Melissa Whaley

Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

ABSTRACT The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.

Global Role and Burden of Influenza in Pediatric Respiratory Hospitalizations, 1982–2012: A Systematic Analysis

Background The global burden of pediatric severe respiratory illness is substantial, and influenza viruses contribute to this burden. Systematic surveillance and testing for influenza among hospitalized children has expanded globally over the past decade. However, only a fraction of the data has been used to estimate influenza burden. In this analysis, we use surveillance data to provide an estimate of influenza-associated hospitalizations among children worldwide. Methods and Findings We aggregated data from a systematic review (n = 108) and surveillance platforms (n = 37) to calculate a pooled estimate of the proportion of samples collected from children hospitalized with respiratory illnesses and positive for influenza by age group (<6 mo, <1 y, <2 y, <5 y, 5–17 y, and <18 y). We applied this proportion to global estimates of acute lower respiratory infection hospitalizations among children aged <1 y and <5 y, to obtain the number and per capita rate of influenza-associated hospitalizations by geographic region and socio-economic status. Influenza was associated with 10% (95% CI 8%–11%) of respiratory hospitalizations in children <18 y worldwide, ranging from 5% (95% CI 3%–7%) among children <6 mo to 16% (95% CI 14%–20%) among children 5–17 y. On average, we estimated that influenza results in approximately 374,000 (95% CI 264,000 to 539,000) hospitalizations in children <1 y—of which 228,000 (95% CI 150,000 to 344,000) occur in children <6 mo—and 870,000 (95% CI 610,000 to 1,237,000) hospitalizations in children <5 y annually. Influenza-associated hospitalization rates were more than three times higher in developing countries than in industrialized countries (150/100,000 children/year versus 48/100,000). However, differences in hospitalization practices between settings are an important limitation in interpreting these findings. Conclusions Influenza is an important contributor to respiratory hospitalizations among young children worldwide. Increasing influenza vaccination coverage among young children and pregnant women could reduce this burden and protect infants <6 mo.

Repeat revaccination with 23-valent pneumococcal polysaccharide vaccine among adults aged 55–74 years living in Alaska: No evidence of hyporesponsiveness☆☆☆

BACKGROUND Older adults are at highest risk of invasive pneumococcal disease (IPD) and are recommended to receive vaccination with 23-valent pneumococcal polysaccharide vaccine (PPV23). Antibody concentrations decline following vaccination. We evaluated the immunogenicity and reactogenicity of revaccination and repeat revaccination. METHODS Adults aged 55-74 years were vaccinated with a 1st to 4th dose of PPV23. Participants were eligible for revaccination if a minimum of 6 years had passed since their last dose of PPV23. Blood collected on the day of vaccination and 30 days later was analyzed by ELISA for IgG to five serotypes. Functional antibody activity was measured using an opsonophagocytic killing (OPK) assay. Reactions to vaccination were documented. RESULTS Subjects were vaccinated with a 1st dose (n=123), 2nd dose (n=121), or 3rd or 4th dose (n=71) of PPV23. The post-vaccination IgG geometric mean concentrations (GMCs) were similar among first-time vaccinees and re-vaccinees for all serotypes with the exception of a lower GMC for serotype 1 in re-vaccinees. The post-vaccination OPK geometric mean titers (GMTs) were similar among first-time vaccinees and re-vaccinees with the exception of a higher GMT for serotype 6B in re-vaccinees. Compared to first-time vaccinees, re-vaccinees reported more joint pain (p=0.004), fatigue (p=0.019), headache (p=0.014), swelling (p=0.006), and moderate limitation in arm movement (p=0.025). CONCLUSIONS Repeat revaccination with PPV23, administered 6 or more years after the prior dose, was immunogenic and generally well tolerated.

Interlaboratory comparison of three multiplexed bead-based immunoassays for measuring serum antibodies to pneumococcal polysaccharides.

ABSTRACT Serotype-specific IgG, as quantified by a standardized WHO enzyme-linked immunosorbent assay (ELISA), is a serologic end point used to evaluate pneumococcal polysaccharide-based vaccine immunogenicity. Antibodies to each vaccine polysaccharide in licensed multivalent vaccines are quantified separately; this is laborious and consumes serum. We compared three bead-based immunoassays: a commercial assay (xMAP Pneumo14; Luminex) and two in-house assays (of the Health Protection Agency [HPA] and Centers for Disease Control and Prevention [CDC]), using the WHO-recommended standard reference and reference sera (n = 11) from vaccinated adults. Multiple comparisons of the IgG concentrations for seven conjugate vaccine serotypes were performed by sample (percent error), serotype (equivalency testing), and laboratory (concordance correlation coefficient [CCC]). When comparing concentrations by sample, bead-based immunoassays generally yielded higher antibody concentrations than the ELISA and had higher variability for serotypes 6B, 18C, and 23F. None of the three assays met the current WHO recommendation of 75% of sera falling within 40% of the assigned antibody concentrations for all seven serotypes. When compared by serotype, the CDC and HPA tests were equivalent for five of seven serotypes, whereas the Luminex assay was equivalent for four of seven serotypes. When overall mean IgG concentrations were compared by laboratory, a higher level of agreement (CCC close to 1) was found among bead-based immunoassays than between the assays and WHO assignments. When compared to WHO assignments, the HPA assay outperformed the other assays (r = 0.920; CCC = 0.894; coefficient of accuracy = 0.972). Additional testing with sera from immunogenicity studies should demonstrate the applicability of this methodology for vaccine evaluation.

Transmission of 2009 Pandemic Influenza A (H1N1) Virus among Healthcare Personnel—Southern California, 2009

OBJECTIVE In April 2009, 2009 pandemic influenza A (H1N1) (hereafter, pH1N1) virus was identified in California, which caused widespread illness throughout the United States. We evaluated pH1N1 transmission among exposed healthcare personnel (HCP) and assessed the use and effectiveness of personal protective equipment (PPE) early in the outbreak. DESIGN Cohort study. SETTING Two hospitals and 1 outpatient clinic in Southern California during March 28-April 24, 2009. PARTICIPANTS Sixty-three HCP exposed to 6 of the first 8 cases of laboratory-confirmed pH1N1 in the United States. METHODS Baseline and follow-up questionnaires were used to collect demographic, epidemiologic, and clinical data. Paired serum samples were obtained to test for pH1N1-specific antibodies by microneutralization and hemagglutination-inhibition assays. Serology results were compared with HCP work setting, role, and self-reported PPE use. RESULTS Possible healthcare-associated pH1N1 transmission was identified in 9 (14%) of 63 exposed HCP; 6 (67%) of 9 seropositive HCP had asymptomatic infection. The highest attack rates occurred among outpatient HCP (6/19 [32%]) and among allied health staff (eg, technicians; 8/33 [24%]). Use of mask or N95 respirator was associated with remaining seronegative (P = .047). Adherence to PPE recommendations for preventing transmission of influenza virus and other respiratory pathogens was inadequate, particularly in outpatient settings. CONCLUSIONS pH1N1 transmission likely occurred in healthcare settings early in the pandemic associated with inadequate PPE use. Organizational support for a comprehensive approach to infectious hazards, including infection prevention training for inpatient- and outpatient-based HCP, is essential to improve HCP and patient safety.

Influenza A(H1N1)pdm09 during air travel

The global spread of the influenza A(H1N1)pdm09 virus (pH1N1) associated with travelers from North America during the onset of the 2009 pandemic demonstrates the central role of international air travel in virus migration. To characterize risk factors for pH1N1 transmission during air travel, we investigated travelers and airline employees from four North American flights carrying ill travelers with confirmed pH1N1 infection. Of 392 passengers and crew identified, information was available for 290 (74%) passengers were interviewed. Overall attack rates for acute respiratory infection and influenza-like illness 1-7 days after travel were 5.2% and 2.4% respectively. Of 43 individuals that provided sera, 4 (9.3%) tested positive for pH1N1 antibodies, including 3 with serologic evidence of asymptomatic infection. Investigation of novel influenza aboard aircraft may be instructive. However, beyond the initial outbreak phase, it may compete with community-based mitigation activities, and interpretation of findings will be difficult in the context of established community transmission.

Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction

Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824–135,982 for 5x Omni, and 168–6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0–99.9%, 97.5–99.9%, and 92.9–99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.

A real-time polymerase chain reaction for the detection of Streptococcus pneumoniae in blood using a mouse model: a potential new “gold standard”

Better diagnostics for pneumococcal disease are urgently needed. In a murine model, real-time polymerase chain reaction was superior to conventional culture in detecting pneumococcus in blood, particularly in early disease and after antibiotic administration, and could distinguish between commensalism and infection.

Concomitant administration of recombinant PsaA and PCV7 reduces Streptococcus pneumoniae serotype 19A colonization in a murine model.

A murine colonization model was used to determine the effect of co-administering 7-valent polysaccharide-protein conjugate vaccine and pneumococcal surface adhesin A. Mice were challenged intranasally with either PCV7 serotypes, 4 or 14, or a non-PCV7 serotype, 19A. Post-challenge samples were evaluated for IgG antibody levels, opsonophagocytic activity, and nasopharyngeal colonization. No interference was observed between immune responses from the concomitant and individual immunizations. Concomitant immunizations reduced carriage for tested serotypes; largest reduction was observed for 19A. From these mouse studies, co-administering pneumococcal antigens appear to expand coverage and reduce colonization against a non-PCV7 serotype without inhibiting immunogenicity to other serotypes.

Meningococcal carriage among a university student population-United States, 2015

OBJECTIVES Several outbreaks of serogroup B meningococcal disease have occurred among university students in recent years. In the setting of high coverage of the quadrivalent meningococcal conjugate vaccine and prior to widespread use of serogroup B meningococcal vaccines among adolescents, we conducted surveys to characterize the prevalence and molecular characteristics of meningococcal carriage among university students. METHODS Two cross-sectional oropharyngeal carriage surveys were conducted among undergraduates at a Rhode Island university. Isolates were characterized using slide agglutination, real-time polymerase chain reaction (rt-PCR), and whole genome sequencing. Adjusted prevalence ratios and 95% confidence intervals were calculated using Poisson regression to determine risk factors for carriage. RESULTS A total of 1837 oropharyngeal specimens were obtained from 1478 unique participants. Overall carriage prevalence was 12.7-14.6% during the two survey rounds, with 1.8-2.6% for capsular genotype B, 0.9-1.0% for capsular genotypes C, W, or Y, and 9.9-10.8% for nongroupable strains by rt-PCR. Meningococcal carriage was associated with being male, smoking, party or club attendance, recent antibiotic use (inverse correlation), and recent respiratory infections. CONCLUSIONS In this university setting, the majority of meningococcal carriage was due to nongroupable strains, followed by serogroup B. Further evaluation is needed to understand the dynamics of serogroup B carriage and disease among university students.