Estimating the burden of vaccine preventable lower respiratory tract disease in primary care, UK: protocol for a prospective surveillance study (AvonCAP GP2).

BACKGROUND: The true burden of acute lower respiratory tract diseases (aLRTD; includes acute lower respiratory tract infection, acute exacerbation of pre-existing heart failure and chronic lung disease) among adults presenting to primary care, and the proportion that are potentially vaccine preventable, is unknown. AIMS: To describe aLRTD incidence in adults presenting to primary care; estimate proportions caused by RSV, SARS-CoV-2 and pneumococcus; and investigate disease burden from patient and NHS perspectives. DESIGN & SETTING: Primary care prospective cohort study conducted in six representative General Practices (total Ì´83 000 registered adults) in Bristol, UK. METHOD: Adults (aged≥18 years) registered at participating General Practices and presenting to primary care (in-hours or out-of-hours) or emergency department (if not admitted) with aLRTD will be eligible and identified by real-time primary care record searches. Researchers will screen electronic GP records, including free text, contact patients to assess eligibility, and offer enrolment in a surveillance study and an enhanced diagnostic study (urine, saliva and respiratory samples; physical examination; and symptom diaries). Data will be collected for all aLRTD episodes, with patients assigned to one of three arms: surveillance, embedded diagnostic, and descriptive dataset. Outcome measures will include clinical and pathogen defined aLRTD incidence rates, symptom severity and duration, NHS contacts and costs, health-related quality of life changes, and mortality (≤30 days post identification). CONCLUSION: This comprehensive surveillance study of adults presenting to primary care with aLRTD, with embedded detailed data and sample collection, will provide an accurate assessment of aLRTD burden due to vaccine preventable infections.

Prolonged T-cell activation and long COVID symptoms independently associate with severe COVID-19 at 3 months.

Coronavirus disease-19 (COVID-19) causes immune perturbations which may persist long term, and patients frequently report ongoing symptoms for months after recovery. We assessed immune activation at 3-12 months post hospital admission in 187 samples from 63 patients with mild, moderate, or severe disease and investigated whether it associates with long COVID. At 3 months, patients with severe disease displayed persistent activation of CD4+ and CD8+ T-cells, based on expression of HLA-DR, CD38, Ki67, and granzyme B, and elevated plasma levels of interleukin-4 (IL-4), IL-7, IL-17, and tumor necrosis factor-alpha (TNF-α) compared to mild and/or moderate patients. Plasma from severe patients at 3 months caused T-cells from healthy donors to upregulate IL-15Rα, suggesting that plasma factors in severe patients may increase T-cell responsiveness to IL-15-driven bystander activation. Patients with severe disease reported a higher number of long COVID symptoms which did not however correlate with cellular immune activation/pro-inflammatory cytokines after adjusting for age, sex, and disease severity. Our data suggests that long COVID and persistent immune activation may correlate independently with severe disease.

Evaluation and deployment of isotype-specific salivary antibody assays for detecting previous SARS-CoV-2 infection in children and adults.

BACKGROUND: Saliva is easily obtainable non-invasively and potentially suitable for detecting both current and previous SARS-CoV-2 infection, but there is limited evidence on the utility of salivary antibody testing for community surveillance. METHODS: We established 6 ELISAs detecting IgA and IgG antibodies to whole SARS-CoV-2 spike protein, to its receptor binding domain region and to nucleocapsid protein in saliva. We evaluated diagnostic performance, and using paired saliva and serum samples, correlated mucosal and systemic antibody responses. The best-performing assays were field-tested in 20 household outbreaks. RESULTS: We demonstrate in test accuracy (N = 320), spike IgG (ROC AUC: 95.0%, 92.8-97.3%) and spike IgA (ROC AUC: 89.9%, 86.5-93.2%) assays to discriminate best between pre-pandemic and post COVID-19 saliva samples. Specificity was 100% in younger age groups (0-19 years) for spike IgA and IgG. However, sensitivity was low for the best-performing assay (spike IgG: 50.6%, 39.8-61.4%). Using machine learning, diagnostic performance was improved when a combination of tests was used. As expected, salivary IgA was poorly correlated with serum, indicating an oral mucosal response whereas salivary IgG responses were predictive of those in serum. When deployed to household outbreaks, antibody responses were heterogeneous but remained a reliable indicator of recent infection. Intriguingly, unvaccinated children without confirmed infection showed evidence of exposure almost exclusively through specific IgA responses. CONCLUSIONS: Through robust standardisation, evaluation and field-testing, this work provides a platform for further studies investigating SARS-CoV-2 transmission and mucosal immunity with the potential for expanding salivo-surveillance to other respiratory infections in hard-to-reach settings.

If a person has been previously infected with SARS-CoV-2 they will produce specific proteins, called antibodies. These are present in the saliva and blood. Saliva is easier to obtain than blood, so we developed and evaluated six tests that detect SARS-CoV-2 antibodies in saliva in children and adults. Some tests detected antibodies to a particular protein made by SARS-CoV-2 called the spike protein, and these tests worked best. The most accurate results were obtained by using a combination of tests. Similar tests could also be developed to detect other respiratory infections which will enable easier identification of infected individuals.

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2.

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

Impact of meningococcal ACWY conjugate vaccines on pharyngeal carriage in adolescents: evidence for herd protection from the UK MenACWY programme.

OBJECTIVE: Serogroup W and Y invasive meningococcal disease increased globally from 2000 onwards. Responding to a rapid increase in serogroup W clonal complex 11 (W:cc11) invasive meningococcal disease, the UK replaced an adolescent booster dose of meningococcal C conjugate vaccine with quadrivalent MenACWY conjugate vaccine in 2015. By 2018, the vaccine coverage in the eligible school cohorts aged 14 to 19 years was 84%. We assessed the impact of the MenACWY vaccination programme on meningococcal carriage. METHODS: An observational study of culture-defined oropharyngeal meningococcal carriage prevalence before and after the start of the MenACWY vaccination programme in UK school students, aged 15 to 19 years, using two cross-sectional studies: 2014 to 2015 "UKMenCar4" and 2018 "Be on the TEAM" (ISRCTN75858406). RESULTS: A total of 10 625 participants preimplementation and 13 438 postimplementation were included. Carriage of genogroups C, W, and Y (combined) decreased from 2.03 to 0.71% (OR 0.34 [95% CI 0.27-0.44], p < 0.001). Carriage of genogroup B meningococci did not change (1.26% vs 1.23% [95% CI 0.77-1.22], p = 0.80) and genogroup C remained rare (n = 7/10 625 vs 17/13 438, p = 0.135). The proportion of serogroup positive isolates (i.e. those expressing capsule) decreased for genogroup W by 53.8% (95% CI -5.0 - 79.8, p = 0.016) and for genogroup Y by 30.1% (95% CI 8.946·3, p = 0.0025). DISCUSSION: The UK MenACWY vaccination programme reduced carriage acquisition of genogroup and serogroup Y and W meningococci and sustained low levels of genogroup C carriage. These data support the use of quadrivalent MenACWY conjugate vaccine for indirect (herd) protection.

Outbreak strain characterisation and pharyngeal carriage detection following a protracted group B meningococcal outbreak in adolescents in South-West England.

Between April 2016 and September 2017, four cases of group B meningococcal disease were reported among sixth-form college students in Bristol, UK. Culture and non-culture whole genome sequencing was utilised and demonstrated that the four genomes of the responsible ST-41 strains clustered closely on a sub-lineage of ST-41/44 clonal complex. The outbreak resulted in two fatalities. A distinct social group associated with one of the cases was selected for vaccination with 4CMenB and pharyngeal swabbing. In vitro culturing, multiple real-time PCR assays (sodC, ctrA and siaDB) and a PorA PCR-sequencing assay were used to detect meningococcal colonisation and a carriage rate of 32.6% was observed. Furthermore, a high proportion of the pharyngeal swabs (78.3%) yielded a Factor H-Binding Protein (fHbp) nucleotide allele suggesting that the antigenic gene is prevalent among non-meningococcal flora, most likely Neisseria commensals. This may have implications for fHbp as a vaccine antigen should it be shown to influence bacterial colonisation.

In Situ Hybridisation Study of Neuronal Neuropeptides Expression in Models of Mandibular Denervation with or without Inflammation: Injury Dependant Neuropeptide Plasticity.

Neuronal expression of neuropeptides is altered following peripheral tissue injury associated with inflammation or nerve injury. This results in neuropathic pain with or without neurogenic inflammation which is a major health problem regularly seen in trigeminal neuralgia. Activation of the trigeminal system results in the release of vasoactive neuropeptides substance P and Calcitonin Gene-related Peptide (CGRP) which contribute to nociception, pain and neurogenic inflammation in injured tissues. AIM: To study the alterations in the neuronal neuropeptides expressions in models of tissue injury associated with either nerve injury or with inflammation and to determine if denervation would alter the neuronal response to inflammation. MATERIAL AND METHODS: Experiments were performed on rat mandibles to produce three models. Firstly, denervation model by sectioning one of the mandibular nerve branches (inferior alveolar nerve). Secondarily, inflammation model by intra-gingival injection of lipopolysaccharide (LPS). Thirdly, combined denervation and inflammation model by sectioning the nerve with subsequent LPS injection. The animals were sacrificed seven days postoperative. Trigeminal ganglia on the operated sides were processed for in situ hybridisation for neuropeptides; substance P and CGRP mRNAs. Images were analysed for morphological and morphometric analysis using Image J software. RESULTS: substance P and CGRP mRNAs were expressed in small and medium-size primary afferent neurons in the mandibular division of the trigeminal ganglia. Both the denervation and the inflammation models showed alteration in neuropeptides expression in the sensory primary afferent neurons innervating the affected mandibular tissues. While, denervation resulted in a significant (substance P=P<0.04, CGRP=P<0.01) downregulation contrarily, inflammation resulted in a significant (P<0.001) upregulation of neuropeptides' mRNAs. Interestingly, denervation prior to induction of inflammation resulted in insignificant changes in neuropeptides levels. There was a strong correlation (Pearson Correlation=0.8) between substance P and CGRP expression. CONCLUSION: We show that tissue damage associated with nerve injury or inflammation results in alteration of neuropeptides levels in the innervating primary afferent neurons. Tissue destruction associated with chronic inflammatory condition such as arthritis and periodontitis are believed to be due to the production of neuromodulators causing neurogenic inflammation. Here we show that denervation abolishes the neuronal response to inflammation. Therefore, tissues denervation could relieve neurogenic inflammation associated with chronic disorders through regulation of neuronal neuropeptide production. Moreover, the current model that combined denervation and inflammation provides a useful animal model to study the contribution of nerve-related mediators in the pathophysiology of tissue injury.

High-density Bacterial Nasal Carriage in Children Is Transient and Associated With Respiratory Viral Infections-Implications for Transmission Dynamics.

BACKGROUND: This longitudinal study describes the associations between respiratory viral infections, rhinitis and the prevalence and density of the common nasopharyngeal bacterial colonizers, Streptococcus pneumoniae (Sp), Moraxella catarrhalis (Mc), Haemophilus influenzae (Hi) and Staphylococcus aureus. METHODS: In an observational cohort study, 161 children attending day care centers in Bristol, United Kingdom, were recruited. Monthly nasopharyngeal swabs were taken and stored frozen in Skim-milk, tryptone, glucose and glycerin broth (STGG) broth. Quantitative polymerase chain reaction was used for detection of respiratory viruses and 4 bacterial species. t tests and logistic regression models were used for analysis. RESULTS: The frequent colonisers, Sp, Mc and Hi were more frequently found at high density in contrast to Staphylococcus aureus although temporally, high-density carriage was short lived. Respiratory viral infections and symptoms of rhinitis were both independently and consistently associated with higher bacterial density with an observed 2-fold increase in density for Sp, Mc and Hi (P = 0.004-0.017). CONCLUSIONS: For Sp and Hi, the association between young age and higher bacterial DNA density was explained by more frequent viral infection and increased nasal discharge, while the associations between some viral specie's and some bacterial species' density appear to be stronger than others. Increased colonization density and rhinitis may promote transmission of these commonly carried organisms.

Viable Neisseria meningitidis is commonly present in saliva in healthy young adults: Non-invasive sampling and enhanced sensitivity of detection in a follow-up carriage study in Portuguese students.

INTRODUCTION AND AIMS: Improved sensitivity and efficiency of detection and quantification of carriage of Neisseria meningitidis (Nm) in young people is important for evaluation of the impact of vaccines upon transmission and associated population-wide effects. Saliva collection is quick, non-invasive and facilitates frequent sampling, but has been reported to yield low sensitivity by culture. We re-evaluated this approach in a follow-up cross sectional study using direct and culture-amplified PCR. MATERIAL/METHODS: In April 2016 we collected paired oropharyngeal swabs (OPS) and saliva samples from 1005 healthy students in Portugal into STGG broth and stored them at -80°C until DNA extraction and batched qPCR analysis. Samples were also cultured on GC agar plates for 72h and PCR done on DNA extracts from overall growth. Nm isolates were also sought from a selection of 50 samples. qPCR amplification targets were superoxide dismutase sodC and capsular locus/genogroup-specific genes (B, C, W, X and Y) and, for cultured isolates only, porA. Cycle threshold values of ≤36 were considered positive. RESULTS: 556 tests (460 samples, 363 subjects, 36.1%) were positive for Nm (sodC) and 65 (45, 36, 3.6%) for MenB. More salivas were positive by direct sodC qPCR (211, 21.0%) than OPS (126, 12.5%) but fewer were positive by culture-amplified qPCR (94 vs. 125). For both sample types, many that were negative on direct qPCR came positive on culture-amplification and Nm was consistently isolated from salivas in which culture amplified the PCR signal. Using both methods on both samples yielded 36.1% Nm and 5.5% encapsulated Nm carriage rates while direct qPCR on OPS alone detected 12.5% and 2.2%. CONCLUSIONS: Detectable MenB carriage rates (2.9%) were lower than 4 years earlier (6.8%) in this population (p = 0.0003). Viable meningococci were often present in saliva. Although evidence of encapsulated Nm was less frequent in saliva than OPS, collection is more acceptable to subjects allowing more frequent sampling. Use of culture-amplification increases detection sensitivity in both sample types, especially when combined with direct PCR. Combining these samples and/or methodologies could greatly enhance the power of carriage studies to detect the impact of vaccines upon carriage and transmission.

Memory B cell response to a PCV-13 booster in 3.5year old children primed with either PCV-7 or PCV-13.

Pneumococcal protein-polysaccharide conjugate vaccines provide direct protection against Streptococcus pneumoniae through the induction of persistent anti-polysaccharide antibodies, and by priming for a rapid secondary antibody response. Memory B cells (BMEM) generated during an initial immune response are responsible for both the more rapid and quantitatively greater secondary antibody response and are also thought to contribute to the ongoing production of plasma cells providing long-term antibody persistence. We recruited 3.5-year-old children who had participated in a previous clinical trial comparing infant immunization with either a 7-valent (PCV-7) or a 13-valent pneumococcal conjugate vaccine (PCV-13) to investigate whether prior priming with pneumococcal antigens influences BMEM responses. Blood was taken before and 1month after a PCV-13 booster. BMEM were quantified using a cultured ELISpot assay for pneumococcal serotypes 1, 3, 4, 14, 19A, 23F, and with diphtheria and tetanus toxoid as controls, and then correlated with serotype-specific IgG concentrations and opsonophagocytic activity (OPA) titers. In total, blood samples from 62 participants were available for analysis. Serotype-specific BMEM frequencies were generally low at baseline (before boost) although for serotypes 14 and 3, they were significantly higher in children primed with PCV-13 than PCV-7 primed children. Following the PCV-13 booster, BMEM frequencies increased and were not different between the groups for all serotypes. A strong inverse correlation was found between antibody concentrations and OPA titers at baseline and BMEM following booster vaccination for serotype 3 but not for other serotypes suggesting that, for this serotype, pre-existing serotype-specific antibodies may inhibit BMEM formation in response to vaccination. Clinicaltrials.gov registration number: NCT01095471.

Pneumococcal Serotypes Colonise the Nasopharynx in Children at Different Densities.

Prevalence of pneumococcal serotypes in carriage and disease has been described but absolute serotype colonisation densities have not been reported. 515 paediatric nasal swab DNA extracts were subjected to lytA qPCR and molecular serotyping by microarray. Absolute serotype densities were derived from total pneumococcal density (qPCR cycle threshold and standard curve) and relative abundance (microarray) and varied widely. Compared to all serotype densities observed, the strongest evidence of differences was seen for serotypes 21 and 35B (higher) and 3, 38 and non-typeables (lower) (p<0.05) with a similar hierarchy when only a single serotype carriage was assessed. There was no evidence of any overall density differences between children with single or multiple serotypes detected but serotypes with mid-range densities were more prevalent. The hierarchy of distinct pneumococcal serotype carriage densities described here for the first time, may help explain the dynamics of transmission between children.

Density Distribution of Pharyngeal Carriage of Meningococcus in Healthy Young Adults: New Approaches to Studying the Epidemiology of Colonization and Vaccine Indirect Effects.

BACKGROUND: Improved understanding of Neisseria meningitidis (Nm) carriage biology and better methods for detection and quantification would facilitate studies of potential impact of new vaccines on colonization and transmission in adolescents. METHODS: We performed plate cultures on 107 oropharyngeal swabs stored frozen in skim milk tryptone glucose glycerol (STGG) broth and previously positive for Nm. We compared quantitative polymerase chain reaction (qPCR) detection of Nm in 601 STGG-swabs with culture. Using qPCR (n = 87), a log-phase broth culture standard curve and semiquantitative plate cultures (n = 68), we measured density of carriage. We compared qPCR genogrouping of DNA extracts from STGG-swabs and from plate culture lawns (n = 110) with purified isolates (n = 80). RESULTS: Swab storage resulted in only 10% loss of culture sensitivity. Direct sodC qPCR Nm detection yielded more positives (87/601, 14.5%) than culture (80/601, 13.3%). Most samples (57/110) positive by culture were also positive by qPCR and vice versa, but discrepancies (single positives) were frequent among low-density samples. sodC qPCR was positive in 79/80 isolates but in only 65 by ctrA qPCR. Density both by culture and qPCR varied across 4 orders of magnitude with the majority being low (<50 bacteria-gene copies/mL) and a minority being high (>1000). Genogrouping qPCRs yielded more positive results when performed on DNA extracts from lawn cultures. CONCLUSIONS: We provide the first description of the distribution of Nm carriage density. This could be important for understanding transmission dynamics and population-level effectiveness of adolescent vaccine programs. Storage of swabs frozen in STGG for batched laboratory analysis facilitates carriage studies and direct sodC qPCR for Nm combined with qPCR genogrouping of lawn culture extracts provides accurate, detailed description of colonization.

The Effects of Live Attenuated Influenza Vaccine on Nasopharyngeal Bacteria in Healthy 2 to 4 Year Olds. A Randomized Controlled Trial.

RATIONALE: Viral infections of the upper respiratory tract may influence the commensal nasopharyngeal bacteria. Changes in the bacterial niche could affect transmission dynamics. Attenuated vaccine viruses can be used to investigate this empirically in humans. OBJECTIVES: To study the effects of mild viral upper respiratory infections on nasopharyngeal bacterial colonization using live attenuated influenza vaccine (LAIV) as a surrogate. METHODS: We used trivalent LAIV to evaluate the effects of viral infection on bacterial carriage and density of Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, and Staphylococcus aureus. A total of 151 healthy children were randomized 1:1 to receive the vaccine starting either at recruitment (n = 74) or 28 days later (n = 77) in a stepped wedge fashion, allowing comparisons between recipients and nonrecipients as well as whole-group comparisons pre- and postvaccination. Bacterial carriage and density were determined using quantitative polymerase chain reaction assays. MEASUREMENTS AND MAIN RESULTS: A total of 151 children were recruited, 77 in the LAIV group and 74 in the control group. LAIV recipients (n = 63 analyzed) showed an apparent transient increase in H. influenzae carriage but no further significant differences in carriage prevalence of the four bacterial species compared with controls (n = 72 analyzed). S. pneumoniae density was substantially higher in vaccine recipients (16,687 vs. 1935 gene copies per milliliter) 28 days after the first dose (P < 0.001). Whole-group multivariable analysis (prevaccine, after one dose, and after two doses) also showed increases in density of other species and H. influenzae carriage prevalence. CONCLUSIONS: In the absence of any safety signals despite widespread use of the vaccine, these findings suggest that bacterial density, and thus transmission rates among children and to people in other age groups, may rise following attenuated influenza infections without associated clinical disease. LAIV could therefore be used as an experimental tool to elucidate the dynamics of transmission of nasopharyngeal bacteria.

Population density profiles of nasopharyngeal carriage of 5 bacterial species in pre-school children measured using quantitative PCR offer potential insights into the dynamics of transmission.

Bacterial vaccines can reduce carriage rates. Colonization is usually a binary endpoint. Real time quantitative PCR (qPCR) can quantify bacterial DNA in mucosal samples over a wide range. Using culture and single-gene species-specific qPCRs for Streptococcus pneumoniae (lytA), Streptococcus pyogenes (ntpC), Moraxella catarrhalis (ompJ), Haemophilus influenzae (hdp) and Staphylococcus aureus (nuc) and standard curves against log-phase reference strain broth cultures we described frequency and peak density distributions of carriage in nasopharyngeal swabs from 161 healthy 2-4 y old children collected into STGG broth. In general, detection by qPCR and culture was consistent. Discordance mostly occurred at lower detection thresholds of both methods, although PCR assays for S. pyogenes and S. aureus were less sensitive. Density varied across 5-7 orders of magnitude for the 5 species with the abundant species skewed toward high values (modes: S. pneumoniae log3-4, M. catarrhalis & H. influenzae log4-5 CFU/ml broth). Wide ranges of bacterial DNA concentrations in healthy children carrying these bacteria could mean that different individuals at different times vary greatly in infectiousness. Understanding the host, microbial and environmental determinants of colonization density will permit more accurate prediction of vaccine effectiveness.

Oropharyngeal Carriage of Meningococcus in Portugal by Group and Clonal Complex 6 Years After Adolescent Vaccine Campaign.

Portugal introduced (2+1) conjugate Meningococcal group C vaccine in 2006 with high coverage catch up to 18 years and has given only 1 dose at 1 year since 2012. Among 601 student oropharyngeal swabs, meningococcal carriage rate was 13.3% (A-0%, B-5.3%, C-0.3%, W-0.2%, X-0.2% and Y-1.7%). C and W strains were of potentially disease-causing clonal complexes (cc) but not the hyperinvasive cc11.

Meningococcal carriage in adolescents in the United Kingdom to inform timing of an adolescent vaccination strategy.

OBJECTIVES: Recent development of serogroup B meningococcal (MenB) vaccines highlights the importance of pharyngeal carriage data, particularly in adolescents and young adults, to inform implementation strategies. We describe current UK carriage prevalence in this high risk population and compare methods of carriage detection. METHODS: In this multisite study, pharyngeal swabs were collected on 3-4 occasions over 6-12 months, from 1040 school and university students, aged 10-25 years. Meningococcal carriage was detected by standard culture combined with seroagglutination or PCR of cultured isolates, or by direct PCR from swab. The factor H binding protein (fHBP) variants present in meningococcal isolates were determined. RESULTS: Meningococcal serogroups B and Y were most common, with carriage up to 6.5% and 5.5% respectively, increasing throughout adolescence. Identification by seroagglutination was often unreliable, and the sensitivity of direct PCR detection was 66% compared to culture combined with PCR. Of MenB isolates, 89.1% had subfamily A variants of fHBP. The acquisition rate of MenB carriage was estimated at 2.8 per 1000 person-months. CONCLUSIONS: If vaccination is to precede the adolescent rise in MenB carriage, these data suggest it should take place in early adolescence. Studies assessing vaccine impact should use molecular methods to detect carriage.

Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot.

The ELISpot assay is used in vaccine studies for the quantification of antigen-specific memory B cells (B(MEM)), and can be performed using cryopreserved samples. The effects of cryopreservation on B(MEM) detection and the consistency of cultured ELISpot assays when performed by different operators or laboratories are unknown. In this study, blood was taken from healthy volunteers, and a cultured ELISpot assay was used to count B(MEM) specific for 2 routine vaccine antigens (diphtheria and tetanus toxoid). Results were assessed for intra- and inter-operator variation, and the effects of cryopreservation. Cryopreserved samples were shipped to a second laboratory in order to assess inter-laboratory variation. B(MEM) frequencies were very strongly correlated when comparing fresh and frozen samples processed by the same operator, and were also very strongly correlated when comparing 2 operators in the same laboratory. Results were slightly less consistent when samples were processed in different laboratories but correlation between the 2 measurements was still very strong. Although cell viability was reduced in some cryopreserved samples due to higher temperatures during transportation, B(MEM) could still be quantified. These results demonstrate the reproducibility of the ELISpot assay across operators and laboratories, and support the use of cryopreserved samples in future B(MEM) studies.

Effect of a quadrivalent meningococcal ACWY glycoconjugate or a serogroup B meningococcal vaccine on meningococcal carriage: an observer-blind, phase 3 randomised clinical trial.

BACKGROUND: Meningococcal conjugate vaccines protect individuals directly, but can also confer herd protection by interrupting carriage transmission. We assessed the effects of meningococcal quadrivalent glycoconjugate (MenACWY-CRM) or serogroup B (4CMenB) vaccination on meningococcal carriage rates in 18-24-year-olds. METHODS: In this phase 3, observer-blind, randomised controlled trial, university students aged 18-24 years from ten sites in England were randomly assigned (1:1:1, block size of three) to receive two doses 1 month apart of Japanese Encephalitis vaccine (controls), 4CMenB, or one dose of MenACWY-CRM then placebo. Participants were randomised with a validated computer-generated random allocation list. Participants and outcome-assessors were masked to the treatment group. Meningococci were isolated from oropharyngeal swabs collected before vaccination and at five scheduled intervals over 1 year. Primary outcomes were cross-sectional carriage 1 month after each vaccine course. Secondary outcomes included comparisons of carriage at any timepoint after primary analysis until study termination. Reactogenicity and adverse events were monitored throughout the study. Analysis was done on the modified intention-to-treat population, which included all enrolled participants who received a study vaccination and provided at least one assessable swab after baseline. This trial is registered with ClinicalTrials.gov, registration number NCT01214850. FINDINGS: Between Sept 21 and Dec 21, 2010, 2954 participants were randomly assigned (987 assigned to control [984 analysed], 979 assigned to 4CMenB [974 analysed], 988 assigned to MenACWY-CRM [983 analysed]); 33% of the 4CMenB group, 34% of the MenACWY-CRM group, and 31% of the control group were positive for meningococcal carriage at study entry. By 1 month, there was no significant difference in carriage between controls and 4CMenB (odds ratio 1·2, 95% CI 0·8-1·7) or MenACWY-CRM (0·9, [0·6-1·3]) groups. From 3 months after dose two, 4CMenB vaccination resulted in significantly lower carriage of any meningococcal strain (18·2% [95% CI 3·4-30·8] carriage reduction), capsular groups BCWY (26·6% [10·5-39·9] carriage reduction), capsular groups CWY (29·6% [8·1-46·0] carriage reduction), and serogroups CWY (28·5% [2·8-47·5] carriage reduction) compared with control vaccination. Significantly lower carriage rates were also noted in the MenACWY-CRM group compared with controls: 39·0% (95% CI 17·3-55·0) carriage reduction for serogroup Y and 36·2% (15·6-51·7) carriage reduction for serogroup CWY. Study vaccines were generally well tolerated, with increased rates of transient local injection pain and myalgia in the 4CMenB group. No safety concerns were identified. INTERPRETATION: Although we detected no significant difference between groups at 1 month after vaccine course, MenACWY-CRM and 4CMenB vaccines reduced meningococcal carriage rates during 12 months after vaccination and therefore might affect transmission when widely implemented. FUNDING: Novartis Vaccines.

Multiple Streptococcus pneumoniae serotypes in aural discharge samples from children with acute otitis media with spontaneous otorrhea.

Among 55 children with cultures positive for acute otitis media with spontaneous otorrhea, 28 (51%) had cultures positive for aural Streptococcus pneumoniae, and in 10 of these, two distinct strains were detected, in which 5 had pairs of strains that were both capsule-bearing serotypes. Such cases were more likely to have cultures positive for other otopathogens than those with only one pneumococcus present.