ISOM 2023 research Panel 4 - Diagnostics and microbiology of otitis media.

OBJECTIVES: To identify and review key research advances from the literature published between 2019 and 2023 on the diagnosis and microbiology of otitis media (OM) including acute otitis media (AOM), recurrent AOM (rAOM), otitis media with effusion (OME), chronic suppurative otitis media (CSOM) and AOM complications (mastoiditis). DATA SOURCES: PubMed database of the National Library of Medicine. REVIEW METHODS: All relevant original articles published in Medline in English between July 2019 and February 2023 were identified. Studies that were reviews, case studies, relating to OM complications (other than mastoiditis), and studies focusing on guideline adherence, and consensus statements were excluded. Members of the panel drafted the report based on these search results. MAIN FINDINGS: For the diagnosis section, 2294 unique records screened, 55 were eligible for inclusion. For the microbiology section 705 unique records were screened and 137 articles were eligible for inclusion. The main themes that arose in OM diagnosis were the need to incorporate multiple modalities including video-otoscopy, tympanometry, telemedicine and artificial intelligence for accurate diagnoses in all diagnostic settings. Further to this, was the use of new, cheap, readily available tools which may improve access in rural and lowmiddle income (LMIC) settings. For OM aetiology, PCR remains the most sensitive method for detecting middle ear pathogens with microbiome analysis still largely restricted to research use. The global pandemic response reduced rates of OM in children, but post-pandemic shifts should be monitored. IMPLICATION FOR PRACTICE AND FUTURE RESEARCH: Cheap, easy to use multi-technique assessments combined with artificial intelligence and/or telemedicine should be integrated into future practice to improve diagnosis and treatment pathways in OM diagnosis. Longitudinal studies investigating the in-vivo process of OM development, timings and in-depth interactions between the triad of bacteria, viruses and the host immune response are still required. Standardized methods of collection and analysis for microbiome studies to enable inter-study comparisons are required. There is a need to target underlying biofilms if going to effectively prevent rAOM and OME and possibly enhance ventilation tube retention.

Amitriptyline at Low-Dose and Titrated for Irritable Bowel Syndrome as Second-Line Treatment in primary care (ATLANTIS): a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Most patients with irritable bowel syndrome (IBS) are managed in primary care. When first-line therapies for IBS are ineffective, the UK National Institute for Health and Care Excellence guideline suggests considering low- dose tricyclic antidepressants as second-line treatment, but their effectiveness in primary care is unknown, and they are infrequently prescribed in this setting. METHODS: This randomised, double-blind, placebo-controlled trial (Amitriptyline at Low-Dose and Titrated for Irritable Bowel Syndrome as Second-Line Treatment [ATLANTIS]) was conducted at 55 general practices in England. Eligible participants were aged 18 years or older, with Rome IV IBS of any subtype, and ongoing symptoms (IBS Severity Scoring System [IBS-SSS] score ≥75 points) despite dietary changes and first-line therapies, a normal full blood count and C-reactive protein, negative coeliac serology, and no evidence of suicidal ideation. Participants were randomly assigned (1:1) to low-dose oral amitriptyline (10 mg once daily) or placebo for 6 months, with dose titration over 3 weeks (up to 30 mg once daily), according to symptoms and tolerability. Participants, their general practitioners, investigators, and the analysis team were all masked to allocation throughout the trial. The primary outcome was the IBS-SSS score at 6 months. Effectiveness analyses were according to intention-to-treat; safety analyses were on all participants who took at least one dose of the trial medication. This trial is registered with the ISRCTN Registry (ISRCTN48075063) and is closed to new participants. FINDINGS: Between Oct 18, 2019, and April 11, 2022, 463 participants (mean age 48·5 years [SD 16·1], 315 [68%] female to 148 [32%] male) were randomly allocated to receive low-dose amitriptyline (232) or placebo (231). Intention-to-treat analysis of the primary outcome showed a significant difference in favour of low-dose amitriptyline in IBS-SSS score between groups at 6 months (-27·0, 95% CI -46·9 to -7·10; p=0·0079). 46 (20%) participants discontinued low-dose amitriptyline (30 [13%] due to adverse events), and 59 (26%) discontinued placebo (20 [9%] due to adverse events) before 6 months. There were five serious adverse reactions (two in the amitriptyline group and three in the placebo group), and five serious adverse events unrelated to trial medication. INTERPRETATION: To our knowledge, this is the largest trial of a tricyclic antidepressant in IBS ever conducted. Titrated low-dose amitriptyline was superior to placebo as a second-line treatment for IBS in primary care across multiple outcomes, and was safe and well tolerated. General practitioners should offer low-dose amitriptyline to patients with IBS whose symptoms do not improve with first-line therapies, with appropriate support to guide patient-led dose titration, such as the self-titration document developed for this trial. FUNDING: National Institute for Health and Care Research Health Technology Assessment Programme (grant reference 16/162/01).

The Respiratory Microbiome in Paediatric Chronic Wet Cough: What Is Known and Future Directions.

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.

Evidence of maternal transfer of antigen-specific antibodies in serum and breast milk to infants at high-risk of S. pneumoniae and H. influenzae disease.

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.

Prevalence and subtyping of biofilms present in bronchoalveolar lavage from children with protracted bacterial bronchitis or non-cystic fibrosis bronchiectasis: a cross-sectional study.

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 104 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.

Predominant Bacterial and Viral Otopathogens Identified Within the Respiratory Tract and Middle Ear of Urban Australian Children Experiencing Otitis Media Are Diversely Distributed.

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.

Australian Aboriginal Otitis-Prone Children Produce High-Quality Serum IgG to Putative Nontypeable Haemophilus influenzae Vaccine Antigens at Lower Titres Compared to Non-Aboriginal Children.

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.

Sleep Disordered Breathing and Recurrent Tonsillitis Are Associated With Polymicrobial Bacterial Biofilm Infections Suggesting a Role for Anti-Biofilm Therapies.

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.

Differences in Pneumococcal and Haemophilus influenzae Natural Antibody Development in Papua New Guinean Children in the First Year of Life.

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.

Optimising a 6-plex tetanus-diphtheria-pertussis fluorescent bead-based immunoassay.

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.

PCV10 elicits Protein D IgG responses in Papua New Guinean children but has no impact on NTHi carriage in the first two years of life.

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.

An observational study of antibody responses to a primary or subsequent pertussis booster vaccination in Australian healthcare workers.

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.

Pneumococcal conjugate vaccine primes mucosal immune responses to pneumococcal polysaccharide vaccine booster in Papua New Guinean children.

INTRODUCTION: Invasive pneumococcal disease remains a major cause of hospitalization and death in Papua New Guinean (PNG) children. We assessed mucosal IgA and IgG responses in PNG infants vaccinated with pneumococcal conjugate vaccine (PCV) followed by a pneumococcal polysaccharide vaccine (PPV) booster. METHODS: Infants received 7-valent PCV (7vPCV) in a 0-1-2 (neonatal) or 1-2-3-month (infant) schedule, or no 7vPCV (control). At age 9 months all children received 23-valent PPV (23vPPV). IgA and IgG to 7vPCV and non-7vPCV (1, 5, 7F, 19A) serotypes were measured in saliva collected at ages 1, 2, 3, 4, 9, 10 and 18 months (131 children, 917 samples). Correlations were studied between salivary and serum IgG at 4, 10 and 18 months. RESULTS: Salivary IgA and IgG responses overall declined in the first 9 months. Compared to non-7vPCV recipients, salivary IgA remained higher in 7vPCV recipients for serotypes 4 at 3 months, 6B at 3 months (neonatal), and 14 at 3 (neonatal), 4 and 9 months (infant); and for salivary IgG for serotypes 4 at 3, 4 and 9 months, 6B at 9 months, 14 at 4 (neonatal) and 9 months, 18C at 3, 4, and 9 (infant) months, and 23F at 4 months. Following 23vPPV, salivary 7vPCV-specific IgA and IgG increased in 7vPCV-vaccinated children but not in controls; and salivary IgA against non-PCV serotypes 5 and 7F increased in 7vPCV recipients and non-recipients. Salivary and serum IgG against 7vPCV-serotypes correlated in 7vPCV-vaccinated children at 4 and 10 months of age. CONCLUSIONS: PCV may protect high-risk children against pneumococcal colonization and mucosal disease by inducing mucosal antibody responses and priming for mucosal immune memory that results in mucosal immune responses after booster PPV. Saliva can be a convenient alternative sample to serum to study PCV-induced systemic IgG responses.

Nasal Delivery of a Commensal Pasteurellaceae Species Inhibits Nontypeable Haemophilus influenzae Colonization and Delays Onset of Otitis Media in Mice.

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 × 104.5 PFU of influenza A virus MEM H3N2, followed by intranasal challenge with 5 × 107 CFU of NTHi R2866 Specr Mice were pretreated or not with an intranasal inoculation of 5 × 107 CFU M. muris 24 h before coinfection. NTHi and M. muris viable counts and inflammatory mediators (gamma interferon [IFN-γ], interleukin-1ß [IL-1ß], 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 × 105 CFU/ml to 9 × 103 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.

Panel 8: Vaccines and immunology.

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.

Bacterial Reservoirs in the Middle Ear of Otitis-prone Children Are Associated With Repeat Ventilation Tube Insertion.

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.

High concentrations of middle ear antimicrobial peptides and proteins and proinflammatory cytokines are associated with detection of middle ear pathogens in children with recurrent acute otitis media.

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 TGFß) 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.

The Contribution of Geogenic Particulate Matter to Lung Disease in Indigenous Children.

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 km2), 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.