prevalent (57%) is also the most prevalent in the USA. CONCLUSIONS: These data, in conjunction with surveillance activities, may impact strategies regarding prevention and control of pertussis in the region, providing useful information for introduction of new vaccination strategies and reduction of risk of transmission to infants less than 6 months of age.

Pertussis Pseudo-outbreak linked to specimens contaminated by Bordetella pertussis DNA From clinic surfaces.

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.

Novel multitarget real-time PCR assay for rapid detection of Bordetella species in clinical specimens.

A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness.

Effectiveness of adolescent and adult tetanus, reduced-dose diphtheria, and acellular pertussis vaccine against pertussis.

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%; P = .092). 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.

A computational genomics pipeline for prokaryotic sequencing projects.

MOTIVATION: New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. RESULTS: We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. AVAILABILITY AND IMPLEMENTATION: The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems.

Pathology and pathogenesis of fatal Bordetella pertussis infection in infants.

BACKGROUND: Each year, Bordetella pertussis infection causes an estimated 294,000 deaths worldwide, primarily among young, nonvaccinated children. Approximately 90% of all deaths due to pertussis in the Unites States occur in young infants. These children often develop intractable pulmonary hypertension; however, the pathophysiologic mechanism responsible for this complication has not been well characterized, and there have been no detailed descriptions of the pathology of this disease since the 1940s. METHODS: Respiratory tissue samples obtained at autopsy from 15 infants aged

Development and evaluation of dual-target real-time polymerase chain reaction assays to detect Bordetella spp.

Novel, highly specific, and sensitive real-time polymerase chain reaction (PCR) assays using 2 targets, insertion sequence (IS481) and pertussis toxin subunit 1 (ptxS1), were developed to detect Bordetella pertussis and to differentiate between relevant Bordetella spp. Sixty-four non-Bordetella isolates were negative by both assays, demonstrating the specificity of the assays. B. pertussis, Bordetella parapertussis, and Bordetella holmesii isolates were specifically identified using the assays. The lower limit of detection was less than 10 genomic equivalents per reaction for the IS481 and ptxS1 assays. These assays were evaluated using 145 human clinical specimens obtained during cough-illness outbreak investigations, and PCR results were compared with Bordetella spp. culture results. Twenty-seven (18.6%) specimens had late positive cycle threshold (Ct) values (35