Risk Factors Associated With Bordetella pertussis Among Infants ≤4 Months of Age in the Pre-Tdap Era: United States, 2002-2005.

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 antipertussis toxin IgG enzyme-linked immunosorbent assay units per milliliter. RESULTS: Enrolled subjects (115 case-patients; 230 control subjects) had 4396 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 that breastfeeding protects against pertussis and warrants recommendation with pertussis prevention strategies, which currently include pertussis vaccination of pregnant mothers and infants' close contacts.

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.

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms.

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (>95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.

Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens.

The large and growing number of viral and bacterial pathogens responsible for respiratory infections poses a challenge for laboratories seeking to provide rapid and comprehensive pathogen identification. We evaluated a novel application of the TaqMan low-density array (TLDA) cards for real-time PCR detection of 21 respiratory-pathogen targets. The performance of the TLDA was compared to that of individual real-time PCR (IRTP) assays with the same primers and probes using (i) nucleic acids extracted from the 21 pathogen strains and 66 closely related viruses and bacteria and (ii) 292 clinical respiratory specimens. With spiked samples, TLDA cards were about 10-fold less sensitive than IRTP assays. By using 292 clinical specimens to generate 2,238 paired individual assays, the TLDA card exhibited 89% sensitivity (95% confidence interval [CI], 86 to 92%; range per target, 47 to 100%) and 98% specificity (95% CI, 97 to 99%; range per target, 85 to 100%) overall compared to IRTP assays as the gold standard with a threshold cycle (C(T)) cutoff of 43. The TLDA card approach offers promise for rapid and simultaneous identification of multiple respiratory pathogens for outbreak investigations and disease surveillance.

Development and analytical validation of an immunoassay for quantifying serum anti-pertussis toxin antibodies resulting from Bordetella pertussis infection.

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 article describes test development, initial evaluation of a prototype kit enzyme-linked immunosorbent assay (ELISA) in an interlaboratory collaborative study, and analytical validation. The data presented here demonstrate that the kit met all prespecified 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/ml. In conclusion, the IgG anti-PT ELISA met all assay validation parameters within the range considered most relevant for serodiagnosis. This ELISA was developed and analytically validated as a user-friendly kit that can be used in both qualitative and quantitative formats. The technology for producing the kit is transferable to public health laboratories.

Circulating Chlamydia pneumoniae DNA and advanced coronary artery disease.

BACKGROUND: Chlamydia pneumoniae (C. pneumoniae) has been linked to atherosclerosis. Detection of this pathogen in peripheral blood cells may be valuable in the diagnosis of disease state. This study aimed to evaluate the prevalence of circulating C. pneumoniae DNA and its relationship with severity and extent of coronary artery disease (CAD). METHODS: Blood samples from 269 patients undergoing coronary angiography were collected. The presence of circulating C. pneumoniae DNA was determined by real-time PCR assay. Data regarding coronary risk factors and severity and extent of CAD were collected. Severity and extent of CAD was defined by the number of major epicardial coronary arteries with >50% stenosis and by the Duke jeopardy score. RESULTS: Sixteen of 269 specimens (5.9%) from the study cohort were positive for C. pneumoniae DNA. Thirteen specimens among 149 samples from patients with multi-vessel disease (8.7%) were positive for C. pneumoniae DNA compared with 3 of 120 (2.5%) among patients without multi-vessel CAD. The prevalence of circulating C. pneumoniae DNA was significantly associated with multi-vessel disease. The odds ratio was 5.1 (P=0.02) after adjustment for conventional risk factors. CONCLUSIONS: Presence of circulating C. pneumoniae DNA is associated with advanced CAD, suggesting C. pneumoniae infection as a contributing factor to progression of coronary atherosclerosis.

Seropositivity to Chlamydia pneumoniae is associated with risk of first ischemic stroke.

BACKGROUND AND PURPOSE: Serologic evidence of infection with Chlamydia pneumoniae has been associated with cardiovascular disease, but its relationship with stroke risk remains uncertain. The objective of this study is to determine whether serological evidence of C pneumoniae infection is associated with risk of ischemic stroke. METHODS: A population-based case-control study was performed in an urban, multiethnic population. Cases (n=246) had first ischemic stroke, and controls (n=474) matched for age, sex, and race-ethnicity were derived through random-digit dialing. Titers of C pneumoniae-specific IgG and IgA antibodies were measured using microimmunofluorescence, and positive titers were prospectively defined. Conditional logistic regression was used to calculate odds ratios (ORs) and 95% CIs adjusting for medical, behavioral, and socioeconomic factors. RESULTS: Mean age among cases was 72.3+/-9.7 years; 50.8% were women. Elevated C pneumoniae IgA titers were associated with increased risk of ischemic stroke after adjusting for hypertension, diabetes mellitus, current cigarette use, atrial fibrillation, and levels of high-density lipoprotein and low-density lipoprotein (adjusted OR, 1.5; 95% CI, 1.0 to 2.2). Elevated IgG titers were not associated with stroke risk (adjusted OR, 1.2; 95% CI, 0.8 to 1.8). There was a trend toward an association of elevated IgA titers with atherosclerotic and lacunar stroke but less so cardioembolic or cryptogenic subtypes. CONCLUSIONS: Serologic evidence of C pneumoniae infection is associated with ischemic stroke risk. IgA titers may be a better marker of risk than IgG. This association is independent of other stroke risk factors and is present for atherosclerotic, lacunar, and cardioembolic subtypes. Further studies of the effect of C pneumoniae on stroke risk are warranted.

Leukocyte count is associated with reduced endothelial reactivity.

BACKGROUND: Leukocyte count has been associated with cardiovascular and cerebrovascular disease in several studies. We hypothesized that white blood cell count is associated with endothelial reactivity. METHODS AND RESULTS: Leukocyte count was measured in a sample of stroke-free community participants undergoing brachial artery testing for endothelial reactivity. Flow-mediated dilation (FMD) during reactive hyperemia was assessed in each subject using high-resolution B-mode ultrasound. Multivariate linear regression was used to calculate the effect of leukocyte count on endothelial reactivity after adjusting for potential confounding factors. Mean age of the 868 participants was 66.7+/-8.8 years; 57% were women. Mean leukocyte count was (6.1+/-1.8)x10(9)/L. Each unit increase in leukocyte count was associated with a mean 0.18% decrease in FMD (p = 0.01). After adjusting for other atherosclerosis risk factors, including age, sex, hypertension, diabetes, hyperlipidemia, and smoking, the relationship persisted (mean decrease in FMD per unit leukocyte count = 0.17%, p = 0.02). There was a linear decrease in FMD by quartile of leukocyte count (p = 0.0014). The effect of leukocyte count on FMD was greater for women, those under age 70, and non-diabetics. CONCLUSIONS: Relative elevations in leukocyte count are associated with a reduction in brachial artery endothelial reactivity. These findings are consistent with current hypotheses regarding the inflammatory or infectious etiology of risk of atherosclerosis and stroke, but also suggest interactions with demographic and other risk factors.

Chlamydia pneumoniae and Mycoplasma pneumoniae in young children from China with community-acquired pneumonia.

Eighty-five cases community-acquired pneumonia (CAP) in children 5 years or younger, confirmed by chest X-ray, and 185 age-matched control patients with diarrhea or dermatitis from the Outpatient Department at Beijing Children's Hospital were enrolled into this study. Nasopharyngeal swab specimens were obtained from all subjects. Real-time PCR-based fluorescence assays were performed for Chlamydia pneumoniae and Mycoplasma pneumoniae. A nested PCR was also run for C. pneumoniae for comparison of assays. C. pneumoniae was found in 3 (3.5%) of CAP cases and in 4 (2.1%) of controls (P = 0.51). M. pneumoniae was found in 6 (7.1%) of CAP cases and in none of the controls (P = 0.001). The agreement rate of the 2 applied PCR methods used for C. pneumoniae detection was 98.5%. Our study demonstrates that M. pneumoniae may play a significant role in CAP affecting children up to 5 years in China, whereas C. pneumoniae in nasopharyngeal specimens was not associated with CAP in this age group.

Identification of Haemophilus influenzae serotypes by standard slide agglutination serotyping and PCR-based capsule typing.

To resolve discrepancies in slide agglutination serotyping (SAST) results from state health departments and the Centers for Disease Control and Prevention (CDC), we characterized 141 of 751 invasive Haemophilus influenzae isolates that were identified in the United States from January 1998 to December 1999 through an active, laboratory-based, surveillance program coordinated by the CDC. We found discrepancies between the results of SAST performed at state health departments and those of PCR capsule typing performed at the CDC for 56 (40%) of the isolates characterized: 54 isolates that were identified as a particular serotype by SAST were shown to be unencapsulated by PCR, and two isolates that were reported as serotypes b and f were found to be serotypes f and e, respectively, by PCR. The laboratory error most likely to affect the perceived efficacy of the conjugate H. influenzae type b (Hib) vaccine was the misidentification of isolates as serotype b: of 40 isolates identified as serotype b by SAST, 27 (68%) did not contain the correlating capsule type genes. The frequency of errors fell substantially when standardized reagents and routine quality control of SAST were used during a study involving three laboratories. An overall 94% agreement between SAST and PCR results showed that slide agglutination could be a valid and reliable method for serotyping H. influenzae if the test was performed correctly, in accordance with standardized and recommended procedures. An ongoing prospective analysis of all H. influenzae surveillance isolates associated with invasive disease in children less than 5 years old will provide more accurate national figures for the burden of invasive disease caused by Hib and other H. influenzae serotypes.

Outbreak of W135 meningococcal disease in 2000: not emergence of a new W135 strain but clonal expansion within the electophoretic type-37 complex.

In 2000, >400 cases of disease caused by Neisseria meningitidis serogroup W135 (MenW135), the largest MenW135 outbreak reported to date, occurred worldwide among Hajj pilgrims and their contacts. To elucidate the origin of the outbreak strains and to investigate their relatedness to major clonal groups, genotypic and phenotypic subtyping was performed on 26 MenW135 outbreak-associated isolates and 50 MenW135 isolates collected worldwide from 1970 through 2000. All outbreak-associated isolates were members of a single clone of the hypervirulent electrophoretic type (ET)-37 complex, designated the "(W)ET-37 clone"; 19 additional MenW135 strains were also members of this clone, and the remaining 31 MenW135 strains were clearly distinct. The 2000 MenW135 outbreak was not caused by emergence of a new MenW135 strain but rather by expansion of the (W)ET-37 clone that has been in circulation at least since 1970; the strains most closely related to those causing the 2000 outbreak have been isolated in Algeria, Mali, and The Gambia in the 1990s.

Development and evaluation of real-time PCR-based fluorescence assays for detection of Chlamydia pneumoniae.

Chlamydia pneumoniae is an important respiratory pathogen recently associated with atherosclerosis and several other chronic diseases. Detection of C. pneumoniae is inconsistent, and standardized PCR assays are needed. Two real-time PCR assays specific for C. pneumoniae were developed by using the fluorescent dye-labeled TaqMan probe-based system. Oligonucleotide primers and probes were designed to target two variable domains of the ompA gene, VD2 and VD4. The limit of detection for each of the two PCR assays was 0.001 inclusion-forming unit. Thirty-nine C. pneumoniae isolates obtained from widely distributed geographical areas were amplified by the VD2 and VD4 assays, producing the expected 108- and 125-bp amplification products, respectively. None of the C. trachomatis serovars, C. psittaci strains, other organisms, or human DNAs tested were amplified. The amplification results of the newly developed assays were compared to the results of culturing and two nested PCR assays, targeting the 16S rRNA and ompA genes. The assays were compared by testing C. pneumoniae purified elementary bodies, animal tissues, 228 peripheral blood mononuclear cell (PBMC) specimens, and 179 oropharyngeal (OP) swab specimens obtained from ischemic stroke patients or matched controls. The real-time VD4 assay and one nested PCR each detected C. pneumoniae in a single, but different, PBMC specimen. Eleven of 179 OP specimens (6.1%) showed evidence of the presence of C. pneumoniae in one or more tests. The real-time VD4 assay detected the most positive results of the five assays. We believe that this real-time PCR assay offers advantages over nested PCR assays and may improve the detection of C. pneumoniae in clinical specimens.