Genomic Insights on Variation Underlying Capsule Expression in Meningococcal Carriage Isolates From University Students, United States, 2015-2016.

In January and February 2015, Neisseria meningitidis serogroup B (NmB) outbreaks occurred at two universities in the United States, and mass vaccination campaigns using MenB vaccines were initiated as part of a public health response. Meningococcal carriage evaluations were conducted concurrently with vaccination campaigns at these two universities and at a third university, where no NmB outbreak occurred. Meningococcal isolates (N = 1,514) obtained from these evaluations were characterized for capsule biosynthesis by whole-genome sequencing (WGS). Functional capsule polysaccharide synthesis (cps) loci belonging to one of seven capsule genogroups (B, C, E, W, X, Y, and Z) were identified in 122 isolates (8.1%). Approximately half [732 (48.4%)] of isolates could not be genogrouped because of the lack of any serogroup-specific genes. The remaining 660 isolates (43.5%) contained serogroup-specific genes for genogroup B, C, E, W, X, Y, or Z, but had mutations in the cps loci. Identified mutations included frameshift or point mutations resulting in premature stop codons, missing or fragmented genes, or disruptions due to insertion elements. Despite these mutations, 49/660 isolates expressed capsule as observed with slide agglutination, whereas 45/122 isolates with functional cps loci did not express capsule. Neither the variable capsule expression nor the genetic variation in the cps locus was limited to a certain clonal complex, except for capsule null isolates (predominantly clonal complex 198). Most of the meningococcal carriage isolates collected from student populations at three US universities were non-groupable as a result of either being capsule null or containing mutations within the capsule locus. Several mutations inhibiting expression of the genes involved with the synthesis and transport of the capsule may be reversible, allowing the bacteria to switch between an encapsulated and non-encapsulated state. These findings are particularly important as carriage is an important component of the transmission cycle of the pathogen, and understanding the impact of genetic variations on the synthesis of capsule, a meningococcal vaccine target and an important virulence factor, may ultimately inform strategies for control and prevention of disease caused by this pathogen.

Epidemiologic, Immunologic, and Virus Characteristics in Patients With Paired Severe Acute Respiratory Syndrome Coronavirus 2 Serology and Reverse-Transcription Polymerase Chain Reaction Testing.

BACKGROUND: The natural history and clinical progression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections can be better understood using combined serological and reverse-transcription polymerase chain reaction (RT-PCR) testing. METHODS: Nasopharyngeal swabs and serum were collected at a single time-point from patients at an urban, public hospital during August-November 2020 and tested for SARS-CoV-2 using RT-PCR, viral culture, and anti-spike pan-immunoglobulin antibody testing. Participant demographics and symptoms were collected through interview. The χ 2 and Fisher exact tests were used to identify associations between RT-PCR and serology results with presence of viable virus and frequency of symptoms. RESULTS: Among 592 participants, 129 (21.8%) had evidence of SARS-CoV-2 infection by RT-PCR or serology. Presence of SARS-CoV-2 antibodies was strongly associated with lack of viable virus (P = .016). COVID-19 symptom frequency was similar for patients testing RT-PCR positive/seronegative and patients testing RT-PCR positive/seropositive. Patients testing RT-PCR positive/seronegative reported headaches, fatigue, diarrhea, and vomiting at rates not statistically significantly different from those testing RT-PCR negative/seropositive. CONCLUSIONS: While patients testing SARS-CoV-2 seropositive were unlikely to test positive for viable virus and were therefore at low risk for forward transmission, coronavirus disease 2019 (COVID-19) symptoms were common. Paired SARS-CoV-2 RT-PCR and antibody testing provides more nuanced understanding of patients' COVID-19 status.

Pregnancy, Birth, Infant, and Early Childhood Neurodevelopmental Outcomes among a Cohort of Women with Symptoms of Zika Virus Disease during Pregnancy in Three Surveillance Sites, Project Vigilancia de Embarazadas con Zika (VEZ), Colombia, 2016-2018.

Project Vigilancia de Embarazadas con Zika (VEZ), an intensified surveillance of pregnant women with symptoms of the Zika virus disease (ZVD) in Colombia, aimed to evaluate the relationship between symptoms of ZVD during pregnancy and adverse pregnancy, birth, and infant outcomes and early childhood neurodevelopmental outcomes. During May-November 2016, pregnant women in three Colombian cities who were reported with symptoms of ZVD to the national surveillance system, or with symptoms of ZVD visiting participating clinics, were enrolled in Project VEZ. Data from maternal and pediatric (up to two years of age) medical records were abstracted. Available maternal specimens were tested for the presence of the Zika virus ribonucleic acid and/or anti-Zika virus immunoglobulin antibodies. Of 1213 enrolled pregnant women with symptoms of ZVD, 1180 had a known pregnancy outcome. Results of the Zika virus laboratory testing were available for 569 (48.2%) pregnancies with a known pregnancy outcome though testing timing varied and was often distal to the timing of symptoms; 254 (21.5% of the whole cohort; 44.6% of those with testing results) were confirmed or presumptive positive for the Zika virus infection. Of pregnancies with a known outcome, 50 (4.2%) fetuses/infants had Zika-associated brain or eye defects, which included microcephaly at birth. Early childhood adverse neurodevelopmental outcomes were more common among those with Zika-associated birth defects than among those without and more common among those with laboratory evidence of a Zika virus infection compared with the full cohort. The proportion of fetuses/infants with any Zika-associated brain or eye defect was consistent with the proportion seen in other studies. Enhancements to Colombia's existing national surveillance enabled the assessment of adverse outcomes associated with ZVD in pregnancy.

Serologic Testing of US Blood Donations to Identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Reactive Antibodies: December 2019-January 2020.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China, in December 2019, with subsequent worldwide spread. The first US cases were identified in January 2020. METHODS: To determine if SARS-CoV-2-reactive antibodies were present in sera prior to the first identified case in the United States on 19 January 2020, residual archived samples from 7389 routine blood donations collected by the American Red Cross from 13 December 2019 to 17 January 2020 from donors resident in 9 states (California, Connecticut, Iowa, Massachusetts, Michigan, Oregon, Rhode Island, Washington, and Wisconsin) were tested at the Centers for Disease Control and Prevention for anti-SARS-CoV-2 antibodies. Specimens reactive by pan-immunoglobulin (pan-Ig) enzyme-linked immunosorbent assay (ELISA) against the full spike protein were tested by IgG and IgM ELISAs, microneutralization test, Ortho total Ig S1 ELISA, and receptor-binding domain/ACE2 blocking activity assay. RESULTS: Of the 7389 samples, 106 were reactive by pan-Ig. Of these 106 specimens, 90 were available for further testing. Eighty-four of 90 had neutralizing activity, 1 had S1 binding activity, and 1 had receptor-binding domain/ACE2 blocking activity >50%, suggesting the presence of anti-SARS-CoV-2-reactive antibodies. Donations with reactivity occurred in all 9 states. CONCLUSIONS: These findings suggest that SARS-CoV-2 may have been introduced into the United States prior to 19 January 2020.

Performance of InBios ZIKV Detect™ 2.0 IgM Capture ELISA in two reference laboratories compared to the original ZIKV Detect™ IgM Capture ELISA.

ZIKV Detect™ 2.0 IgM Capture ELISA (InBios International, Seattle, WA) recently replaced the ZIKV Detect™ IgM Capture ELISA and a number of significant changes have been made to the original version. This study compares data generated from the ZIKV Detect™ 2.0 IgM Capture ELISA, to data generated using the original version of the kit. The same sample sets were used in this comparison, and reference test results for these samples were used to assess sensitivity, specificity, accuracy and concordance of results across two laboratories. Average sensitivity increased from 90.4% to 92.5% with the updated kit where the increase was not statistically different, and specificity increased from 79.5% to 97.4%, a statistically-significant difference. Accuracy of the ZIKV Detect™ 2.0 IgM Capture ELISA was 89% compared to 63.9% for the original version of the kit, and agreement across the laboratories increased from 79.5% to 97.4%. With secondary dengue virus infections, specificity increased from 9.3% to 82.6% with the updated kit, primarily due to the change in interpretation criteria that no longer includes "Possible Zika positive."

Meningococcal Carriage Following a Vaccination Campaign With MenB-4C and MenB-FHbp in Response to a University Serogroup B Meningococcal Disease Outbreak-Oregon, 2015-2016.

Background: Limited data exist on the impact of the serogroup B meningococcal (MenB) vaccines MenB-FHbp and MenB-4C on meningococcal carriage and herd protection. We therefore assessed meningococcal carriage following a MenB vaccination campaign in response to a university serogroup B meningococcal disease outbreak in 2015. Methods: A convenience sample of students recommended for vaccination provided oropharyngeal swab specimens and completed questionnaires during 4 carriage surveys over 11 months. Isolates were tested by real-time polymerase chain reaction analysis, slide agglutination, and whole-genome sequencing. Vaccination history was verified via university records and the state immunization registry. Results: A total of 4225 oropharyngeal swab specimens from 3802 unique participants were analyzed. Total meningococcal and genotypically serogroup B carriage prevalence among sampled students were stable, at 11%-17% and 1.2%-2.4% during each round, respectively; no participants carried the outbreak strain. Neither 1-3 doses of MenB-FHbp nor 1-2 doses of MenB-4C was associated with decreased total or serogroup B carriage prevalence. Conclusions: While few participants completed the full MenB vaccination series, limiting analytic power, these data suggest that MenB-FHbp and MenB-4C do not have a large, rapid impact on meningococcal carriage and are unlikely to provide herd protection in the context of an outbreak response.

Validation of high throughput screening of human sera for detection of anti-PA IgG by Enzyme-Linked Immunosorbent Assay (ELISA) as an emergency response to an anthrax incident.

To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r2), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from -4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 µg/mL. The regression analysis results for dilutional linearity data were r2 = 0.952, slope = 1.02 and intercept = -0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate.

Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses.

Background. Influenza disproportionately impacts older adults while current vaccines have reduced effectiveness in the older population. Methods. We conducted a comprehensive evaluation of cellular and humoral immune responses of adults aged 50 years and older to the 2008-2009 seasonal trivalent inactivated influenza vaccine and assessed factors influencing vaccine response. Results. Vaccination increased hemagglutination inhibition and neutralizing antibody; however, 66.3% of subjects did not reach hemagglutination inhibition titers ≥ 40 for H1N1, compared with 22.5% for H3N2. Increasing age had a minor negative impact on antibody responses, whereas prevaccination titers were the best predictors of postvaccination antibody levels. Preexisting memory B cells declined with age, especially for H3N2. However, older adults still demonstrated a significant increase in antigen-specific IgG(+) and IgA(+) memory B cells postvaccination. Despite reduced frequency of preexisting memory B cells associated with advanced age, fold-rise in memory B cell frequency in subjects 60+ was comparable to subjects age 50-59. Conclusions. Older adults mounted statistically significant humoral and cell-mediated immune responses, but many failed to reach hemagglutination inhibition titers ≥40, especially for H1N1. Although age had a modest negative effect on vaccine responses, prevaccination titers were the best predictor of postvaccination antibody levels, irrespective of age.

Severe acute respiratory infections caused by 2009 pandemic influenza A (H1N1) among American Indians--southwestern United States, May 1-July 21, 2009.

BACKGROUND: During April-July 2009, U.S. hospitalization rates for 2009 pandemic influenza A (H1N1) virus (H1N1pdm09) infection were estimated at 4·5/100 000 persons. We describe rates and risk factors for H1N1pdm09 infection among American Indians (AIs) in four isolated southwestern U.S. communities served by the Indian Health Service (IHS). METHODS: We reviewed clinical and demographic information from medical records of AIs hospitalized during May 1-July 21, 2009 with severe acute respiratory infection (SARI). Hospitalization rates were determined using denominator data provided by IHS. H1N1pdm09 infection was confirmed with polymerase chain reaction, rapid tests, or convalescent serology. Risk factors for more severe (SARI) versus milder [influenza-like illness (ILI)] illness were determined by comparing confirmed SARI patients with outpatients with ILI. RESULTS: Among 168 SARI-hospitalized patients, 52% had confirmed H1N1pdm09 infection and 93% had >1 high-risk condition for influenza complications. The H1N1pdm09 SARI hospitalization rate was 131/100 000 persons [95% confidence interval (CI), 102-160] and was highest among ages 0-4 years (353/100 000; 95% CI, 215-492). Among children, asthma (adjusted odds ratio [aOR] 3·2; 95% CI, 1·2-8·4) and age<2 years (aOR 3·8; 95% CI, 1·4-10·0) were associated with H1N1pdm09 SARI-associated hospitalization, compared with outpatient ILI. Among adults, diabetes (aOR 3·1; 95% CI, 1·5-6·4) was associated with hospitalization after controlling for obesity. CONCLUSIONS: H1N1pdm09 hospitalization rates among this isolated AI population were higher than reported for other U.S. populations. Almost all case patients had high-risk health conditions. Prevention strategies for future pandemics should prioritize AIs, particularly in isolated rural areas.

Quantitative assessment of anthrax vaccine immunogenicity using the dried blood spot matrix.

The collection, processing and transportation to a testing laboratory of large numbers of clinical samples during an emergency response situation present significant cost and logistical issues. Blood and serum are common clinical samples for diagnosis of disease. Serum preparation requires significant on-site equipment and facilities for immediate processing and cold storage, and significant costs for cold-chain transport to testing facilities. The dried blood spot (DBS) matrix offers an alternative to serum for rapid and efficient sample collection with fewer on-site equipment requirements and considerably lower storage and transport costs. We have developed and validated assay methods for using DBS in the quantitative anti-protective antigen IgG enzyme-linked immunosorbent assay (ELISA), one of the primary assays for assessing immunogenicity of anthrax vaccine and for confirmatory diagnosis of Bacillus anthracis infection in humans. We have also developed and validated high-throughput data analysis software to facilitate data handling for large clinical trials and emergency response.

Seropositivity for influenza A(H1N1)pdm09 virus among frontline health care personnel.

Seroprevalence of antibodies to influenza A(H1N1)pdm09 virus among 193 emergency department health care personnel was similar among 147 non-health care personnel (odds ratio 1.4, 95% CI 0.8-2.4). Working in an acute care setting did not substantially increase risk for virus infection above risk conferred by community-based exposures.

Sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic H1N1 virus in U.S. populations.

Swine origin 2009 H1N1 influenza virus has spread globally to cause the first influenza pandemic of the 21st century. Serological studies can improve our understanding of the extent of human infection and risk factors associated with the transmission of this pandemic virus. The "gold standard" for serodiagnosis of human influenza virus infection is the detection of seroconversion between acute- and convalescent-stage samples. However, the timing of seroepidemiological investigations often precludes the collection of truly acute-phase sera, requiring development of serological criteria for evaluating convalescent-phase sera that optimize detection of true positives and true negatives. To guide seroepidemiological investigations into the spread of the novel 2009 pandemic H1N1 virus, we characterized serum antibody responses to 2009 H1N1 virus in 87 individuals with confirmed viral infection and 227 nonexposed U.S. individuals using microneutralization (MN) and hemagglutination inhibition (HI) assays. Sensitivity and specificity were determined for each assay alone and in combination for detection of 2009 H1N1 virus-specific antibodies in convalescent-phase sera. Although the HI assay was more specific for detecting antibody to 2009 H1N1, the MN assay was more sensitive, particularly for detecting low-titer seroconversions. A combination of titers (MN ≥ 40 and HI ≥ 20) provided the highest sensitivity (90%) and specificity (96%) for individuals aged <60 years and 92% specificity for adults aged ≥ 60 years for detection of serologically confirmed 2009 H1N1 infections in U.S. populations during the first pandemic waves. These studies provide an approach to optimize timely serological investigations for future pandemics or outbreaks of novel influenza viruses among humans.

Immune responses to Bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax.

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for >1 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested. Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested. Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83). PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested. Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.