Shyan Vijayasekaran

Predominance of nontypeable Haemophilus influenzae in children with otitis media following introduction of a 3 + 0 pneumococcal conjugate vaccine schedule

In Australia the 7-valent pneumococcal conjugate vaccine (PCV7) is administered at 2, 4 and 6 months of age, with no booster dose. Information on bacterial carriage and the aetiology of recurrent acute otitis media (rAOM) after introduction of PCV7 using the 3+0 schedule is required to evaluate the potential impact of second generation pneumococcal vaccines. We found that 2-4 years after introduction of PCV7 in the National Immunisation Program, nontypeable Haemophilus influenzae (NTHi) was the predominant pathogen isolated from the nasopharynx and middle ear of children with a history of rAOM. Compared with healthy controls (n=81), NTHi and Streptococcus pneumoniae carriage rates were significantly higher in children with a history of rAOM (n=186) (19% vs. 56% p<0.0001 and 26% vs. 41%, p=0.02, respectively). Carriage of PCV7 pneumococcal serotypes was rare, whereas PCV7-related and non-PCV7 serotypes were isolated of 38% of cases and 24% of controls. Serotype 19A was the most common serotype isolated from the nasopharynx and middle ear and accounted for 36% (14/39) of total pneumococcal isolates with reduced susceptibility to cotrimoxazole. Of the 119 children carrying NTHi, 17% of isolates were β-lactamase positive. The scarcity of PCV7 serotypes in children with and without a history of rAOM indicates that the 3+0 PCV7 schedule is preventing carriage and rAOM from PCV7 serotypes. Introduction of new vaccines in Australia with increased pneumococcal serotype and pathogen coverage, including 19A and NTHi, should decrease the circulation of antibiotic-resistant bacteria and reduce the burden of rAOM.

Multi-species bacterial biofilm and intracellular infection in otitis media

BackgroundBacteria 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.MethodsMiddle 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.ResultsNo 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.ConclusionUsing 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.

The role of chronic infection in children with otitis media with effusion: Evidence for intracellular persistence of bacteria

Objective Demonstrate mucosal bacterial infection in children with otitis media with effusion (OME). Study Design and Setting Middle ear mucosal biopsies from 11 children with OME were examined for bacteria utilizing transmission electron microscopy. This was correlated with standard culture and polymerase chain reaction (PCR) of middle ear effusions. Results Gram-positive coccal bacteria were demonstrated in middle ear mucosal epithelial cells of 4 of 11 (36%) children. Morphological appearance of bacteria and detection of pneumolysin DNA by PCR in middle ear fluid suggests a role for persistent intracellular infection with Streptococcus pneumoniae and other gram-positive cocci in some cases of OME. Conclusion Intracellular bacterial infection of middle ear mucosal epithelial cells in children with OME may be an important mechanism for bacterial persistence, and contribute to inflammation and mucus production in the pathogenesis of this condition. Significance Persistent intracellular infection is a novel paradigm for OME pathogenesis in children and may influence antibiotic effectiveness in treatment of this condition.

Neutrophil Extracellular Traps and Bacterial Biofilms in Middle Ear Effusion of Children with Recurrent Acute Otitis Media – A Potential Treatment Target

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.

Genome-Wide Association Study to Identify the Genetic Determinants of Otitis Media Susceptibility in Childhood

Background Otitis media (OM) is a common childhood disease characterised by middle ear inflammation and effusion. Susceptibility to recurrent acute OM (rAOM; ≥3 episodes of AOM in 6 months) and chronic OM with effusion (COME; MEE ≥3 months) is 40–70% heritable. Few underlying genes have been identified to date, and no genome-wide association study (GWAS) of OM has been reported. Methods and Findings Data for 2,524,817 single nucleotide polymorphisms (SNPs; 535,544 quality-controlled SNPs genotyped by Illumina 660W-Quad; 1,989,273 by imputation) were analysed for association with OM in 416 cases and 1,075 controls from the Western Australian Pregnancy Cohort (Raine) Study. Logistic regression analyses under an additive model undertaken in GenABEL/ProbABEL adjusting for population substructure using principal components identified SNPs at CAPN14 (rs6755194: OR = 1.90; 95%CI 1.47–2.45; Padj-PCA = 8.3×10−7) on chromosome 2p23.1 as the top hit, with independent effects (rs1862981: OR = 1.60; 95%CI 1.29–1.99; Padj-PCA = 2.2×10−5) observed at the adjacent GALNT14 gene. In a gene-based analysis in VEGAS, BPIFA3 (PGene = 2×10−5) and BPIFA1 (PGene = 1.07×10−4) in the BPIFA gene cluster on chromosome 20q11.21 were the top hits. In all, 32 genomic regions show evidence of association (Padj-PCA<10−5) in this GWAS, with pathway analysis showing a connection between top candidates and the TGFβ pathway. However, top and tag-SNP analysis for seven selected candidate genes in this pathway did not replicate in 645 families (793 affected individuals) from the Western Australian Family Study of Otitis Media (WAFSOM). Lack of replication may be explained by sample size, difference in OM disease severity between primary and replication cohorts or due to type I error in the primary GWAS. Conclusions This first discovery GWAS for an OM phenotype has identified CAPN14 and GALNT14 on chromosome 2p23.1 and the BPIFA gene cluster on chromosome 20q11.21 as novel candidate genes which warrant further analysis in cohorts matched more precisely for clinical phenotypes.

Objective assessment of pediatric voice disorders with the acoustic voice quality index

OBJECTIVES/HYPOTHESIS Instrumental measures of voice allow practitioners to assess the severity of voice disorders and objectively measure treatment outcomes. Instrumental measures should be calculated on both sustained vowel and connected speech samples to ensure ecological validity. However, there is a lack of appropriate, validated acoustic measurements for use in the pediatric population. The Acoustic Voice Quality Index (AVQI) is a multivariate acoustic measure of dysphonia that has been found to be reliable, valid, and have diagnostic accuracy and response to change in an adult population. This study aimed to evaluate the AVQI in a pediatric population. STUDY DESIGN This study was a prospective observational study of a sample of dysphonic and normophonic children. METHODS Sixty-seven preterm participants (born at less than 25 weeks gestation) aged between 6 and 15 years were recruited. Participants were excluded because of either inability to comply with task requirements or other speech-related factors that affected acoustic measurement. Forty normophonic term-born participants aged between 5 and 15 years were also recruited. AVQI analysis was conducted on a prolonged vowel sample and a sample of continuous speech. RESULTS The AVQI was found to have diagnostic accuracy and specificity in this population of children with and without dysphonia. It was moderately correlated with ratings of severity on the GRBAS (overall grade of hoarseness (G), roughness (R), breathiness (B), aesthenicity (A), and strain (S)), a subjective rating scale. The threshold for pathology of this sample of 3.46 showed strong sensitivity, specificity, and accuracy, with good-to-excellent likelihood ratios. CONCLUSIONS This study found that the AVQI has diagnostic accuracy in a pediatric population, suggesting that it is an appropriate assessment tool to determine the presence and severity of pediatric voice disorders.

A review of the burden of disease due to otitis media in the Asia-Pacific.

OBJECTIVE The burden of disease due to otitis media (OM) in Asia Pacific countries was reviewed to increase awareness and raise understanding within the region. METHODS Published literature and unpublished studies were reviewed. RESULTS In school-age children, OM prevalence varied between 3.25% (Thailand) and 12.23% (Philippines) being highest (42%) in Aboriginal Australian children. OME prevalence at school age varied between 1.14% (Thailand) and 13.8% (Malaysia). Higher prevalence was reported in children with hearing impairment, HIV, pneumonia and rhinitis. CSOM prevalence was 5.4% in Indonesia (all ages), 15% in Aboriginal Australian children and 2-4% in Thailand, Philippines, Malaysia and Vietnam (WHO estimate). OM prevalence/incidence and service utilisation were highest in children 2-5 years of age. The disease burden was substantially higher in Pacific Island children living in New Zealand (25.4% with OME), and was highest in indigenous Australians (>90% with any OM). Streptococcus pneumoniae and Haemophilus influenzae dominated as primary causes of AOM in all studies. Few studies examined pneumococcal serotype distribution. Health-related cost estimates for OM, when available, were substantial. In developing countries, significant investment is needed to provide facilities for detection and treatment of ear disease in children, if long term hearing deficits and other sequelae are to be prevented. CONCLUSION The available evidence suggests an important burden of disease and economic cost associated with OM in most Asia Pacific countries and a potential benefit of prevention through vaccination. Large, prospective community-based studies are needed to better define the prevalence of ear disease in children, and to predict and track pneumococcal conjugate vaccine impacts. AOM prevention through vaccination may also provide a means of reducing antibiotic use and controlling antibiotic-resistant disease in children. This review highlights the need for additional research, and provides a basis on which to build and develop regional guidelines for OM management.

Objective assessment of supraglottoplasty outcomes using polysomnography

OBJECTIVE Supraglottoplasty is well documented as an effective procedure in the management of moderate to severe laryngomalacia. Traditionally assessed outcomes in the treatment of laryngomalacia include: reduced stridor, improved feeding and a resumption of weight-gain. Previous authors have documented the use of polysomnography in assessing the severity of paediatric laryngeal abnormalities. The aim of the current study was to objectively assess the efficacy of supraglottoplasty for laryngomalacia by comparing data from pre- and post-operative polysomnography. METHODS A retrospective review was performed of patients undergoing supraglottoplasty for laryngomalacia at a tertiary referral paediatric hospital over a 35-month period. Patients were required to have undergone full overnight polysomnography both before and after supraglottoplasty. Pre- and post-operative polysomnographic results were compared and the statistical significance between mean values was determined. RESULTS From a total of 46 patients, 10 were suitable for inclusion into the study. Mean age at first presentation was 2 months and 19 days (range 30-134 days). The surgical procedure performed was bilateral division of aryepiglottic folds alone in 1 patient (1/10), bilateral division of aryepiglottic folds and bilateral trimming of arytenoid mucosa in 5 patients (5/10) and bilateral aryepiglottic fold division, bilateral arytenoid mucosal trimming and epiglottic trimming or epiglottopexy in 4 patients (4/10). Statistically significant improvements occurred in mean values for Total Sleep Time (TST) (P=0.049), Lowest Oxygen Saturation Levels (SpO2 nadir) (P=0.006), Obstructive Apnoea Hypopnoea Index (OAHI) (P=0.009) and Respiratory Disturbance Index (RDI) (P=0.002), following supraglottoplasty. An improvement in mean Transcutaneous Carbon Dioxide (TcCO2) value occurred, but did not achieve statistical significance (57.1 vs. 52.8) (P=0.259). The mean age at which post-operative polysomnography confirmed a reversal of abnormal respiratory parameters following surgery was 5 months and 18 days. CONCLUSIONS Polysomnography is an effective method for objectively assessing the efficacy of supraglottoplasty for laryngomalacia. Supraglottoplasty effectively reverses the abnormal respiratory parameters occurring in moderate to severe laryngomalacia.

High detection rates of nucleic acids of a wide range of respiratory viruses in the nasopharynx and the middle ear of children with a history of recurrent acute otitis media

Both bacteria and viruses play a role in the development of acute otitis media, however, the importance of specific viruses is unclear. In this study molecular methods were used to determine the presence of nucleic acids of human rhinoviruses (HRV; types A, B, and C), respiratory syncytial viruses (RSV; types A and B), bocavirus (HBoV), adenovirus, enterovirus, coronaviruses (229E, HKU1, NL63, and OC43), influenza viruses (types A, B, and C), parainfluenza viruses (types 1, 2, 3, 4A, and 4B), human metapneumovirus, and polyomaviruses (KI and WU) in the nasopharynx of children between 6 and 36 months of age either with (n = 180) or without (n = 66) a history of recurrent acute otitis media and in 238 middle ear effusion samples collected from 143 children with recurrent acute otitis media. The co‐detection of these viruses with Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis was analyzed. HRV (58.3% vs. 42.4%), HBoV (52.2% vs. 19.7%), polyomaviruses (36.1% vs. 15.2%), parainfluenza viruses (29.4% vs. 9.1%), adenovirus (25.0% vs. 6.1%), and RSV (27.8% vs. 9.1%) were detected significantly more often in the nasopharynx of children with a history of recurrent acute otitis media compared to healthy children. HRV was predominant in the middle ear and detected in middle ear effusion of 46% of children. Since respiratory viruses were detected frequently in the nasopharynx of both children with and without a history of recurrent acute otitis media, the etiological role of specific viruses in recurrent acute otitis media remains uncertain, however, anti‐viral therapies may be beneficial in future treatment and prevention strategies for acute otitis media. J. Med. Virol. 83:2008–2017, 2011.

Voice abnormalities at school age in children born extremely preterm

BACKGROUND AND OBJECTIVES: Voice abnormality is a frequent finding in school age children born at <25 weeks’ gestation in Western Australia. The objective of this study was to determine the frequency of voice abnormality, voice-related quality of life, and demographic and intubation factors in this population. METHODS: Survivors <25 weeks’ gestational age in Western Australia born from 1996 to 2004 were included. Voice assessments (auditory perceptual assessment scale and Pediatric Voice Handicap Index) were carried out by speech pathologists. Intubation history was obtained by retrospective chart review. RESULTS: Of 251 NICU admissions, 154 (61%) survived. Exclusions were based on severe disability (11) or distant residence (13). Of 70 assessed, 67 completed assessments, 4 (6%) were in the normal range and 39 (58%) showed moderate-severe hoarseness. Simultaneous modeling of demographic and intubation characteristics showed an increased odds of moderate-severe voice disorder for children who had more than 5 intubations (odds ratio 6.96, 95% confidence interval 2.07–23.40, P = .002) and for girls relative to boys (odds ratio 3.46, 95% confidence interval 1.12–10.62, P = .030). Tube size and duration of intubation were not significant in the multivariable model. Median scores of parent-reported voice quality of life on the Pediatric Voice Handicap Index were markedly different for preterm (22) and term (3) groups, P < .001. CONCLUSIONS: Voice disorders in this population were much more frequent than expected. Further studies are required to assess voice across a broader range of gestational ages, and to investigate voice-protective strategies in infants requiring multiple episodes of intubation.