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Highlights • Influenza-like illness (ILI) in children is associated with high pneumococcal carriage density. • Children with ILI harboured more viral organisms than asymptomatic healthy children. • Children with ILI patients had higher IL-8 levels in nasal aspirates than asymptomatic healthy children., Summary Background High pneumococcal carriage density is a risk factor for invasive pneumococcal disease (IPD) and transmission, but factors that increase pneumococcal carriage density are still unclear. Methods We undertook a cross-sectional study to evaluate the microbial composition, cytokine levels and pneumococcal carriage densities in samples from children presenting with an influenza-like illness (ILI) and asymptomatic healthy controls (HC). Results The proportion of children harbouring viral organisms (Relative risk (RR) 1.4, p = 0.0222) or ≥ 4 microbes at a time (RR 1.9, p

Streptococcus pneumoniae is the main bacterial pathogen involved in pneumonia. Pneumococcal acquisition and colonization density is probably affected by viral co-infections, the local microbiome composition and mucosal immunity. Here, we report the interactions between live-attenuated influenza vaccine (LAIV), successive pneumococcal challenge, and the healthy adult nasal microbiota and mucosal immunity using an experimental human challenge model. Nasal microbiota profiles at baseline are associated with consecutive pneumococcal carriage outcome (non-carrier, low-dense and high-dense pneumococcal carriage), independent of LAIV co-administration. Corynebacterium/Dolosigranulum-dominated profiles are associated with low-density colonization. Lowest rates of natural viral co-infection at baseline and post-LAIV influenza replication are detected in the low-density carriers. Also, we detected the fewest microbiota perturbations and mucosal cytokine responses in the low-density carriers compared to non-carriers or high-density carriers. These results indicate that the complete respiratory ecosystem affects pneumococcal behaviour following challenge, with low-density carriage representing the most stable ecological state., Using a Streptococcus pneumoniae human challenge model, the authors here show that baseline nasal microbiota profiles are associated with pneumococcal acquisition and density, independent of live-attenuated influenza vaccine (LAIV) administration, and with mucosal cytokine responses to LAIV.

T Follicular helper cells (TFH) are considered critical for B cell antibody response, and recent efforts have focused on promoting TFH in order to enhance vaccine efficacy. We studied the frequency and function of TFH in nasopharynx-associated lymphoid tissues (NALT) from children and adults, and its role in anti-influenza antibody response following stimulation by a live-attenuated influenza vaccine (LAIV) or an inactivated seasonal virus antigen (sH1N1). We further studied whether CpG-DNA promotes TFH and by which enhances anti-influenza response. We showed NALT from children aged 1.5-10 years contained abundant TFH, suggesting efficient priming of TFH during early childhood. Stimulation by LAIV induced a marked increase in TFH that correlated with a strong production of anti-hemagglutinin (HA) IgA/IgG/IgM antibodies in tonsillar cells. Stimulation by the inactivated sH1N1 antigen induced a small increase in TFH which was markedly enhanced by CpG-DNA, accompanied by enhanced anti-HA antibody responses. In B cell co-culture experiment, anti-HA responses were only seen in the presence of TFH, and addition of plasmacytoid dendritic cell to TFH-B cell co-culture enhanced the TFH-mediated antibody production following CpG-DNA and sH1N1 antigen stimulation. Induction of TFH differentiation from naïve T cells was also shown following the stimulation. Our results support a critical role of TFH in human mucosal anti-influenza antibody response. Use of an adjuvant such as CpG-DNA that has the capacity to promote TFH by which to enhance antigen-induced antibody responses in NALT tissue may have important implications for future vaccination strategies against respiratory pathogens.

Controlled human infection (CHI) trials, in which healthy volunteers are experimentally infected, can accelerate the development of novel drugs and vaccines for infectious diseases of global importance. The use of CHI models is expanding from around 60 studies in the 1970s to more than 120 publications in this decade, primarily for influenza, rhinovirus, and malaria. CHI trials have provided landmark data for several registered drugs and vaccines, and have generated unprecedented scientific insights. Because of their invasive nature, CHI studies demand critical ethical review according to established frameworks. CHI-associated serious adverse events are rarely reported. Novel CHI models need standardised safety data from comparable CHI models to facilitate evidence-based risk assessments, as well as funds to produce challenge inoculum according to regulatory requirements. Advances such as the principle of controlled colonisation, the expansion of models to endemic areas, and the use of genetically attenuated strains will further broaden the scope of CHI trials.

Data on the epidemiology of severe acute respiratory illness (SARI) in adults from low-income, high human immunodeficiency virus (HIV) prevalence African settings are scarce. We conducted adult SARI surveillance in Blantyre, Malawi. From January 2011 to December 2013, individuals aged ≥ 15 years with SARI (both inpatients and outpatients) were enrolled at a large teaching hospital in Blantyre, Malawi. Nasopharyngeal aspirates were tested for influenza and other respiratory viruses by polymerase chain reaction. We estimated hospital-attended influenza-positive SARI incidence rates and assessed factors associated with influenza positivity and clinical severity (Modified Early Warning Score > 4). We enrolled 1,126 SARI cases; 163 (14.5%) were positive for influenza. Human immunodeficiency virus prevalence was 50.3%. Annual incidence of hospital-attended influenza-associated SARI was 9.7-16.8 cases per 100,000 population. Human immunodeficiency virus was associated with a 5-fold greater incidence (incidence rate ratio 4.91, 95% confidence interval [CI]: 3.83-6.32). On multivariable analysis, female gender, as well as recruitment in hot, rainy season (December to March; adjusted odds ratios (aOR): 2.82, 95% CI: 1.57-5.06) and cool, dry season (April to August; aOR: 2.47, 95% CI: 1.35-4.15), was associated with influenza positivity, whereas influenza-positive patients were less likely to be HIV-infected (aOR: 0.59, 95% CI: 0.43-0.80) or have viral coinfection (aOR: 0.51, 95% CI: 0.36-0.73). Human immunodeficiency virus infection (aOR: 1.86; 95% CI: 1.35-2.56) and recruitment in hot, rainy season (aOR: 4.98, 95% CI: 3.17-7.81) were independently associated with clinical severity. In this high HIV prevalence population, influenza was associated with nearly 15% of hospital-attended SARI. Human immunodeficiency virus infection is an important risk factor for clinical severity in all-cause and influenza-associated SARI. Expanded access to HIV testing and antiretroviral treatment, as well as targeted influenza vaccination, may reduce the burden of SARI in Malawi and other high HIV prevalence settings.

In temperate countries, influenza and other viral respiratory diseases often have distinct seasonal peaks occurring during colder, wintertime months. However, little is known about the dynamics of influenza and viral respiratory disease dynamics in the tropics, despite high morbidity and a clear epidemiological link between tropical and temperate countries. In temperate countries, the dynamics of influenza and other respiratory diseases are often analyzed using syndromic surveillance data describing influenza-like illness (ILI) as ILI is highly correlated with virological surveillance for influenza. To obtain a detailed picture of respiratory disease incidence patterns in a large tropical city, we established an mHealth study in community outpatient clinics in Ho Chi Minh City, Vietnam (11N latitude). From August 2009 through December 2015, clinics reported daily case numbers of ILI using standard mobile-phone SMS messaging. A subset of these clinics performed molecular diagnostics for influenza A and B viruses. Unlike the annual patterns seen in temperate countries, ILI activity in Ho Chi Minh City exhibited strong non-annual periodicity and was not correlated with PCR-confirmed influenza. The dominant periodicity in the data was approximately 200 days. This was confirmed by a time series decomposition, a step-wise regression analysis on annual and non-annual covariates, and a forecasting exercise showing that forecasting was 30% to 40% more accurate when a 200-day non-annual cycle was included in the forecast. This suggests, for the first-time, that a non-annual cycle may be an essential driver of ILI dynamics in the tropics. This raises new questions about the seasonality and drivers of respiratory disease transmission in tropical countries.

There are no contemporary data available describing human immunity to novel influenza A/H7N9. Using 1723 prospectively collected serum samples in southern Vietnam, we tested for antibodies to 5 avian influenza virus antigens, using a protein microarray. General-population antibody titers against subtype H7 virus are higher than antibody titers against subtype H5 and lower than titers against H9. The highest titers were observed for human influenza virus subtypes. Titers to avian influenza virus antigens increased with age and with geometric mean antibody titer to human influenza virus antigens. There were no titer differences between the urban and the rural location in our study.

Seroepidemiological studies aim to understand population-level exposure and immunity to infectious diseases. Results from serological assays are normally presented as binary outcomes describing the presence or absence of pathogen-specific antibody, despite the fact that many assays measure continuous quantities. A population’s natural distribution of antibody titers to an endemic infectious disease may in fact include information on multiple serological states – e.g. naiveté, recent infection, non-recent infection – depending on the disease in question and the acquisition and waning patterns of host immunity. In this study, we investigate a collection of 20,152 general-population serum samples from southern Vietnam collected between 2009 and 2013 from which we report antibody titers to the influenza virus HA1 protein using a continuous titer measurement from a protein microarray assay. We describe the distributions of antibody titers to subtypes 2009 H1N1 and H3N2. Using a model selection approach to fit mixture distributions, we show that 2009 H1N1 antibody titers fall into four titer subgroups and that H3N2 titers fall into three subgroups. For H1N1, our interpretation is that the two highest-titer subgroups correspond to recent infection and historical infection, which is consistent with 2009 pandemic attack rates. For H3N2, observations censored at the highest titer dilutions make similar interpretations difficult to validate.

Background: \ud The UK Department of Health recommends annual influenza vaccination for healthcare workers, but uptake remains low. For staff, there is uncertainty about the rationale for vaccination and evidence underpinning the recommendation.\ud \ud Objectives: \ud Clarify the rationale, and evidence-base, for influenza vaccination of healthcare workers from the occupational health, employer, and patient safety perspectives.\ud \ud Design: \ud Systematic appraisal of published systematic reviews\ud \ud Results: \ud The quality of the 11 included reviews was variable; some included exactly the same trials but made conflicting recommendations. Three reviews assessed vaccine effects in healthcare workers and found one trial reporting a vaccine efficacy of 88%. Six reviews assessed vaccine effects in healthy adults and vaccine efficacy was consistent with a median of 62% (95% CI 56 to 67). Two reviews assessed effects on working days lost in healthcare workers (three trials), and three reported effects in healthy adults (four trials). The meta-analyses presented by the most recent reviews do not reach standard levels of statistical significance, but may be misleading as individual trials suggest benefit with wide variation in size of effect.\ud The 2013 Cochrane review reported absolute effects close to zero for laboratory-confirmed influenza, and hospitalization for patients, but excluded data on clinically-suspected influenza and all-cause mortality which had shown potentially important effects in previous editions. A more recent systematic review reports these effects as a 42% reduction in clinically-suspected influenza (95% CI 27 to 54), and a 29% reduction in all-cause mortality (95% CI 15 to 41).\ud \ud Conclusions: \ud The evidence for employer and patient safety benefits of influenza vaccination is not straightforward, and has been interpreted differently by different systematic review authors. Future uptake of influenza vaccination among healthcare workers may benefit from a fully transparent guideline process by a panel representing all relevant stakeholders, which clearly communicates the underlying rationale, evidence-base, and judgements made.

To the Editor—In this issue of the Journal and in a recent study, Pawar et al reported results of serologic tests performed on a high-risk group of 446 poultry workers and 162 individuals from the general population in Maharashtra and Jamshedpur states, India [1, 2]. None of the 608 samples tested positive for antibodies to influenza A virus subtypes H5N1 or H7N1 by hemagglutination-inhibition (HI) or microneutralization (MN) assays. None of the 162 individuals from the general population tested positive for H9N2 antibodies by HI or MN assay. A total of 4.7% and 10% of high-risk individuals in Pune and Jamshedpur, respectively, tested positive by the HI assay for influenza A(H9N2); 3.8% and 4.7%, respectively, had positive results of the MN assay. The authors suggest that this higher rate of seropositivity could be related to the circulation of influenza A(H9N2) in Jamshedpur in poultry, with resultant zoonotic spread to humans, and reference our publication showing presence of antibodies to avian influenza virus antigens in Vietnam. As noted by the authors, H9 titers were highest among all antibodies to avian influenza virus strains in our general population sample [3]. Other publications looking at high-risk individuals in South East Asia have demonstrated a higher seropositivity rate for influenza A(H9) strains, compared with influenza A(H5) and/or A(H7) strains [4–6], but this is not a consistent finding globally, even in high-risk groups [7, 8]. Vaccine studies conducted in locations thought to have a low risk of avian influenza exposure (ie, the United Kingdom and United States) found that up to a third of participants were seropositive for H9 [9–11]. Of those positive for H9 antibodies (>1:40 by the HI assay), a greater proportion were born before 1968, and it was postulated that this was related to cross-reactivity from influenza A virus subtype H2N2, which was circulating in humans from 1957 to 1968. Within our data set, there was a significantly higher seropositivity (and, accordingly, mean titer) for all avian strains in individuals born before 1968 (multivariate analysis of variance [MANOVA]: F = 4.7, P < .0005, Pillai 0.1256), with the H9 titer being the highest. However, when we investigate other birth year cutoffs (±20 years), we find the same trend, suggesting that this effect is more related to an increase in age rather than to a specific exposure event. In the Vietnam sample set (n = 1424; slightly reduced due to quality checks from our original sample set), antibody titers to avian influenza A virus antigens increased with age. The optimal fitted regression curve among nonlinear models was a fifth-order polynomial (ANOVA: P < .001), but this curve provided no additional qualitative explanations of the data than the second best fit model, a simple quadratic regression (Figure ​(Figure1).1). Titers to H9 antigen increase more rapidly with age than titers to H7 or H5. One hypothesis is that this difference is caused by varying levels of exposure to avian influenza viruses. A second hypothesis is that this is caused by differences in cross-reactivity between human influenza antibodies and each of the subtypes H9, H7, and H5. Our analysis provides more support for the second hypothesis (supplementary figures 1 and 2 in the article by Boni et al [3]). Figure 1. Scatterplot of avian strain titer against age with polynomial regressions lines. The black line denotes the second-order polynomial, and the red line denotes the fifth-order polynomial. As noted in our article, the microarray assay used in this analysis is more sensitive than traditional HI and MN tests when comparing titers for the homologous antigens. A problem in doing studies to evaluate zoonotic exposure is that the exact antigenic composition of the infecting virus may be unknown. Therefore, HI/MN tests may yield false-negative results, a problem that is less evident with the microarray, which measures antibodies to the head of hemagglutinin and is, therefore, more broadly reactive within subtype. The differences we see in response between H9 and H5 avian strains (and to a lesser extent between H7 and H5 strains) are robust even when a much higher titer cutoff is used (up to 1:320, using microarray). With a cutoff titer of 1:20, 76% of individuals aged ≥50 years test positive for H9 antibodies (compared with 33% aged

BACKGROUND\ud \ud Seasonal influenza has been associated with greater morbidity and mortality in AIDS patients. Highly-active antiretroviral therapy (HAART) has led to some reduction in influenza-related complications but the nature of naturally-acquired T-cell immunity to influenza virus in an African setting, and how this changes with immune reconstitution following HAART is unknown. We measured influenza-specific CD4(+) T-cell immunity in unimmunized HIV-infected Malawian adults and then investigated immune reconstitution following HAART.\ud \ud METHODS\ud \ud Peripheral blood mononuclear cells were isolated from HIV-infected and HIV-uninfected Malawian adults. CFSE proliferation and CD154 expression flow cytometry-based assays were used to measure influenza-specific CD4(+) T-cell immunity.\ud \ud RESULTS\ud \ud We found lower naturally-acquired proliferative influenza-specific CD4(+) T-cell responses in AIDS patients that was also present in asymptomatic HIV-infected adults with relatively high CD4 counts (>350 cells/µl). Influenza-specific CD4(+) T-cell immune reconstitution in HIV-infected patients on HAART for 12 months was poor despite a marked reduction in viral load and an increase in CD4 count. This poor immune reconstitution was characterised by a low influenza-specific proliferative CD4(+) T-cell response and reduced proportions of CD154-expressing influenza-specific CD4(+) T-cells in peripheral blood.\ud \ud CONCLUSION\ud \ud Our data suggest that asymptomatic HIV-infected adults may also be at risk of influenza-related complications and that HAART alone may not circumvent this risk in AIDS patients. This study highlights the need to identify possible interventions early in HIV infection to reduce the risk of influenza and to intensify influenza surveillance in these susceptible African populations.

Background: \ud Pandemic influenza has potential to overwhelm healthcare resources. There is uncertainty over performance of existing triage tools for hospital admission and discharge decisions.\ud Aim: \ud Our aim was to identify clinical criteria that predict safe discharge from hospital and develop a pragmatic triage tool to guide physician decision-making.\ud Design: \ud We retrospectively examined an existing database of patients who presented to the Royal Liverpool University Hospital during the 2010-2011 influenza pandemic.\ud Methods: \ud Inclusion criteria: patients ≥18 years, with PCR confirmed H1N1 influenza. Exclusion criteria: died in the emergency department or case notes unavailable. Successful discharge was defined as discharge within 24 hours of presentation and no readmission within seven days.\ud Results:\ud Eighty-six patients were included and 16 were successfully discharged. Estimated P/F ratio and C-reactive protein predicted safe discharge in a multivariable logistic regression model (AUC 0.883). A composite univariate predictor (estimated P/F minus C-reactive protein, AUC 0.877) was created to calculate specific cut off points for sensitivity and specificity. A pragmatic decision tool was created to incorporate these thresholds and relevant guidelines. Discharge: SpO2 (in air) ≥ 94% and CRP 50 or SpO2 ≤ 93% and CRP 50.\ud Conclusions: \ud We identified that oxygen exchange and CRP, a marker of acute inflammation, were the most important predictors of safe discharge. Our proposed simple triage model requires validation but has the potential to aid clinical decisions in the event of a future pandemic, and potentially for seasonal influenza.