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Key Points Choice of cell line is critical for functional glycosylation of human IgG1-Fc. Sialylated IgG1-Fc monomers block influenza B only when made by CHO-K1 cells., Abs are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG plays a major role in this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary–K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B–mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary–K1, but not in human endothelial kidney 293-F cells.

Background\ud During the first wave of the influenza A(H1N1)pdm09 pandemic in England in 2009, morbidity and mortality were higher in patients of South Asian (Indian, Pakistani or Bangladeshi) ethnic minority groups.\ud \ud Objectives\ud This study aims to provide insights in the representation of this group among reported cases, indicating susceptibility and exposure.\ud \ud Methods\ud All laboratory‐confirmed cases including basic demographic and limited clinical information that were reported to the FluZone surveillance system between April and October 2009 were retrieved. Missing ethnicity data were imputed using the previously developed and validated South Asian Names and Group Recognition Algorithm (SANGRA). Differences between ethnic groups were calculated using chi‐square, log‐rank and t tests and rate ratios. Geographic clustering was compared using Ripley's K functions.\ud \ud Results\ud SANGRA identified 2447 (28%) of the total of 8748 reported cases as South Asian. South Asian cases were younger (P < .001), more often male (P = .002) and more often from deprived areas (P < .001) than cases of other ethnic groups. Time between onset of symptoms and laboratory sampling was longer in this group (P < .001), and they were less often advised antiviral treatment (P < .001), however, declined treatment less. The highest cumulative incidence was seen in the West Midlands region (32.7/10 000), London (7.0/10 000) and East of England region (5.7/10 000).\ud \ud Conclusions\ud People of South Asian ethnic groups were disproportionally affected by the first wave of the influenza pandemic in England in 2009. The findings presented contribute to further understanding of demographic, socioeconomic and ethnic factors of the outbreak and inform future influenza preparedness to ensure appropriate prevention and care.

Influenza virus infections affect millions of people annually. Current available vaccines provide varying rates of protection. There is a knowledge gap on how the nasal microbiota, particularly established pneumococcal colonization, shapes the response to influenza vaccination. In this study, we inoculated healthy adults with live S. pneumoniae and vaccinated them three days later with either TIV or LAIV. Vaccine-induced immune responses were assessed in nose, blood and lung. Nasal pneumococcal colonization had no impact upon TIV-induced antibody responses to influenza, which manifested in all compartments. However, experimentally induced pneumococcal colonization dampened LAIV-mediated mucosal antibody responses, primarily IgA in the nose and IgG in the lung. Pulmonary influenza-specific cellular responses were more apparent in the LAIV group compared to either TIV or an unvaccinated group. These results indicate that TIV and LAIV elicit differential immunity to adults and that LAIV immunogenicity is diminished by the nasal presence of S. pneumoniae. Therefore, nasopharyngeal pneumococcal colonization may affect LAIV efficacy.

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

Background\ud \ud Nasopharyngeal colonisation by S. pneumoniae is a prerequisite for invasive pneumococcal infections. Influenza co-infection leads to increased susceptibility to secondary pneumonia and mortality during influenza epidemics. Increased bacterial load and impaired immune responses to pneumococcus caused by influenza play a role in this increased susceptibility. Using an Experimental Human Challenge Model and influenza vaccines, we examined symptoms experienced by healthy adults during nasal co-infection with S. pneumoniae and live attenuated influenza virus.\ud Methods\ud \ud Randomised, blinded administration of Live Attenuated Influenza Vaccine (LAIV) or Tetravalent Inactivated Influenza Vaccine (TIV) either preceded bacterial inoculation or followed it, separated by a 3-day interval. The presence and density of S. pneumoniae was determined from nasal washes. Participants completed a symptom questionnaire from the first intervention until 6 days post second intervention.\ud Results\ud \ud The timing and type of influenza vaccination and presence of S. pneumoniae in the nasopharynx significantly affected symptom reporting. In the study where influenza vaccination preceded bacterial inoculation: nasal symptoms were less common in the LAIV group than the TIV group (OR 0.57, p < 0.01); with colonisation status only affecting the TIV group where more symptoms were reported by colonised participants compared to non-colonised participants following inoculation (n = 12/23 [52.17%] vs n = 13/38 [34.21%], respectively; p < 0.05). In the study where influenza vaccination followed bacterial inoculation: no difference was seen in the symptoms reported between the LAIV and TIV groups following inoculation and subsequent vaccination; and symptoms were unaffected by colonisation status.\ud Conclusion\ud \ud Symptoms experienced during live viral vaccination and bacterial co-infection in the nasopharynx are directly affected by the precedence of the pathogen acquisition. Symptoms were directly affected by nasal pneumococcal colonisation but only when TIV was given prior to bacterial exposure.

Streptococcus pneumoniae is one of the world’s leading bacterial pathogens, responsible for pneumonia, septicaemia and meningitis. Asymptomatic colonisation of the nasopharynx is considered to be a prerequisite for these severe infections, however little is understood about the biological changes that permit the pneumococcus to switch from asymptomatic coloniser to invasive pathogen. A phase variable type I restriction-modification (R-M) system (SpnIII) has been linked to a change in capsule expression and to the ability to successfully colonise the murine nasopharynx. Using our laboratory data, we have developed a Markov change model that allows prediction of the expected level of phase variation within a population, and as a result measures when populations deviate from those expected at random. Using this model, we have analysed samples from the Experimental Human Pneumococcal Carriage (EHPC) project. Here we show, through mathematical modelling, that the patterns of dominant SpnIII alleles expressed in the human nasopharynx are significantly different than those predicted by stochastic switching alone. Our inter-disciplinary work demonstrates that the expression of alternative methylation patterns should be an important consideration in studies of pneumococcal colonisation.

Objectives Severe lower respiratory tract infection caused by adenovirus is well described in immunocompromised hosts and can cause significant morbidity and mortality. We compare and contrast the clinical presentation, radiological, and virological features of two rare cases in immunocompetent adults admitted to an intensive care unit in a large, teaching hospital in North West England. We then provide a concise, comprehensive literature review. Methods The first case was a 35-year old female asthmatic who presented with respiratory distress and pneumonitis during peak influenza season, and recovered after a prolonged hospital stay. The second case was a 73-year old male who presented with diarrhoea, vomiting, and general malaise outside of influenza season, developed respiratory compromise, and died. Adenovirus type 7 was identified in bronchoalveolar lavages and plasma samples of both patients, each of whom received cidofovir. No other infectious aetiology was identified. Results Clinical and radiological features of severe lower respiratory tract adenoviral infection are similar to other infectious causes of pneumonia and ARDS, including severe influenza. This can create diagnostic uncertainty, especially during influenza season. Positive adenovirus polymerase chain reaction results can support a diagnosis of severe lower respiratory tract adenovirus infection in patients with a clinically compatible syndrome and no other identified aetiology, with higher viral loads being associated with worse prognosis. Although treatment is predominantly supportive, early use of cidofovir may improve outcomes. Conclusions These rare cases highlight that severe lower respiratory tract adenoviral infection should be considered in the differential diagnoses of immunocompetent patients presenting with pneumonia and ARDS., Highlights • Severe lower respiratory tract adenovirus infection requiring critical care admission is rare in immunocompetent adults. • Evidence concerning diagnosis and management of immunocompetent adults with adenovirus severe lower respiratory tract infection (sLRTI) is limited. • We present rare cases of severe lower respiratory tract adenovirus infection in immunocompetent adults, on a critical care unit. • Immunocompetent patients with adenovirus sLRTI may present with pneumonitis and acute respiratory distress syndrome (ARDS) • Clinical features of sLRTI are difficult to distinguish clinically and radiologically from other causes of pneumonitis, including severe influenza.

Background. Maternal influenza vaccination protects infants against influenza virus infection. Impaired transplacental transfer of influenza antibodies may reduce this protection. \ud Methods. We conducted a cross-sectional study of influenza vaccine-naïve pregnant women recruited at delivery from Blantyre (urban, low malaria transmission) and Chikwawa (rural, high malaria transmission) in Southern Malawi. HIV-infected mothers were excluded in Chikwawa. Maternal and cord blood antibodies against circulating influenza strains A/California/7/2009, A/Victoria/361/2011, B/Brisbane/60/2008 and B/Wisconsin/1/2010 were measured by hemagglutination inhibition (HAI). We studied the impact of maternal HIV infection and placental malaria on influenza antibody levels in mother-infant pairs in Blantyre and Chikwawa, respectively.\ud Results. We included 454 mother-infant pairs (Blantyre, n=253; Chikwawa, n=201). HIV-infected mothers and their infants had lower seropositivity (HAI titer >1:40) against influenza A(H1N1)pdm09 (mothers, 24.3 vs. 45.4%, p=0.02; infants 24.3 vs. 50.5%, p=0.003) and A(H3N2) (mothers, 37.8% vs. 63.9%, p=0.003; infants 43.2 vs. 64.8%, p=0.01), whereas placental malaria had inconsistent effect on maternal and infant seropositivity. In multivariable analyses, maternal HIV infection was associated with reduced infant seropositivity (A(H1N1)pdm09 aOR 0.34, 95%CI 0.15-0.79; A(H3N2) aOR 0.43, 95%CI 0.21-0.89). Transplacental transfer was not impaired by maternal HIV or placental malaria.\ud Discussion. Maternal HIV infection influenced maternal antibody response to influenza A virus infection and therefore antibody levels in newborns, but did not affect transplacental antibody transfer.

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.

The human nasopharynx is frequently colonized by Streptococcus pneumoniae (the pneumococcus), serving as the reservoir for transmission, a state which necessarily precedes invasive pneumococcal infection. Influenza infection increases pneumococcal colonization density and dysregulates host immune responses, increasing the risk of secondary bacterial pneumonia and death.

Influenza virus infections cause between 291 243 and 645 832 deaths annually, with the highest burden in low-income settings. Research in high-income countries has examined public understanding of influenza, but there is little information on views and behaviours about influenza in low-income countries. We explored communities’ ideas about the severity, causes, prevention and treatment of influenza in Chikwawa district, Malawi. Methods We conducted 64 in-depth interviews with parents of children aged

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein-Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A-mediated agglutination of human erythrocytes.

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.

Secondary bacterial pneumonia following influenza infection is a significant cause of mortality worldwide. Upper respiratory tract pneumococcal carriage is important as both determinants of disease and population transmission. The immunological mechanisms that contain pneumococcal carriage are well-studied in mice but remain unclear in humans. Loss of this control of carriage following influenza infection is associated with secondary bacterial pneumonia during seasonal and pandemic outbreaks. We used a human type 6B pneumococcal challenge model to show that carriage acquisition induces early degranulation of resident neutrophils and recruitment of monocytes to the nose. Monocyte function associated with clearance of pneumococcal carriage. Prior nasal infection with live attenuated influenza virus induced inflammation, impaired innate function and altered genome-wide nasal gene responses to pneumococcal carriage. Levels of the cytokine IP-10 promoted by viral infection at the time of pneumococcal encounter was positively associated with bacterial density. These findings provide novel insights in nasal immunity to pneumococcus and viral-bacterial interactions during co-infection.

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.

Background\ud Year‐round transmission of influenza has been detected in Vietnam through both national surveillance and other epidemiological studies. Understanding the demographic and clinical features of influenza‐like‐illness (ILI) presenting to primary care in urban Vietnam is vital to understand these transmission dynamics.\ud \ud Methods\ud A prospective, observational study of patients with ILI in Ho Chi Minh City, Vietnam was conducted between August 2013 and November 2015 in a mix of public and private primary care settings. Molecular testing for Influenza A & B and 12 other respiratory viruses was performed.\ud \ud Results\ud 1152 ILI patients were recruited. 322 and 136 subjects tested positive for influenza A and B, respectively. 193 subjects tested positive for another respiratory virus; most commonly rhinovirus and parainfluenza virus 3. Influenza was detected in 81% of the 116 study weeks. Three peaks of influenza activity were detected; an H3N2 peak April‐June 2014, an influenza B peak July‐December 2014, and a mixed H3N2 and H1N1 peak March‐September 2015. Subjects recruited from private clinics were more likely to have higher income, and to have reported previous influenza vaccination. Antibiotic use was common (50.3%) despite limited evidence of bacterial infection.\ud \ud Conclusion\ud Influenza in southern Vietnam has complex transmission dynamics including periods of intense influenza activity of alternating types and subtypes. Broadening surveillance from hospital to the community in tropical settings is feasible and a valuable for improving our understanding of the global spread and evolution of the virus.

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.

There is increasing interest recently in developing intranasal vaccines against respiratory tract infections. Antibody response is critical in vaccine-induced protection and T is considered important in mediating antibody response. Most data supporting the role for T in antibody response are from animal studies, and direct evidence from humans is limited, apart from T -like cells in blood. We studied activation and induction of T and its role on anti-influenza antibody response by live-attenuated influenza vaccine(LAIV) in human nasopharynx-associated lymphoid tissue(NALT). T activation in adenotonsillar tissues were analysed by flow-cytometry, and anti-hemagglutinin(HA) antibodies examined following LAIV stimulation of tonsillar mononuclear cells(MNC). Induction of antigen-specific T by LAIV was studied by flow-cytometry for induced T and CD154 expression. LAIV induced T proliferation which correlated with anti-HA antibody production, and T was shown critical for antibody response. Induction of T from naïve T cells by LAIV was shown in newly induced T expressing BCL6 and CD21, which was followed by the detection of anti-HA antibodies. Antigen specificity of LAIV-induced T was demonstrated by the expression of antigen-specific T cell activation marker CD154 upon challenge by H1N1 virus antigen or HA. LAIV-induced T differentiation was inhibited by BCL6, IL21, ICOS and CD40 signalling blocking respectively, and that diminished anti-HA antibody production. We demonstrate for the first time the induction of antigen-specific T by LAIV in human NALT that provide critical support for anti-influenza antibody response. Promoting antigen-specific T in NALT by intranasal vaccines may provide an effective vaccination strategy against respiratory infections in humans. Airway infection such as influenza is common in humans. Intranasal vaccination has been considered a more biologically relevant and effective way of immunization against airway infection. Vaccine-induced antibody response is crucial for protection against infection. Recent data from animal studies suggest one type of T cells, named T is important for the antibody response. However, data on whether this T -mediated help for antibody production operates in humans is limited, due to the lack of access to human immune tissue containing the T In this study, we demonstrated the induction of T cells by an intranasal influenza vaccine in human immune tissue that provide critical support for anti-influenza antibody response. Our findings provide direct evidence that T cells play a critical role in vaccine-induced immunity in humans, and suggest a novel strategy to promote such cells by intranasal vaccines against respiratory infections. [Abstract copyright: Copyright © 2018 Aljurayyan et al.]

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.

The World Health Organisation estimates that approximately one billion people are infected and up to 500 000 people die from influenza each year.1 The greatest burden of illness usually occurs among children, while the highest burden of severe disease (in terms of hospitalisation and death) occurs in those with underlying medical conditions, infants and young children, and elderly people.2 Current circulating influenza strains in humans include influenza A(H1N1)pdm09, influenza A(H3N2), and both influenza B viruses (B/Victoria and B/Yamagata).3 4 This article provides non-specialists with information on how to diagnose, manage, and prevent flu.

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.

Objective \ud \ud To assess whether retail sales of non-prescription products can be used for syndromic surveillance and whether it can detect influenza activity at different spatial scales. A secondary objective was to assess whether changes in purchasing behaviour were related to public health advice or levels of media or public interest.\ud \ud Setting \ud \ud The UK.\ud \ud Participants \ud \ud National and regional influenza case estimates and retail sales from a major British supermarket.\ud \ud Outcome measures \ud \ud Weekly, seasonally adjusted sales of over-the-counter symptom remedies and non-pharmaceutical products; recommended as part of the advice offered by public health agencies; were compared with weekly influenza case estimates. Comparisons were made at national and regional spatial resolutions. We also compared sales to national measures of contemporaneous media output and public interest (Internet search volume) related to the pandemic.\ud \ud Results \ud \ud At a national scale there was no significant correlation between retail sales of symptom remedies and cases for the whole pandemic period in 2009. At the regional scale, a minority of regions showed statistically significant positive correlations between cases and sales of adult ‘cold and flu’ remedies and cough remedies (3.2%, 5/156, 3.8%, 6/156), but a greater number of regions showed a significant positive correlation between cases and symptomatic remedies for children (35.6%, 55/156). Significant positive correlations between cases and sales of thermometers and antiviral hand gels/wash were seen at both spatial scales (Cor 0.477 (95% CI 0.171 to 0.699); 0.711 (95% CI 0.495 to 0.844)). We found no significant association between retail sales and media reporting or Internet search volume.\ud \ud Conclusions \ud \ud This study provides evidence that the British public responded appropriately to health messaging about hygiene. Non-prescription retail sales at a national level are not useful for the detection of cases. However, at finer spatial scales, in particular age-groups, retail sales may help augment existing surveillance and merit further study.

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.