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Objectives: PICOBOO is a randomised, adaptive trial evaluating the immunogenicity, reactogenicity, and safety of COVID-19 booster strategies. We report data for second boosters among individuals 50-<70 years old primed with AZD1222 (50-<70y-AZD1222) until Day 84., Methods: Immunocompetent adults who received any first booster ≥three months prior were eligible. Participants were randomly allocated to BNT162b2, mRNA-1273 or NVX-CoV2373 1:1:1. The concentrations of ancestral anti-spike immunoglobulin were summarised as the geometric mean concentrations (GMC). Reactogenicity and safety outcomes were captured. Additional analyses including neutralising antibodies were performed on a subset. ACTRN12622000238774., Results: Between Mar 2022 and Aug 2023, 743 participants were recruited and had D28 samples; 155 belonged to the 50-<70y-AZD1222 stratum. The mean adjusted GMCs (95% credible intervals) were 20,690 (17 555-23 883), 23,867 (20 144-27 604) and 8654 (7267-9962) U/mL at D28 following boosting with BNT162b2, mRNA-1273 and NVX-CoV2372, respectively, and 10,976 (8826-13 196), 15,779 (12 512-19 070) and 6559 (5220-7937) U/mL by D84. IgG against Omicron BA.5 was 2.7-2.9 times lower than the ancestral strain. Limited neutralisation against Omicron subvariants was found following all vaccines. Severe reactogenicity events were <4%., Conclusions: All vaccines were immunogenic with more rapid waning after mRNA vaccines. These data support boosting with vaccines with greater specificity for circulating Omicron subvariants., Competing Interests: Declaration of Competing Interest KF and TS are members of the Australian Technical Advisory Group on Immunisation (ATAGI), which advises the government on vaccine policy; their involvement as investigators on this trial has been declared to ATAGI. MP is involved in an ovarian cancer clinical trial that received funding from AstraZeneca. MP was involved in performing immunological assays on biological specimens obtained from participants in this trial, but was not involved in participant recruitment, data collection or the analysis of results. SNF leads the UK National Institute for Health and Care Research funded trial of third and fourth-dose COVID-19 boosters. SNF acts on behalf of University Hospital Southampton NHS Foundation Trust, UK, as an Investigator and/or providing consultative advice on clinical trials and studies of vaccines funded or sponsored by vaccine manufacturers including Moderna, Sanofi, Janssen, Pfizer, AstraZeneca, GlaxoSmithKline, Novavax, Seqirus, Medimmune, Merck and Valneva vaccines and antimicrobials. UW is an investigator on clinical trials of vaccines funded or sponsored by vaccine manufactures including Moderna, Sanofi, Pfizer, Merck and GlaxoSmithKline. PR also reports acting on behalf of University of Western Australia, as an Investigator and/or providing consultative advice on clinical trials and studies of vaccines funded or sponsored by vaccine manufacturers including Moderna, Sanofi, Janssen, Pfizer, AstraZeneca, GlaxoSmithKline, Novavax, Seqirus, Merck and Clover Biopharmaceutical vaccines. They receive no personal financial payment for this work. The other authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: One third of children require repeat ventilation tube insertion (VTI) for otitis media. Disease recurrence is associated with persistent middle ear bacterial biofilms. With demonstration that Dornase alfa (a DNase) disrupts middle ear effusion biofilms ex vivo, we identified potential for this as an anti-biofilm therapy to prevent repeat VTI. First, safety and tolerability needed to be measured., Methods: This was a phase 1B double-blinded randomized control trial conducted in Western Australia. Children between 6 months and 5 years undergoing VTI for bilateral middle ear effusion were recruited between 2012 and 2014 and followed for two years. Children's ears were randomized to receive either Dornase alfa (1 mg/mL) or 0.9 % sodium chloride (placebo) at time of surgery. Children were followed up at 2 weeks post-VTI and at 3-monthly intervals for 2 years. Outcomes assessed were: 1) safety and tolerability, 2) otorrhoea frequency, 3) blocked or extruded ventilation tube (VT) frequency, 4) time to blockage or extrusion, 5) time to infection recurrence and/or need for repeat VTI., Results: Sixty children (mean age 2.3 years) were enrolled with 87 % reaching study endpoint. Treatment did not change otorrhoea frequency. Hearing improved in all children following VTI, with no indication of ototoxicity. Dornase alfa had some effect on increasing time until VT extrusion (p = 0.099); and blockage and/or extrusion (p = 0.122). Frequency of recurrence and time until recurrence were similar. Fourteen children required repeat VTI within the follow-up period., Conclusion: A single application of Dornase alfa into the middle ear at time of VTI was safe, non-ototoxic, and well-tolerated., Trial Registration: ACTRN12623000504617., Competing Interests: Declaration of competing interest There are no financial interests. Prof. Richmond serves on scientific advisory boards, and together with A/Prof Kirkham has received institutional funding for investigator-initiated grants, from GSK, Pfizer and MSD – all are unrelated to this work., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Chronic wet cough for longer than 4 weeks is a hallmark of chronic suppurative lung diseases (CSLD), including protracted bacterial bronchitis (PBB), and bronchiectasis in children. Severe lower respiratory infection early in life is a major risk factor of PBB and paediatric bronchiectasis. In these conditions, failure to clear an underlying endobronchial infection is hypothesised to drive ongoing inflammation and progressive tissue damage that culminates in irreversible bronchiectasis. Historically, the microbiology of paediatric chronic wet cough has been defined by culture-based studies focused on the detection and eradication of specific bacterial pathogens. Various 'omics technologies now allow for a more nuanced investigation of respiratory pathobiology and are enabling development of endotype-based models of care. Recent years have seen substantial advances in defining respiratory endotypes among adults with CSLD; however, less is understood about diseases affecting children. In this review, we explore the current understanding of the airway microbiome among children with chronic wet cough related to the PBB-bronchiectasis diagnostic continuum. We explore concepts emerging from the gut-lung axis and multi-omic studies that are expected to influence PBB and bronchiectasis endotyping efforts. We also consider how our evolving understanding of the airway microbiome is translating to new approaches in chronic wet cough diagnostics and treatments.

Background: The need for coronavirus 2019 (COVID-19) vaccination in different age groups and populations is a subject of great uncertainty and an ongoing global debate. Critical knowledge gaps regarding COVID-19 vaccination include the duration of protection offered by different priming and booster vaccination regimens in different populations, including homologous or heterologous schedules; how vaccination impacts key elements of the immune system; how this is modified by prior or subsequent exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and future variants; and how immune responses correlate with protection against infection and disease, including antibodies and effector and T cell central memory., Methods: The Platform Trial In COVID-19 priming and BOOsting (PICOBOO) is a multi-site, multi-arm, Bayesian, adaptive, randomised controlled platform trial. PICOBOO will expeditiously generate and translate high-quality evidence of the immunogenicity, reactogenicity and cross-protection of different COVID-19 priming and booster vaccination strategies against SARS-CoV-2 and its variants/subvariants, specific to the Australian context. While the platform is designed to be vaccine agnostic, participants will be randomised to one of three vaccines at trial commencement, including Pfizer's Comirnaty, Moderna's Spikevax or Novavax's Nuvaxovid COVID-19 vaccine. The protocol structure specifying PICOBOO is modular and hierarchical. Here, we describe the Core Protocol, which outlines the trial processes applicable to all study participants included in the platform trial., Discussion: PICOBOO is the first adaptive platform trial evaluating different COVID-19 priming and booster vaccination strategies in Australia, and one of the few established internationally, that is designed to generate high-quality evidence to inform immunisation practice and policy. The modular, hierarchical protocol structure is intended to standardise outcomes, endpoints, data collection and other study processes for nested substudies included in the trial platform and to minimise duplication. It is anticipated that this flexible trial structure will enable investigators to respond with agility to new research questions as they arise, such as the utility of new vaccines (such as bivalent, or SARS-CoV-2 variant-specific vaccines) as they become available for use., Trial Registration: Australian and New Zealand Clinical Trials Registry ACTRN12622000238774. Registered on 10 February 2022., (© 2023. Crown.)

Introduction: Children in low-mid income countries, and First Nations children in high-income countries, experience disproportionately high rates of Streptococcus pneumoniae and Haemophilus influenzae infections and diseases including pneumonia and otitis media. We previously observed that infants from Papua New Guinea had no evidence of waning maternal immunity for H. influenzae -specific antibodies. In this study, we assessed S. pneumoniae and H. influenzae antibody titres in Australian First Nation mothers and infants to determine antigen-specific antibody ontogenies and whether H. influenzae antibody titres in infants were due to low maternal antibody titres or lack of placental transfer., Methods: Breast milk, infant nasopharyngeal swabs and ear assessment data were collected 1-, 2-, 7-months post-birth as well as maternal, cord and 7-month-old infant sera, from 85 Australian Aboriginal and Torres Strait Islander mother-infant pairs. Serum IgG and breast milk IgG and IgA antibody titres to S. pneumoniae antigens (PspA1, PspA2, CbpA, Ply) and H. influenzae antigens (PD, ChimV4, OMP26, rsPilA) were measured., Results: IgG titres in maternal and cord sera were similar for all antigens, except Ply (higher in cord; p=0.004). Sera IgG titres at 7-months of age were lower than cord sera IgG titres for all S. pneumoniae antigens (p<0.001). Infant sera IgG titres were higher than cord sera for H. influenzae PD (p=0.029), similar for OMP26 (p=0.817) and rsPilA (p=0.290), and lower for ChimV4 (p=0.004). Breast milk titres were similar for all antigens at 1, 2 and 7-months except OMP26 IgA (lower at 7-months than 1-month; p=0.035), PspA2 IgG (p=0.012) and Ply IgG that increased by 7-months (p=0.032). One third of infants carried nontypeable Haemophilus influenzae (NTHi), 45% carried S. pneumoniae and 52% had otitis media (OM) observed at least once over the 7-months. 73% of infants who carried either S. pneumoniae or NTHi, also had otitis media observed., Conclusions: Similarities between maternal and cord IgG titres, and absence of waning, support a lack of maternal H. influenzae IgG antibodies available for cross-placental transfer. Increased maternal anti-PD IgG could offer some protection from early carriage with NTHi, and maternal immunisation strategies should be considered for passive-active immunisation of infants to protect against S. pneumoniae and H. influenzae diseases., Trial Registration: ClinicalTrials.gov NCT00714064 and NCT00310349., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Martinovich, Seppanen, Bleakley, Clark, Andrews, Richmond, Binks, Thornton and Kirkham.)

Background: Nontypeable Haemophilus influenzae (NTHi) is the most common bacterial otopathogen associated with otitis media (OM). NTHi persists in biofilms within the middle ears of children with chronic and recurrent OM. Australian Aboriginal children suffer exceptionally high rates of chronic and recurrent OM compared to non-Aboriginal children. NTHi protein vaccines comprised of antigens associated with both adhesion and persistence in a biofilm are under development and could be beneficial for children with chronic and recurrent OM. Understanding the ontogeny of natural antibody development to these antigens provides insight into the value of vaccinating with particular antigens., Methods: An in-house multiplex fluorescent bead immunoassay was used to measure serum IgG titres and avidity for three putative vaccine antigens: recombinant soluble PilA (rsPilA), ChimV4, and outer membrane protein 26 (OMP26) in sera from Australian Aboriginal otitis-prone children (n=77), non-Aboriginal otitis-prone children (n=70) and non-otitis-prone children (n=36). Serum IgG titres were adjusted for age, and geometric mean concentrations (GMCs) were compared between groups using a univariate analysis model. Antibody avidity was calculated as a relative avidity index and compared between groups using ANOVA., Results: Australian Aboriginal otitis-prone children had lower serum IgG titres to rsPilA and ChimV4 than non-Aboriginal otitis-prone children (p<0.001), and non-otitis-prone children (p<0.020). No differences were observed between serum IgG titres from non-Aboriginal otitis-prone children and non-otitis-prone children. There were also no differences in the proportion of high avidity IgG specific for these antigens between these groups. Serum IgG titres to OMP26 were similar between all groups (p>0.670) although otitis-prone children had a higher proportion of high avidity antibodies to this antigen., Conclusions: Australian Aboriginal otitis-prone children had lower serum IgG titres to 2/3 major NTHi vaccine candidate antigens, suggesting these children are unable to develop persistent IgG responses due to repeated NTHi exposure. These reduced IgG titres may relate to earlier and more frequent exposure to diverse NTHi strains in Aboriginal children through carriage or infection. These data suggest that Aboriginal children may benefit from immunisation with vaccines containing these antigens to increase titres of protective antibodies., Competing Interests: LB is an inventor of technology related to PilA-derived immunogens that is licensed to GlaxoSmithKline Biologicals. PR reports receiving funds to his institution from GlaxoSmithKline for investigator led research and participation in vaccine scientific advisory boards outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Clark, Seppanen, Kirkham, Novotny, Bakaletz, Cripps, Corscadden, Coates, Vijayasekaran, Richmond and Thornton.)

Background: Otitis media (OM) is one of the most common infections in young children, arising from bacterial and/or viral infection of the middle ear. Globally, Streptococcus pneumoniae and non-typeable Haemophilus influenzae (NTHi) are the predominant bacterial otopathogens. Importantly, common upper respiratory viruses are increasingly recognized contributors to the polymicrobial pathogenesis of OM. This study aimed to identify predominant bacteria and viruses in the nasopharynx, adenoids and middle ears of peri-urban/urban South-East Queensland Australian children, with and without clinical history of chronic otitis media with effusion (COME) and/or recurrent acute otitis media (RAOM)., Methods: Sixty children, 43 diagnosed with OM and 17 controls with no clinical history of OM from peri-urban/urban South-East Queensland community were recruited to the study. Respiratory tract bacterial and viral presence were examined within nasopharyngeal swabs (NPS), middle ear effusions (MEE) and adenoids, using real-time polymerase chain reaction (RT-PCR) and bacterial culture., Results: At least one otopathogen present was observed in all adenoid samples, 86.1% and 82.4% of NPS for children with and without OM, respectively, and 47.1% of the MEE from the children with OM. NTHi was the most commonly detected bacteria in both the OM and control cohorts within the adenoids (90.0% vs 93.8%), nasopharynx (67.4% vs 58.8%) respectively, and in the MEE (OM cohort 25.9%). Viruses were detected in all adenoid samples, 67.4% vs 47.1% of the NPS from the OM and control cohorts, respectively, and 37% of the MEE. Rhinovirus was the predominant virus identified in the adenoids (85.0% vs 68.8%) and nasopharynx (37.2% vs 41.2%) from the OM and control cohorts, respectively, and the MEE (19.8%)., Conclusions: NTHi and rhinovirus are predominant otopathogens within the upper respiratory tract of children with and without OM from peri-urban and urban South-East Queensland, Australia. The presence of bacterial otopathogens within the middle ear is more predictive of concurrent URT infection than was observed for viruses, and the high otopathogen carriage within adenoid tissues confirms the complex polymicrobial environment in children, regardless of OM history., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ngo, Massa, McMonagle, Perry, Nissen, Sloots, Thornton and Cripps.)

Background: Lower airway biofilms are hypothesised to contribute to poor treatment outcomes among children with chronic lung disease; however, data are scarce. We aimed to determine the presence and prevalence of biofilm in bronchoalveolar lavage from children with protracted bacterial bronchitis (PBB) or bronchiectasis; whether biofilm was associated with signs of lower airway infection; and whether biofilms were consistent with an upper or lower airway origin., Methods: In this cross-sectional study, fluorescent microscopy techniques were used to detect biofilm in archived bronchoalveolar lavage specimens from a paediatric cohort (age <18 years) with PBB or bronchiectasis who were prospectively recruited to observational studies of chronic cough at Royal Children's Hospital (Brisbane, Australia) or Royal Darwin Hospital (Darwin, Australia). Children with cystic fibrosis were excluded. Lower airway infection was defined as bronchoalveolar lavage neutrophil percentage of 15% or more, or a culture of a bacterial pathogen at 10 4 colony-forming units per mL or more, or both. Biofilms were subtyped as either of lower airway origin (unrelated to squamous epithelial cells) or of upper airway origin (observed in close association with squamous epithelial cells). Bronchoalveolar lavages were considered contaminated with upper airway secretions if the squamous cell proportion was more than ten cells per 1000 nucleated cells (>1%). Primary outcomes were the prevalence of each biofilm subtype among children with PBB compared with children with bronchiectasis. Secondary outcomes were the prevalence of each biofilm subtype among children with signs of lower airway infection compared to children without., Findings: Biofilm testing was performed on 144 bronchoalveolar lavage specimens collected between Jan 1, 2011, and Dec 16, 2014, and preserved at -80°C before biofilm testing (69 children with PBB from Brisbane and 75 children with bronchiectasis from Darwin). The prevalence of lower airway biofilms (unrelated to squamous epithelial cells) was similar among the children with PBB (25 [36%] of 69) and children with bronchiectasis (31 [41%] of 75; odds ratio [OR] 1·24, 95% CI 0·63-2·43), but higher among children with signs of lower airway infection (46 [48%] of 95) than children without (eight [19%] of 43; OR 4·11, 95% CI 1·73-9·78), irrespective of the underlying diagnosis. By contrast, upper airway biofilms (associated with squamous epithelial cells) were more prevalent among children with bronchiectasis (32 [43%] of 75) than children with PBB (16 [23%] of 69; OR 2·47, 95% CI 1·20-5·08) and were unrelated to lower airway infection. Upper airway contamination was uncommon (eight [11%] of 71) and was not evident in 23 (79%) of 29 bronchoalveolar lavages that were positive for upper airway biofilms., Interpretation: Lower airway biofilms are prevalent, but not ubiquitous, in bronchoalveolar lavage from children with PBB or bronchiectasis, suggesting anti-biofilm therapies might be beneficial for some children. Detection of upper airway biofilms in bronchoalveolar lavage that did not have signs of contamination suggests that microaspiration might be important in some children. Specimen quality measures are recommended for future studies to account for the presence of upper airway biofilms., Funding: Financial Markets for Children Project Grant, National Health and Medical Research Council of Australia, Rebecca L Cooper Medical Research Foundation, Queensland Children's Hospital Foundation, and BrightSpark Foundation., Competing Interests: Declaration of interests RLM reports grants from Financial Markets for Children, National Health and Medical Research Council of Australia (NHMRC), and the Rebecca L Cooper Foundation during the conduct of this study. HCS-V reports grants from Financial Markets for Children, during the conduct of the study. ABC reports grants from NHMRC during the conduct of this study; and institutional fees relating to advising the study design for an unlicensed molecule for chronic cough (Merck) and for serving as an Independent Data Monitoring Committee member for an unlicensed vaccine (GSK) and a COVID-19 vaccine for children (Moderna), outside the submitted work. RBT reports grants from the Financial Markets for Children and the BrightSpark Foundation, during the conduct of the study. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: The underlying pathogenesis of pediatric obstructive sleep disordered breathing (SDB) and recurrent tonsillitis (RT) are poorly understood but need to be elucidated to develop less invasive treatment and prevention strategies., Methods: Children aged between 1- and 16-years undergoing adenoidectomy, tonsillectomy or adenotonsillectomy for SDB (n=40), RT alone (n=18), or both SDB and RT (SDB+RT) (n=17) were recruited with age-matched healthy controls (n=33). Total bacterial load and species-specific densities of nontypeable Haemophilus influenzae (NTHi) , Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Moraxella catarrhalis were measured by qPCR in nasopharyngeal swabs, oropharyngeal swabs, adenoid and tonsillar tissue from children with SDB, SDB+RT and RT, and in naso- and oro- pharyngeal swabs from healthy children. A subset of tonsil biopsies were examined for biofilms using 16S rRNA FISH (n=3/group)., Results: The 5 bacterial species were detected in naso- and oro- pharyngeal samples from all children. These species were frequently detected in adenotonsillar tissue (except S. aureus , which was absent in adenoids) from children with SDB, SDB+RT and RT. NTHi and S. aureus were observed in tonsils from 66.7-88.2% and 33.3-58.8% of children respectively. Similar total and species-specific bacterial densities were observed in adenotonsillar tissue from children with SDB, SDB+RT or RT. Nasopharyngeal and oropharyngeal swabs were more likely to have multiple bacterial species co-detected than adenotonsillar tissue where one or two targeted species predominated. Polymicrobial biofilms and intracellular bacteria were observed in tonsils from children with adenotonsillar disease., Conclusions: Antimicrobials, particularly anti-biofilm therapies, may be a strategy for managing children with SDB., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mateus, Seppanen, de Gier, Clark, Coates, Vijayasekaran, Prosser, Wiertsema, Fuery, Kirkham, Richmond and Thornton.)

Background: Development of vaccines to prevent disease and death from Streptococcus pneumoniae , and nontypeable Haemophilus influenzae (NTHi), the main pathogens that cause otitis media, pneumonia, meningitis and sepsis, are a global priority. Children living in low and lower-middle income settings are at the highest risk of contracting and dying from these diseases. Improved vaccines with broader coverage are required. Data on the natural development of antibodies to putative vaccine antigens, especially in high-risk settings, can inform the rational selection of the best antigens for vaccine development., Methods: Serum IgG titres to four pneumococcal proteins (PspA1, PspA2, CbpA, and Ply) and five NTHi antigens (P4, P6, OMP26, rsPilA and ChimV4) were measured in sera collected from 101 Papua New Guinean children at 1, 4, 9, 10, 23 and 24 months of age using multiplexed bead-based immunoassays. Carriage density of S. pneumoniae and H. influenzae were assessed by quantitative PCR on genomic DNA extracted from nasopharyngeal swabs using species-specific primers and probes. All data were log-transformed for analysis using Student's unpaired t-tests with geometric mean titre (GMT) or density (GMD) calculated with 95% confidence intervals (CI)., Results: Serum -pneumococcal protein-specific IgG titres followed a "U" shaped pattern, with a decrease in presumably maternally-derived IgG titres between 1 and 4 months of age and returning to similar levels as those measured at 1 month of age by 24 months of age. In contrast, NTHi protein-specific IgG titres steadily increased with age. There was no correlation between antibody titres and carriage density for either pathogen., Conclusion: This longitudinal study indicates that the waning of maternally- derived antibodies that is usually observed in infants, after infants does not occur for NTHi antigens in Papua New Guinean infants. Whether NTHi antigen IgG can be transferred maternally remains to be determined. Vaccines that are designed to specifically increase the presence of protective NTHi antibodies in the first few months of life may be most effective in reducing NTHi disease., Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT01619462., Competing Interests: WP has received funding from Pfizer Australia to attend a conference. AB conducts part-time consultancy work for vaccine companies on projects not related to this study. L-AK has received investigator-initiated research grants, educational grants and travel support from Pfizer, GlaxoSmithKline and Merck, Sharp & Dohme, and is an inventor on patents for a pneumococcal protein vaccine antigen. DL is an investigator on an investigator-initiated research grant that was funded by Pfizer Australia. PR has received nonfinancial support from Pfizer, grants from GlaxoSmithKline and Pfizer, and nonfinancial support from GlaxoSmithKline for work outside the submitted work. The Papua New Guinea Institute of Medical Research received sponsorship from Pfizer Australia to host a national Medical Symposium in 2014. AC is a member of the Seqirus Australia Pneumococcal Advisory Board and the Merck & Co Global Pneumococcal Advisory Board. LB has received grants from GlaxoSmithKline to develop NTHi type IV pilus-derived vaccine antigens. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martinovich, Rahman, de Gier, Seppanen, Orami, Granland, Francis, Yoannes, Corscadden, Ford, Jacoby, van den Biggelaar, Bakaletz, Cripps, Lehmann, Richmond, Pomat, Kirkham and Thornton.)

Background: Nasopharyngeal colonisation with nontypeable Haemophilus influenzae (NTHi) is associated with development of infections including pneumonia and otitis media. The 10-valent pneumococcal conjugate vaccine (PCV10) uses NTHi Protein D (PD) as a carrier. Papua New Guinean children have exceptionally early and dense NTHi carriage, and high rates of NTHi-associated disease. Vaccination with PCV10 could potentially reduce NTHi carriage and disease in this population by inducing a NTHi PD immune response., Methods: Serum and nasopharyngeal swabs were collected from 101 Papua New Guinean children at 1, 4, 9, 10, 23 and 24 months of age. Children received PCV10 (n = 55) or PCV13 (not containing NTHi PD) (n = 46) at 1, 2 and 3 months of age. NTHi carriage density was measured in swabs by qPCR. Serum PD-IgG levels were measured by bead-based immunoassay., Results: Papua New Guinean children did naturally develop PD-IgG antibodies whose levels were increased at 4 months of age with PCV10 vaccination at 1-2-3 months. Despite this, most children were colonised with NTHi by 4 months of age (~95%) regardless of being vaccinated with PCV10 or PCV13, and PCV10 had no impact on NTHi carriage density., Conclusion: Early vaccination of infants with PCV10 elicited a robust PD antibody response but this had no impact on NTHi carriage., Trial Registration: ClinicalTrials.gov CTN NCT01619462., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: W. S. P. has received funding from Pfizer Australia to attend a conference. A. H. J. v. d. B. was previously an employee of Janssen Pharmaceuticals, Johnson and Johnson, and conducts part-time consultancy work for vaccine companies on projects not related to this study. L. A. K. has received investigator-initiated research grants, educational grants and travel support from Pfizer, GlaxoSmithKline and Merck Sharp & Dohme, and is an inventor on patents for a pneumococcal protein vaccine antigen. D. L. has received support from Pfizer Australia to attend conferences, an honorarium from Merck Vaccines to give a seminar at their offices in Pennsylvania and support from Merck Vaccines to attend a conference; and is an investigator on an investigator-initiated research grant that was funded by Pfizer Australia. P. C. R. has received nonfinancial support from Pfizer, grants from GlaxoSmithKline and Pfizer, and nonfinancial support from GlaxoSmithKline for work outside the submitted work. The Papua New Guinea Institute of Medical Research received sponsorship from Pfizer Australia to host a national Medical Symposium in 2014. All other authors declare no competing interests., (Copyright © 2021. Published by Elsevier Ltd.)

Small volume assays are required for large-scale research studies and in particular paediatric trials, where multiple measures are required from a single sample. Fluorescent bead-based technology (Bioplex/Luminex) allows high through-put and simultaneous quantification of multiple analytes in a single test. This technology uses sets of microspheres, each with a unique spectral address that can be coated with a different antigen of interest. Following the addition of a detector antibody, specific for the isotype of interest and labelled with R-Phycoerythrin, the bioplex reader determines the amounts of antigen-specific antibodies in each test sample relative to a reference standard. Here we outline the optimisations undertaken to establish a 6-plex fluorescent bead-based immunoassay that can accurately measure human IgG to individual tetanus-diphtheria-acellular pertussis (Tdap) antigens from 2 to 4 ul of human serum/ plasma. This protocol was adapted from previously published methods and aligns with current recommendations for developing pertussis-serological assays. To our knowledge, this is the first Tdap-specific multiplex immunoassay (MIA) established in Australia. All components were optimised and validated in-house including: microsphere preparation conditions, reference serum and QC development, and assay running.•Determining optimal antigen coating dose and conjugation method.•Optimising an in-house reference serum with clinically relevant titres.•Determining assay specificity and reproducibility., (© 2021 The Authors.)

Adult pertussis vaccination is increasingly recommended to control pertussis in the community. However, there is little data on the duration and kinetics of immunity to pertussis boosters in adults. We compared IgG responses to vaccination with a tetanus, low-dose diphtheria, low-dose acellular pertussis (Tdap) booster at 1 week, 1 month and 1 year post-vaccination in whole-cell (wP)-primed Australian paediatric healthcare workers who had received an adult Tdap booster 5-12 years previously, to those who received their first Tdap booster. Tdap vaccination was well tolerated in both groups. Previously boosted adults had significantly higher pre-vaccination IgG concentrations for all vaccine-antigens, and more were seropositive for pertussis toxin (PT)-specific IgG (≥ 5 IU/mL) (69.5%; 95% confidence interval (CI) 59.5-79.5) than adults in the naïve group (45.2%; 95% CI 32.8-57.5). Tdap vaccination significantly increased IgG responses 1 month post-vaccination in both groups. This increase was more rapid in previously boosted than in naïve adults, with geometric mean fold-increases in PT-IgG at 1 week post vaccination of 3.6 (95% CI 2.9-4.3) and 2.6 (95% CI 2.2-3.2), respectively. Antibody waning between 1 month and 1 year post-vaccination was similar between groups for IgG specific to PT and filamentous haemagglutinin (FHA), but was faster for IgG against pertactin (PRN) in the naïve group (GMC ratio 0.36; 95% CI 0.31-0.42) than the previously boosted group (GMC ratio 0.45; 95% CI 0.39-0.50). At baseline, all but one adult had protective IgG titres against tetanus toxin (TT) (≥ 0.1 IU/mL), and 75.6% in the previously boosted and 61.3% in the naïve group had protective IgG titres against diphtheria toxoid (DT) of ≥ 0.1 IU/mL. This study shows that pertussis immune memory is maintained up to 12 years after Tdap vaccination in wP-primed Australian adults. There was no evidence that pertussis immune responses waned faster after a booster dose. These findings support current recommendations of repeating Tdap booster vaccination in paediatric healthcare workers at least every 10 years. Clinical trials registry: ACTRN12615001262594., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Peter Richmond (PR) has been an investigator on vaccine trials sponsored by Industry (GSK, Sanofi, Merck, Janssen). PR’s institution has received funding for investigator led studies from Industry (Pfizer, GSK, Technovalia). PR has been a member of scientific vaccine advisory boards for industry (GSK, Sanofi, Pfizer, Novavax, Merck) but has not received any personal payments from Industry. Anita van den Biggelaar is a consultant to BioNet-Asia Co., Ltd. All other authors confirm no conflicts of interest to declare., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nasopharyngeal colonization with nontypeable Haemophilus influenzae (NTHi) is a prerequisite for developing NTHi-associated infections, including otitis media. Therapies that block NTHi colonization may prevent disease development. We previously demonstrated that Haemophilus haemolyticus , a closely related human commensal, can inhibit NTHi colonization and infection of human respiratory epithelium in vitro We have now assessed whether Muribacter muris (a rodent commensal from the same family) can prevent NTHi colonization and disease in vivo using a murine NTHi otitis media model. Otitis media was modeled in BALB/c mice using coinfection with 1 × 10 4.5 PFU of influenza A virus MEM H3N2, followed by intranasal challenge with 5 × 10 7 CFU of NTHi R2866 Spec r Mice were pretreated or not with an intranasal inoculation of 5 × 10 7 CFU M. muris 24 h before coinfection. NTHi and M. muris viable counts and inflammatory mediators (gamma interferon [IFN-γ], interleukin-1β [IL-1β], IL-6, keratinocyte chemoattractant [KC], and IL-10) were measured in nasal washes and middle ear tissue homogenate. M. muris pretreatment decreased the median colonization density of NTHi from 6 × 10 5 CFU/ml to 9 × 10 3 CFU/ml ( P  = 0.0004). Only 1/12 M. muris -pretreated mice developed otitis media on day 5 compared to 8/15 mice with no pretreatment (8% versus 53%, P  = 0.0192). Inflammation, clinical score, and weight loss were also lower in M. muris -pretreated mice. We have demonstrated that a single dose of a closely related commensal can delay onset of NTHi otitis media in vivo Human challenge studies investigating prevention of NTHi colonization are warranted to reduce the global burden of otitis media and other NTHi diseases., (Copyright © 2020 Granland et al.)

Objective: To review and highlight significant advances made towards vaccine development and understanding of the immunology of otitis media (OM) since the 19th International Symposium on Recent Advances in Otitis Media (ISOM) in 2015, as well as identify future research directions and knowledge gaps., Data Sources: PubMed database, National Library of Medicine., Review Methods: Key topics were assigned to each panel member for detailed review. Draft reviews were collated, circulated, and thoroughly discussed when the panel met at the 20th ISOM in June 2019. The final manuscript was prepared with input from all panel members., Conclusions: Since 2015 there have been a number of studies assessing the impact of licensed pneumococcal vaccines on OM. While these studies have confirmed that these vaccines are effective in preventing carriage and/or disease caused by vaccine serotypes, OM caused by non-vaccine serotype pneumococci and other otopathogens remains a significant health care burden globally. Development of multi-species vaccines is challenging but essential to reducing the global burden of OM. Influenza vaccination has been shown to prevent acute OM, and with novel vaccines against nontypeable Haemophilus influenzae (NTHi), Moraxella catarrhalis and Respiratory Syncytial Virus (RSV) in clinical trials, the potential to significantly prevent OM is within reach. Research into alternative vaccine delivery strategies has demonstrated the power of maternal and mucosal vaccination for OM prevention. Future OM vaccine trials must include molecular diagnostics of middle ear effusion, for detection of viruses and bacteria that are persisting in biofilms and to enable accurate assessment of vaccine impact on OM etiology. Understanding population differences in natural and vaccine-induced immune responses to otopathogens is also important for development of the most effective OM vaccines. Improved understanding of the interaction between otopathogens will also advance development of effective therapies and encourage the assessment of the indirect benefits of vaccination., Implications for Practice: While NTHi and M. catarrhalis are the predominant otopathogens, funding opportunities to drive vaccine development for these species are limited due to a focus on prevention of childhood mortality rather than morbidity. Delivery of a comprehensive report on the high financial and social costs of OM, including the potential for OM vaccines to reduce antibiotic use and subsequent development of antimicrobial resistance (AMR), would likely assist in engaging stakeholders to recognize the value of prevention of OM and increase support for efforts on OM vaccine development. Vaccine trials with OM prevention as a clinical end-point are challenging, however a focus on developing assays that measure functional correlates of protection would facilitate OM vaccine development., Competing Interests: Declaration of competing interest LSK has received investigator-initiated grants and travel support from Pfizer and GSK to attend conferences that are not related to this work. LLH has received institutional funding from GSK, Pfizer, and Merck. TFM has patents for Moraxella catarrhalis vaccines and LSK holds patents for pneumolysin as a vaccine antigen. SIP has received investigator-initiated research grants through Boston Medical Center from Merck Vaccines and Pfizer and personal honorarium for participation in advisory boards, expert consultation and symposia from GlaxoSmithKline, Pfizer, and Merck Vaccines. All other authors declare no competing interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: To perform a comprehensive review of the literature from July 2015 to June 2019 on the pathogenesis of otitis media. Bacteria, viruses and the role of the microbiome as well as the host response are discussed. Directions for future research are also suggested., Data Sources: PubMed database of the National Library of Medicine., Review Methods: PubMed was searched for any papers pertaining to OM pathogenesis between July 2015 and June 2019. If in English, abstracts were assessed individually for their relevance and included in the report. Members of the panel drafted the report based on these searches and on new data presented at the 20th International Symposium on Recent Advances in Otitis Media., Conclusions: The main themes that arose in OM pathogenesis were around the need for symptomatic viral infections to develop disease. Different populations potentially having different mechanisms of pathogenesis. Novel bacterial otopathogens are emerging and need to be monitored. Animal models need to continue to be developed and used to understand disease pathogenesis., Implications for Practice: The findings in the pathogenesis panel have several implications for both research and clinical practice. The most urgent areas appear to be to continue monitoring the emergence of novel otopathogens, and the need to develop prevention and preventative therapies that do not rely on antibiotics and protect against the development of the initial OM episode., Competing Interests: Declaration of competing interest Ruth B Thornton: Nothing to disclose. Johanna Nokso-Koivisto: None. Kristian Riesbeck has received recent funding from Pfizer (Sweden), and has taken part in scientific advisory boards arranged by GSK (Belgium) and Merck (USA). Peter Richmond has served on vaccine scientific advisory boards and received institutional funding for investigator-initiated grants, from GlaxoSmithKline and Pfizer. Kenneth L Brockman: Nothing to disclose., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Repeat ventilation tube insertion (VTI) is common in children with recurrent acute otitis media (rAOM). Identifying risk factors associated with repeat surgery will improve clinical management and prevent repeat VTI., Methods: Surgical records were assessed at 8 years following VTI surgery for rAOM in children 6-36 months of age. Children were grouped according to detection of bacterial otopathogen in their middle ear effusion (MEE) at the time of VTI, and outcomes for future otorhinolaryngology surgery compared., Results: Age, gender, pneumococcal vaccination status, antibiotic usage, day-care attendance, number of siblings and number of AOM episodes were similar between groups. Of the 63 children who had PCR +ve MEE, 58.7% required repeat VTI compared with 31.4% of the 51 children with no otopathogen detected in their MEE (odds ratio = 3.1, 95% confidence interval [1.4-6.8]; P = 0.004). Nontypeable Haemophilus influenzae (NTHi) was the predominant otopathogen in MEE (79% of all PCR +ve MEE). Respiratory virus detection was not associated with repeat VTI., Conclusions: Presence of bacterial otopathogen, specifically nontypeable H. influenzae, in the middle ear during VTI was a predictor of children at-risk of repeat VTI. Here, we identify a modifiable microbiologic factor for repeat VTI that can be targeted to improve clinical management of rAOM.

Recurrent and chronic otitis media (OM) are often refractory to antibiotics due to bacterial persistence in biofilm within the middle ear. In vitro and in vivo studies have demonstrated that antimicrobial proteins and peptides (AMPs) are bactericidal against otopathogens, indicating potential therapeutic value for recalcitrant OM. We measured concentrations of 6 AMPs and 14 cytokines in middle ear effusion (MEE) from 67 children undergoing ventilation tube insertion for recurrent acute OM. Sixty one percent of children had bacterial otopathogens detected in their MEE, 39% by PCR and 22% by PCR and culture. Groups were defined as: PCR-negative/culture-negative (absence of bacterial otopathogen), n = 26; PCR-positive/culture-negative (presence of nonculturable bacterial otopathogen), n = 26; PCR-positive/culture-positive (presence of culturable bacterial otopathogen), n = 15. Age, antibiotic usage, day-care attendance, presence of respiratory viruses in MEE and number of AOM episodes were similar between groups. AMP and cytokine concentrations were higher in children with bacterial otopathogens in their MEE compared to those with no bacterial otopathogens. Median concentrations of AMPs (except HBD2) were 3 to 56-fold higher in MEE from children with bacterial otopathogens detected in their MEE (P ≤ 0.01). Similarly, median cytokine concentrations (except TGFβ) were >16-fold higher in MEE with bacterial otopathogens detected (P ≤ 0.001). This is the first study to measure AMPs in MEE and together with the cytokine data, results suggest that elevated AMPs and cytokines in MEE are a marker of inflammation and bacterial persistence. AMPs may play an important role in OM pathogenesis., Competing Interests: Professor Peter Richmond has served on vaccine scientific advisory boards, and together with Dr Kirkham has received institutional funding for investigator-initiated grants from GlaxoSmithKline and Pfizer that are unrelated to this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Indigenous children have much higher rates of ear and lung disease than non-Indigenous children, which may be related to exposure to high levels of geogenic (earth-derived) particulate matter (PM). The aim of this study was to assess the relationship between dust levels and health in Indigenous children in Western Australia (W.A.). Data were from a population-based sample of 1077 Indigenous children living in 66 remote communities of W.A. (>2,000,000 km 2 ), with information on health outcomes derived from carer reports and hospitalisation records. Associations between dust levels and health outcomes were assessed by multivariate logistic regression in a multi-level framework. We assessed the effect of exposure to community sampled PM on epithelial cell (NuLi-1) responses to non-typeable Haemophilus influenzae (NTHi) in vitro. High dust levels were associated with increased odds of hospitalisation for upper (OR 1.77 95% CI [1.02-3.06]) and lower (OR 1.99 95% CI [1.08-3.68]) respiratory tract infections and ear disease (OR 3.06 95% CI [1.20-7.80]). Exposure to PM enhanced NTHi adhesion and invasion of epithelial cells and impaired IL-8 production. Exposure to geogenic PM may be contributing to the poor respiratory health of disadvantaged communities in arid environments where geogenic PM levels are high.

Background: Differentiating bacterial from viral pneumonia is important for guiding targeted management and judicious use of antibiotics. We assessed if clinical characteristics and blood inflammatory biomarkers could be used to distinguish bacterial from viral pneumonia., Methods: Western Australian children (≤17 years) hospitalized with radiologically-confirmed community-acquired pneumonia were recruited and clinical symptoms and management data were collected. C-reactive protein (CRP), white cell counts (WCC) and absolute neutrophil counts (ANC) were measured as part of routine care. Clinical characteristics and biomarker levels were compared between cases with definite bacterial pneumonia (clinical empyema and/or bacteria detected in blood or pleural fluid), presumed viral pneumonia (presence of ≥1 virus in nasopharyngeal swab without criteria for definite bacterial pneumonia), and other pneumonia cases (pneumonia in the absence of criteria for either definite bacterial or presumed viral pneumonia). The area-under-curve (AUC) of the receiver operating characteristic (ROC) curve for varying biomarker levels were used to characterise their utility for discriminating definite bacterial from presumed viral pneumonia. For biomarkers with AUC > 0.8 (fair discriminator), Youden index was measured to determine the optimal cut-off threshold, and sensitivity, specificity, predictive values (positive and negative) were calculated. We investigated whether better discrimination could be achieved by combining biomarker values with the presence/absence of symptoms., Results: From May 2015 to October 2017, 230 pneumonia cases were enrolled: 30 with definite bacterial pneumonia, 118 with presumed viral pneumonia and 82 other pneumonia cases. Differences in clinical signs and symptoms across the groups were noted; more definite bacterial pneumonia cases required intravenous fluid and oxygen supplementation than presumed viral or other pneumonia cases. CRP, WCC and ANC were substantially higher in definite bacterial cases. For a CRP threshold of 72 mg/L, the AUC of ROC was 0.82 for discriminating definite bacterial pneumonia from presumed viral pneumonia. Combining the CRP with either the presence of fever (≥38 ο C) or the absence of rhinorrhea improved the discrimination., Conclusions: Combining elevated CRP with the presence or absence of clinical signs/ symptoms differentiates definite bacterial from presumed viral pneumonia better than CRP alone. Further studies are required to explore combination of biomarkers and symptoms for use as definitive diagnostic tool.

Introduction: Respiratory pathogens associated with childhood pneumonia are often detected in the upper respiratory tract of healthy children, making their contribution to pneumonia difficult to determine. We aimed to determine the contribution of common pathogens to pneumonia adjusting for rates of asymptomatic detection to inform future diagnosis, treatment and preventive strategies., Methods: A case-control study was conducted among children <18 years in Perth, Western Australia. Cases were children hospitalised with radiologically confirmed pneumonia; controls were healthy children identified from outpatient and local immunisation clinics. Nasopharyngeal swabs were collected and tested for 14 respiratory viruses and 6 bacterial species by Polymerase chain reaction (PCR). For each pathogen, adjusted odds ratio (aOR; 95% CI) was calculated using multivariate logistic regression and population-attributable fraction (95% CI) for pneumonia was estimated., Results: From May 2015 to October 2017, 230 cases and 230 controls were enrolled. At least one respiratory virus was identified in 57% of cases and 29% of controls (aOR: 4.7; 95% CI: 2.8 to 7.8). At least one bacterial species was detected in 72% of cases and 80% of controls (aOR: 0.7; 95% CI: 0.4 to 1.2). Respiratory syncytial virus (RSV) detection was most strongly associated with pneumonia (aOR: 58.4; 95% CI: 15.6 to 217.5). Mycoplasma pneumoniae was the only bacteria associated with pneumonia (aOR: 14.5; 95% CI: 2.2 to 94.8). We estimated that RSV, human metapneumovirus (HMPV), influenza, adenovirus and Mycoplasma pneumoniae were responsible for 20.2% (95% CI: 14.6 to 25.5), 9.8% (5.6% to 13.7%), 6.2% (2.5% to 9.7%), 4% (1.1% to 7.1%) and 7.2% (3.5% to 10.8%) of hospitalisations for childhood pneumonia, respectively., Conclusions: Respiratory viruses, particularly RSV and HMPV, are major contributors to pneumonia in Australian children., Competing Interests: Competing interests: PCR receives grants from GlaxoSmithKline, Novavax, Medimmune and Janssen outside the submitted work; L-ASK has a patent WO2005108580A1 licensed to Pfizer., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Otitis media (OM) is a major reason for antibiotic consumption and surgery in children. Nasopharyngeal carriage of otopathogens, Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi), is a prerequisite for development of OM, and increased nasopharyngeal otopathogen density correlates with disease onset. Vaccines can reduce or eliminate otopathogen carriage, as demonstrated for pneumococcal serotypes included in pneumococcal conjugate vaccines (PCV). The 10-valent PCV (PCV10) includes an NTHi carrier protein, and in 2011 superseded 7-valent PCV on the New Zealand Immunisation Program. Data are conflicting on whether PCV10 provides protection against NTHi carriage or disease. Assessing this in otitis-prone cohorts is important for OM prevention. We compared otopathogen density in the nasopharynx and middle ear of New Zealand PCV7-vaccinated and PCV10-vaccinated otitis-prone and non-otitis-prone children to determine PCV10 impact on NTHi and S. pneumoniae carriage. We applied qPCR to specimens collected from 217 PCV7-vaccinated children (147 otitis-prone and 70 non-otitis-prone) and 240 PCV10-vaccinated children (178 otitis-prone and 62 non-otitis-prone). After correcting for age and day-care attendance, no difference was observed between NTHi density in the nasopharynx of PCV7-vaccinated versus PCV10-vaccinated otitis-prone ( p = 0.563) or non-otitis-prone ( p = 0.513) children. In contrast, pneumococcal nasopharyngeal density was higher in PCV10-vaccinated otitis-prone children than PCV7-vaccinated otitis-prone children ( p = 0.003). There was no difference in otopathogen density in middle ear effusion from PCV7-vaccinated versus PCV10-vaccinated otitis-prone children (NTHi p = 0.918; S. pneumoniae p = 0.415). When pneumococcal carriage was assessed by vaccine serotypes (VT) and non-vaccine serotypes (NVT), there was no difference in VT density ( p = 0.546) or NVT density ( p = 0.315) between all PCV7-vaccinated versus all PCV10-vaccinated children. In summary, PCV10 did not reduce NTHi density in the nasopharynx or middle ear, and was associated with increased pneumococcal nasopharyngeal density in otitis-prone children in New Zealand. Development of therapies that prevent or reduce otopathogen colonisation density in the nasopharynx are warranted to reduce the burden of OM.

Exposure to environmental geogenic (or earth-derived) dust can lead to more frequent and severe infections in the human airway. Particulate matter < 10 µm (PM 10 ) is the component of air pollution that is commonly associated with the exacerbation of respiratory diseases. We have previously demonstrated that mice exposed to geogenic dust PM 10 experienced an exacerbation of inflammatory responses to influenza A virus. Whether geogenic dust PM 10 also exacerbates respiratory bacterial infection is not yet known, nor are the components of the dust that drive these responses. We treated airway bronchial epithelial cells (NuLi-1) with UV-irradiated geogenic dust PM 10 from six remote Western Australian towns. High levels of IL-6 and IL-8 production were observed, as well as persistent microbial growth. 16 S rRNA sequencing of the growth identified the microbe as Bacillus licheniformis, a spore-forming, environmentally abundant bacterium. We next investigated the interaction of B. licheniformis with respiratory epithelium in vitro to determine whether this exacerbated infection with a bacterial respiratory pathogen (non-typeable Haemophilus influenzae, NTHi). Heat treatment (100 °C) of all PM 10 samples eliminated B. licheniformis contamination and reduced epithelial inflammatory responses, suggesting that heat-labile and/or microbial factors were involved in the host response to geogenic dust PM 10 . We then exposed NuLi-1 epithelium to increasing doses of the isolated Bacillus licheniformis (multiplicity of infection of 10:1, 1:1 or 0.1:1 bacteria: cells) for 1, 3, and 24 h. B. licheniformis and NTHi infection (association and invasion) was assessed using a standard gentamicin survival assay, and epithelial release of IL-6 and IL-8 was measured using a bead based immunoassay. B. licheniformis was cytotoxic to NuLi-1 cells at 24 h. At 3 h post-challenge, B. licheniformis elicited high IL-6 and IL-8 inflammatory responses from NuLi-1 cells compared with cells treated with heat-treated geogenic dust PM 10 (p < 0.0001). Whilst treatment of cells with B. licheniformis increased inflammation, this did not make the cells more susceptible to NTHi infection. This study highlights that geogenic dust PM 10 can harbour viable bacterial spores that induce inflammation in respiratory epithelium. The impact on respiratory health from inhalation of bacterial spores in PM 10 in arid environments may be underestimated. Further investigation into the contribution of B. licheniformis and the wider dust microbiome to respiratory infection is warranted., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Otitis media (OM) remains a common paediatric disease, despite advances in vaccinology. Susceptibility to recurrent acute OM (rAOM) has been postulated to involve defective cell-mediated immune responses to common otopathogenic bacteria. We compared the composition of peripheral blood mononuclear cells (PBMC) from 20 children with a history of rAOM (otitis-prone) and 20 healthy non-otitis-prone controls, and assessed innate and cell-mediated immune responses to the major otopathogen nontypeable Haemophilus influenzae (NTHi). NTHi was a potent stimulator of inflammatory cytokine secretion from PBMC within 4 hours, with no difference in cytokine levels produced between PBMC from cases or controls. In the absence of antigen stimulation, otitis-prone children had more circulating Natural Killer (NK) cells (p<0.01), particularly NKdim (CD56lo) cells (p<0.01), but fewer CD4+ T cells (p<0.01) than healthy controls. NTHi challenge significantly increased the proportion of activated (CD107a+) NK cells in otitis-prone and non-otitis-prone children (p<0.01), suggesting that NK cells from otitis-prone children are functional and respond to NTHi. CD8+ T cells and NK cells from both cases and controls produced IFNγ in response to polyclonal stimulus (Staphylococcal enterotoxin B; SEB), with more IFNγ+ CD8+ T cells present in cases than controls (p<0.05) but similar proportions of IFNγ+ NK cells. Otitis-prone children had more circulating IFNγ-producing NK cells (p<0.05) and more IFNγ-producing CD4+ (p<0.01) or CD8+ T-cells (p<0.05) than healthy controls. In response to SEB, more CD107a-expressing CD8+ T cells were present in cases than controls (p<0.01). Despite differences in PBMC composition, PBMC from otitis-prone children mounted innate and T cell-mediated responses to NTHi challenge that were comparable to healthy children. These data provide evidence that otitis-prone children do not have impaired functional cell mediated immunity.

Background: Children receiving immunosuppressive treatment for cancer are at high risk for invasive pneumococcal disease. The 13-valent pneumococcal conjugate vaccine (PCV13) can prevent pneumococcal disease in healthy children; however, there is an absence of literature regarding the benefit of PCV13 in immunocompromised children with cancer., Methods: A prospective, open-label cohort study recruited children between ages 1 and 18 years who were receiving active immunosuppressive therapy (AIT) or were within 12 months after completing immunosuppressive therapy (CIT). Blood samples were taken before and 4 weeks after the administration of single-dose PCV13. Serotype-specific immunoglobulin G antibody titers were measured, and titers ≥0.35 μg/mL were considered protective. Solicited side effects were recorded in a 7-day diary after vaccination., Results: Eighty-five children were recruited. At baseline, ≤50% had protective antibody titers against Streptococcus pneumoniae for 10 serotypes in the AIT group and for 8 serotypes in the CIT group. Postvaccination, ≥70% had protective antibody titers for 9 and 11 serotypes in the AIT and CIT groups, respectively. Both groups had comparable responses to PCV7 serotypes, whereas a significantly higher proportion in the CIT group achieved protective antibody titers to PCV13 serotypes. There was a low rate of serious adverse events (3.5%)., Conclusions: A single-dose of PCV13 is safe and immunogenic in children diagnosed with cancer. All children who are receiving therapy for cancer should receive a single dose of PCV13 as soon as possible after diagnosis, regardless of prior PCV exposure. The current data support the recommendation for an additional dose of PCV13 after the completion of immunosuppressive therapy to provide additional protection against invasive pneumococcal disease. Cancer 2017;123:4215-4223. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Inflammation of the middle ear, known clinically as chronic otitis media, presents in different forms, such as chronic otitis media with effusion (COME; glue ear) and chronic suppurative otitis media (CSOM). These are highly prevalent diseases, especially in childhood, and lead to significant morbidity worldwide. However, much remains unclear about this disease, including its aetiology, initiation and perpetuation, and the relative roles of mucosal and leukocyte biology, pathogens, and Eustachian tube function. Chronic otitis media is commonly modelled in mice but most existing models only partially mimic human disease and many are syndromic. Nevertheless, these models have provided insights into potential disease mechanisms, and have implicated altered immune signalling, mucociliary function and Eustachian tube function as potential predisposing mechanisms. Clinical studies of chronic otitis media have yet to implicate a particular molecular pathway or mechanism, and current human genetic studies are underpowered. We also do not fully understand how existing interventions, such as tympanic membrane repair, work, nor how chronic otitis media spontaneously resolves. This Clinical Puzzle article describes our current knowledge of chronic otitis media and the existing research models for this condition. It also identifies unanswered questions about its pathogenesis and treatment, with the goal of advancing our understanding of this disease to aid the development of novel therapeutic interventions., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Indigenous populations experience high rates of otitis media (OM), with increased chronicity and severity, compared to those experienced by their nonindigenous counterparts. Data on immune responses to otopathogenic bacteria in these high-risk populations are lacking. Nontypeable Haemophilus influenzae (NTHi) is the predominant otopathogen in Australia. No vaccines are currently licensed to target NTHi; however, protein D (PD) from NTHi is included as a carrier protein in the 10-valent pneumococcal polysaccharide conjugate vaccine (PHiD10-CV), and other promising protein vaccine candidates exist, including outer membrane protein 4 (P4) and protein 6 (P6). We measured the levels of serum and salivary IgA and IgG against PD, P4, and P6 in Aboriginal and non-Aboriginal children with chronic OM who were undergoing surgery and compared the levels with those in healthy non-Aboriginal children (controls). We found that Aboriginal cases had lower serum IgG titers to all NTHi proteins assessed, particularly PD. In contrast, serum IgA and salivary IgA and IgG titers to each of these 3 proteins were equivalent to or higher than those in both non-Aboriginal cases and healthy controls. While serum antibody levels increased with age in healthy controls, no changes in titers were observed with age in non-Aboriginal cases, and a trend toward decreasing titers with age was observed in Aboriginal cases. This suggests that decreased serum IgG responses to NTHi outer membrane proteins may contribute to the development of chronic and severe OM in Australian Aboriginal children and other indigenous populations. These data are important for understanding the potential benefits of PHiD10-CV implementation and the development of NTHi protein-based vaccines for indigenous populations., (Copyright © 2017 Thornton et al.)

The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group ( P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children., (Copyright © 2017 Kirkham et al.)

Background: The Australian Aboriginal population experiences disproportionately high rates of otitis media (OM). Streptococcus pneumoniae is one of the main pathogens responsible for OM and currently no vaccine offering cross strain protection exists. Vaccines consisting of conserved antigens to S. pneumoniae may reduce the burden of OM in high-risk populations; however no data exists examining naturally acquired antibody in Aboriginal children with OM., Methods: Serum and salivary IgA and IgG were measured against the S. pneumoniae antigens PspA1 and 2, CbpA and Ply in a cross sectional study of 183 children, including 36 non-Aboriginal healthy control children and 70 Aboriginal children and 77 non-Aboriginal children undergoing surgery for OM using a multiplex bead assay., Results: Significant differences were observed between the 3 groups for serum anti-PspA1 IgA, anti-CbpA and anti-Ply IgG and for all salivary antibodies assessed. Aboriginal children with a history of OM had significantly higher antibody titres than non-Aboriginal healthy children with no history of OM and non-Aboriginal children with a history of OM for several proteins in serum and saliva. Non-Aboriginal children with a history of OM had significantly higher salivary anti-PspA1 IgG than healthy children, while all other titres were comparable between the groups., Conclusions: Conserved vaccine candidate proteins from S. pneumoniae induce serum and salivary antibody responses in Aboriginal and non-Aboriginal children with a history of OM. Aboriginal children do not have an impaired antibody response to the antigens measured from S. pneumoniae and they may represent vaccine candidates in Indigenous populations., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

We have developed a specific Haemophilus influenzae quantitative PCR (qPCR) that also identifies fucose-negative and protein D-negative strains. Analysis of 100 H. influenzae isolates, 28 Haemophilus haemolyticus isolates, and 14 other bacterial species revealed 100% sensitivity (95% confidence interval [CI], 96% to 100%) and 100% specificity (95% CI, 92% to 100%) for this assay. The evaluation of 80 clinical specimens demonstrated a strong correlation between semiquantitative culture and the qPCR (P < 0.001)., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that resides in the upper respiratory tract and contributes to a significant burden of respiratory related diseases in children and adults. Haemophilus haemolyticus is a respiratory tract commensal that can be misidentified as NTHi due to high levels of genetic relatedness. There are reports of invasive disease from H. haemolyticus, which further blurs the species boundary with NTHi. To investigate differences in pathogenicity between these species, we optimized an in vitro epithelial cell model to compare the interaction of 10 H. haemolyticus strains with 4 NTHi and 4 H. influenzae-like haemophili. There was inter- and intra-species variability but overall, H. haemolyticus had reduced capacity to attach to and invade nasopharyngeal and bronchoalveolar epithelial cell lines (D562 and A549) within 3 h when compared with NTHi. H. haemolyticus was cytotoxic to both cell lines at 24 h, whereas NTHi was not. Nasopharyngeal epithelium challenged with some H. haemolyticus strains released high levels of inflammatory mediators IL-6 and IL-8, whereas NTHi did not elicit an inflammatory response despite higher levels of cell association and invasion. Furthermore, peripheral blood mononuclear cells stimulated with H. haemolyticus or NTHi released similar and high levels of IL-6, IL-8, IL-10, IL-1β, and TNFα when compared with unstimulated cells but only NTHi elicited an IFNγ response. Due to the relatedness of H. haemolyticus and NTHi, we hypothesized that H. haemolyticus may compete with NTHi for colonization of the respiratory tract. We observed that in vitro pre-treatment of epithelial cells with H. haemolyticus significantly reduced NTHi attachment, suggesting interference or competition between the two species is possible and warrants further investigation. In conclusion, H. haemolyticus interacts differently with host cells compared to NTHi, with different immunostimulatory and cytotoxic properties. This study provides an in vitro model for further investigation into the pathogenesis of Haemophilus species and the foundation for exploring whether H. haemolyticus can be used to prevent NTHi disease.

Background: Otitis media (OM) is amongst the most common childhood diseases and is associated with multiple microbial pathogens within the middle ear. Global and temporal monitoring of predominant bacterial pathogens is important to inform new treatment strategies, vaccine development and to monitor the impact of vaccine implementation to improve progress toward global OM prevention., Methods: A systematic review of published reports of microbiology of acute otitis media (AOM) and otitis media with effusion (OME) from January, 1970 to August 2014, was performed using PubMed databases., Results: This review confirmed that Streptococcus pneumoniae and Haemophilus influenzae, remain the predominant bacterial pathogens, with S. pneumoniae the predominant bacterium in the majority reports from AOM patients. In contrast, H. influenzae was the predominant bacterium for patients experiencing chronic OME, recurrent AOM and AOM with treatment failure. This result was consistent, even where improved detection sensitivity from the use of polymerase chain reaction (PCR) rather than bacterial culture was conducted. On average, PCR analyses increased the frequency of detection of S. pneumoniae and H. influenzae 3.2 fold compared to culture, whilst Moraxella catarrhalis was 4.5 times more frequently identified by PCR. Molecular methods can also improve monitoring of regional changes in the serotypes and identification frequency of S. pneumoniae and H. influenzae over time or after vaccine implementation, such as after introduction of the 7-valent pneumococcal conjugate vaccine., Conclusions: Globally, S. pneumoniae and H. influenzae remain the predominant otopathogens associated with OM as identified through bacterial culture; however, molecular methods continue to improve the frequency and accuracy of detection of individual serotypes. Ongoing monitoring with appropriate detection methods for OM pathogens can support development of improved vaccines to provide protection from the complex combination of otopathogens within the middle ear, ultimately aiming to reduce the risk of chronic and recurrent OM in vulnerable populations.

Background: The presence of Pseudomonas aeruginosa biofilms in lower airway specimens from cystic fibrosis (CF) patients is well established. To date, biofilm has not been demonstrated in bronchoalveolar lavage (BAL) from people with non-CF bronchiectasis. The aim of this study was to determine (i) if biofilm was present in BAL from children with and without bronchiectasis, and (ii) if biofilm detection differed between sequentially collected BAL., Methods: Testing for biofilm in two sequentially collected BAL from children with and without bronchiectasis was done using BacLight™ live-dead staining and lectin staining for extracellular polymeric biofilm matrices. Bacterial culture and cytological measures were performed on the first and second lavages, respectively. Clinically important BAL infection was defined as >10 4  cfu of respiratory pathogens/ml BAL., Results: Biofilm was detected in BAL from seven of eight (87.5%) children with bronchiectasis (aged 0.8-6.9 years), but was not detected in any of three controls (aged 1.3-8.6 years). The biofilms contained both live and dead bacteria irrespective of antibiotic use prior to bronchoscopy. Biofilm was detected more frequently in the second lavage than the first. Three of the seven biofilm-positive BAL were culture-positive for respiratory pathogens at clinically important levels., Conclusions: Biofilm is present in BAL from children with non-CF bronchiectasis even when BAL-defined clinically important infection was absent. Studies to characterize lower airway biofilms and determine how biofilm contributes to bronchiectasis disease progression and treatment outcomes are necessary. Pediatr Pulmonol. 2015; 50:284-292. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)

Background: Otitis media (OM) is a common childhood disease characterised by middle ear effusion and inflammation. Susceptibility to recurrent acute OM and chronic OM with effusion is 40-70% heritable. Linkage studies provide evidence for multiple putative OM susceptibility loci. This study attempts to replicate these linkages in a Western Australian (WA) population, and to identify the etiological gene(s) in a replicated region., Methods: Microsatellites were genotyped in 468 individuals from 101 multicase families (208 OM cases) from the WA Family Study of OM (WAFSOM) and non-parametric linkage analysis carried out in ALLEGRO. Association mapping utilized dense single nucleotide polymorphism (SNP) data extracted from Illumina 660 W-Quad analysis of 256 OM cases and 575 controls from the WA Pregnancy Cohort (Raine) Study. Logistic regression analysis was undertaken in ProbABEL. RT-PCR was used to compare gene expression in paired adenoid and tonsil samples, and in epithelial and macrophage cell lines. Comparative genomics methods were used to identify putative regulatory elements and transcription factor binding sites potentially affected by associated SNPs., Results: Evidence for linkage was observed at 10q26.3 (Zlr = 2.69; P = 0.0036; D10S1770) with borderline evidence for linkage at 10q22.3 (Zlr = 1.64; P = 0.05; D10S206). No evidence for linkage was seen at 3p25.3, 17q12, or 19q13.43. Peak association at 10q26.3 was in the intergenic region between TCERG1L and PPP2R2D (rs7922424; P = 9.47 × 10-6), immediately under the peak of linkage. Independent associations were observed at DOCK1 (rs9418832; P = 7.48 × 10-5) and ADAM12 (rs7902734; P = 8.04 × 10-4). RT-PCR analysis confirmed expression of all 4 genes in adenoid samples. ADAM12, DOCK1 and PPP2R2D, but not TCERG1L, were expressed in respiratory epithelial and macrophage cell lines. A significantly associated polymorphism (rs7087384) in strong LD with the top SNP (rs7922424; r2 = 0.97) alters a transcription factor binding site (CREB/CREBP) in the intergenic region between TCERG1L and PPP2R2D., Conclusions: OM linkage was replicated at 10q26.3. Whilst multiple genes could contribute to this linkage, the weight of evidence supports PPP2R2D, a TGF-β/Activin/Nodal pathway modulator, as the more likely functional candidate lying immediately under the linkage peak for OM susceptibility at chromosome 10q26.3.

Background: Indigenous Australian children living in remote communities experience high rates of acute otitis media with tympanic membrane perforation (AOMwiP). Otitis media in this population is associated with dense nasopharyngeal colonization of three primary otopathogens; Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis. Little is known about the relative abundance of these pathogens during infection. The objective of this study was to estimate the abundance and concordance of otopathogens in ear discharge and paired nasopharyngeal swabs from children with AOMwiP (discharge of not more than 6 weeks' duration and perforation size <2%)., Methods: Culture and quantitative PCR (qPCR) estimation of H. influenzae, S. pneumoniae, M. catarrhalis and total bacterial load were performed on paired nasopharyngeal and ear discharge swabs from 55 Indigenous children with AOMwiP aged 3.5 - 45.6 months and resident in remote communities., Results: By culture, H. influenzae, S. pneumoniae, and M. catarrhalis were detected in 80%, 84% and 91% of nasopharyngeal swabs, and 49%, 33% and 4% of ear discharge swabs, respectively. Using qPCR, H. influenzae, S. pneumoniae, and M. catarrhalis were detected in 82%, 82%, and 93% of nasopharyngeal swabs, and 89%, 41% and 18% of ear discharge swabs, respectively. Relative abundance of H. influenzae in ear discharge swabs was 0-68% of the total bacterial load (median 2.8%); whereas S. pneumoniae and M. catarrhalis relative abundances were consistently <2% of the total bacterial load. S. pneumoniae and M. catarrhalis abundances were significantly lower in ear discharge compared with nasopharyngeal swabs (p = 0.001, p < 0.001); no significant difference was observed in H. influenzae mean abundance at the two sites., Conclusions: H. influenzae was the dominant otopathogen detected in ear discharge swabs collected from children with AOMwiP. High prevalence and abundance of S. pneumoniae and M. catarrhalis in the nasopharynx did not predict ear discharge prevalence and abundances of these pathogens. PCR was substantially more sensitive than culture for ear discharge, and a necessary adjunct to standard microbiology. Quantitative methods are required to understand species abundance in polymicrobial infections and may be needed to measure accurately the microbiological impact of interventions and to provide a better understanding of clinical failure in these children.

Recurrent acute otitis media (AOM), frequently caused by Streptococcus pneumoniae, is a major paediatric health problem. A reduced antibody response against pneumococcal polysaccharides may contribute to an increased susceptibility to AOM. Using a multiplex bead-based assay we measured IgG, IgG1 and IgG2 levels against 11 pneumococcal polysaccharides in serum samples from 166 children below 3 years of age with a history of at least 3 episodes of acute otitis media receiving ventilation tubes, and 61 healthy controls. Pneumococcal serotype specific IgG was also determined in 144 middle ear effusion samples. Pneumococcal serotype specific IgG, IgG1 and IgG2 levels were similar in children with or without AOM, except for IgG and IgG1 levels against serotype 5, which were significantly higher in children with a history of frequent AOM (IgG: 137.5 μg/ml vs. 84.0 μg/ml; p=0.02; IgG1: 24.5 μg/ml vs. 18.2 μg/ml; p=0.05). The age-related development of pneumococcal serotype-specific IgG, IgG1 and IgG2 levels was similar in children with or without a history of AOM. Pneumococcal serotype specific IgG was present in middle ear effusion and these levels correlated significantly with serum titres. Children with a history of frequent AOM receiving ventilation tubes do not have a deficient IgG, IgG1 or IgG2 response against pneumococcal polysaccharides, either induced by vaccination or due to natural exposure. The strong correlation between IgG levels in serum and the middle ear suggests parenteral pneumococcal conjugate vaccination induces antibodies in the middle ear which may therefore contribute to reducing the burden of AOM., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Bacteria persist within biofilms on the middle ear mucosa of children with recurrent and chronic otitis media however the mechanisms by which these develop remain to be elucidated. Biopsies can be difficult to obtain from children and their small size limits analysis., Methods: In this study we aimed to investigate biofilm presence in middle ear effusion (MEE) from children with recurrent acute otitis media (rAOM) and to determine if these may represent infectious reservoirs similarly to those on the mucosa. We examined this through culture, viability staining and fluorescent in situ hybridisation (FISH) to determine bacterial species present. Most MEEs had live bacteria present using viability staining (32/36) and all effusions had bacteria present using the universal FISH probe (26/26). Of these, 70% contained 2 or more otopathogenic species. Extensive DNA stranding was also present. This DNA was largely host derived, representing neutrophil extracellular traps (NETs) within which live bacteria in biofilm formations were present. When treated with the recombinant human deoxyribonuclease 1, Dornase alfa, these strands were observed to fragment., Conclusions: Bacterial biofilms, composed of multiple live otopathogenic species can be demonstrated in the MEEs of children with rAOM and that these contain extensive DNA stranding from NETs. The NETs contribute to the viscosity of the effusion, potentially contributing to its failure to clear as well as biofilm development. Our data indicates that Dornase alfa can fragment these strands and may play a role in future chronic OM treatment.

It has been suggested that otitis-prone children have an impaired antibody response. To investigate this in the context of pneumococcal vaccination, we used a multiplex bead-based assay to measure serum IgG and IgA levels against pneumococcal serotypes included in the 7-valent pneumococcal conjugate vaccine (PCV7; serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) and 4 non-PCV7 serotypes (1, 5, 7F and 19A) in healthy (n=43) and otitis-prone children (n=75) before, 6 weeks after and 1 year after vaccination with one dose of PCV7. Pre-vaccination, otitis-prone children had significantly higher serum IgG levels against serotypes 4, 9V and 23F and against all non-PCV7 serotypes. One year following vaccination, there was no difference in IgG or IgA levels between healthy and otitis-prone children. The effect of the administration of one or two doses of PCV7 was investigated in otitis-prone children. After a second dose of PCV7, pneumococcal serotype specific IgG levels, but not IgA titres, were higher compared to the levels measured after the initial dose of PCV7. One year post PCV7 vaccination there was no difference in either IgG or IgA antibody levels to any of the PCV7 serotypes between children who received either one or two doses of PCV7. The finding that otitis-prone children do not have an impaired pneumococcal serotype-specific serum IgG or IgA response suggests that new pneumococcal conjugate vaccines may be immunogenic in otitis-prone children, however, further investigations are necessary to determine the clinical impact of such vaccines against the development of recurrent acute otitis media., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Bacteria which are metabolically active yet unable to be cultured and eradicated by antibiotic treatment are present in the middle ear effusion of children with chronic otitis media with effusion (COME) and recurrent acute otitis media (rAOM). These observations are suggestive of biofilm presence or intracellular sequestration of bacteria and may play a role in OM pathogenesis. The aim of this project is to provide evidence for the presence of otopathogenic bacteria intracellularly or within biofilm in the middle ear mucosa of children with COME or rAOM., Methods: Middle ear mucosal biopsies from 20 children with COME or rAOM were examined for otopathogenic bacteria (either in biofilm or located intracellularly) using transmission electron microscopy (TEM) or species specific fluorescent in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM). One healthy control biopsy from a child undergoing cochlear implant surgery was also examined., Results: No bacteria were observed in the healthy control sample. In 2 of the 3 biopsies imaged using TEM, bacteria were observed in mucus containing vacuoles within epithelial cells. Bacterial species within these could not be identified and biofilm was not observed. Using FISH with CLSM, bacteria were seen in 15 of the 17 otitis media mucosal specimens. In this group, 11 (65%) of the 17 middle ear mucosal biopsies showed evidence of bacterial biofilm and 12 demonstrated intracellular bacteria. 52% of biopsies were positive for both biofilm and intracellular bacteria. At least one otopathogen was identified in 13 of the 15 samples where bacteria were present. No differences were observed between biopsies from children with COME and those with rAOM., Conclusion: Using FISH and CLSM, bacterial biofilm and intracellular infection with known otopathogens are demonstrated on/in the middle ear mucosa of children with COME and/or rAOM. While their role in disease pathogenesis remains to be determined, this previously undescribed infection pattern may help explain the ineffectiveness of current treatment strategies at preventing or resolving COME or rAOM.

Background: There is evidence that the specificity of the IgE binding in allergy tests can vary for different populations., Objective: We aimed to examine the allergenic specificity of IgE binding in sera from house dust mite (HDM)-atopic subjects in a tropical Australian Aboriginal community., Methods: Sera shown to contain IgE antibodies to an HDM extract of Dermatophagoides pteronyssinus were examined for IgE binding to a panel of nine purified HDM allergens from this mite species by quantitative microtitre assays. IgG antibody binding (IgG1 and IgG4) was also measured., Results: The IgE-binding activity in the sera from the Aboriginal community was not directed to the expected major groups 1 and 2 HDM allergens but instead to the group 4 amylase allergen. There was also little IgE binding to the potentially cross-reactive tropomyosin (Der p 10) or arginine kinase (Der p 20) allergens. The IgG4 antibody was rarely detected and limited to the Der p 4 allergen. IgG1 antibody binding was frequently measured to all the allergens regardless of an individual's atopic status, whereas in urban communities it is restricted to the major allergens and to atopic subjects., Conclusion: The high IgE anti-HDM response of Australian Aboriginals predominantly bound Der p 4 and not the Der p 1 and 2 allergens, showing a distinctive allergy that could affect the disease outcome and diagnosis.

The Lifting Stress Calculator is a microprocessor-based device, programmed to select from a group of workers the one best suited for a specified lifting task. It also prompts this worker with the ideal position for him to lift the load in question. The concept is based on a theoretical study that deals with lifting as a total body exertion. The goal of this device is to educate the worker for better prevention of lifting injuries by minimizing the loads on the body.