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).

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.

The contribution of viruses and bacteria to community-acquired pneumonia in vaccinated children: a case-control study.

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.

Combination of clinical symptoms and blood biomarkers can improve discrimination between bacterial or viral community-acquired pneumonia in children.

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 (≥38οC) 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.

Bacillus licheniformis in geogenic dust induces inflammation in respiratory epithelium.

Exposure to environmental geogenic (or earth-derived) dust can lead to more frequent and severe infections in the human airway. Particulate matter < 10 µm (PM10) 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 PM10 experienced an exacerbation of inflammatory responses to influenza A virus. Whether geogenic dust PM10 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 PM10 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 PM10 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 PM10. 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 PM10 (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 PM10 can harbour viable bacterial spores that induce inflammation in respiratory epithelium. The impact on respiratory health from inhalation of bacterial spores in PM10 in arid environments may be underestimated. Further investigation into the contribution of B. licheniformis and the wider dust microbiome to respiratory infection is warranted.

Immunogenicity and safety of single-dose, 13-valent pneumococcal conjugate vaccine in pediatric and adolescent oncology patients.

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.

Influences on patient satisfaction in healthcare centers: a semi-quantitative study over 5 years.

BACKGROUND: Knowledge of ambulatory patients' satisfaction with clinic visits help improve communication and delivery of healthcare. The goal was to examine patient satisfaction in a primary care setting, identify how selected patient and physician setting and characteristics affected satisfaction, and determine if feedback provided to medical directors over time impacted patient satisfaction. METHODS: A three-phase, semi-quantitative analysis was performed using anonymous, validated patient satisfaction surveys collected from 889 ambulatory outpatients in 6 healthcare centers over 5-years. Patients' responses to 21 questions were analyzed by principal components varimax rotated factor analysis. Three classifiable components emerged: Satisfaction with Physician, Availability/Convenience, and Orderly/Time. To study the effects of several independent variables (location of clinics, patients' and physicians' age, education level and duration at the clinic), data were subjected to multivariate analysis of variance (MANOVA).. RESULTS: Changes in the healthcare centers over time were not significantly related to patient satisfaction. However, location of the center did affect satisfaction. Urban patients were more satisfied with their physicians than rural, and inner city patients were less satisfied than urban or rural on Availability/Convenience and less satisfied than urban patients on Orderly/Time. How long a patient attended a center most affected satisfaction, with patients attending >10 years more satisfied in all three components than those attending <1-5 years. Level of education affected patients' satisfaction only in the component Orderly/Time; patients without a high school education were significantly less satisfied than those with more. Patients in their 40's were significantly less satisfied in Availability/Convenience than those >60 years old. Patients were significantly more satisfied with their 30-40 year-old physicians compared with those over 60. On Orderly/Time, patients were more satisfied with physicians who were in their 50's than physicians >60. CONCLUSIONS: Improvement in patient satisfaction includes a need for immediate, specific feedback. Although Medical Directors received feedback yearly, we found no significant changes in patient satisfaction over time. Our results suggest that, to increase satisfaction, patients with lower education, those who are sicker, and those who are new to the center likely would benefit from additional high quality interactions with their physicians.

Duplex Quantitative PCR Assay for Detection of Haemophilus influenzae That Distinguishes Fucose- and Protein D-Negative Strains.

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).

Genetic and functional evidence for a locus controlling otitis media at chromosome 10q26.3.

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-ß/Activin/Nodal pathway modulator, as the more likely functional candidate lying immediately under the linkage peak for OM susceptibility at chromosome 10q26.3.

Dominance of Haemophilus influenzae in ear discharge from Indigenous Australian children with acute otitis media with tympanic membrane perforation.

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.

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.

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.

Influenza virus induces bacterial and nonbacterial otitis media.

Otitis media (OM) is one of the most common childhood diseases. OM can arise when a viral infection enables bacteria to disseminate from the nasopharynx to the middle ear. Here, we provide the first infant murine model for disease. Mice coinfected with Streptococcus pneumoniae and influenza virus had high bacterial load in the middle ear, middle ear inflammation, and hearing loss. In contrast, mice colonized with S. pneumoniae alone had significantly less bacteria in the ear, minimal hearing loss, and no inflammation. Of interest, infection with influenza virus alone also caused some middle ear inflammation and hearing loss. Overall, this study provides a clinically relevant and easily accessible animal model to study the pathogenesis and prevention of OM. Moreover, we provide, to our knowledge, the first evidence that influenza virus alone causes middle ear inflammation in infant mice. This inflammation may then play an important role in the development of bacterial OM.

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.

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.

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.

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.

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.

Multi-species bacterial biofilm and intracellular infection in otitis media.

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.

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.