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Respiratory syncytial virus (RSV) reinfection in children is poorly understood. We examined the incidence, characteristics, and outcomes of hospital-attended RSV reinfections in children <16 years in Western Australia between 2012 and 2022. Individuals with repeat RSV detections ≥56 days apart were identified using laboratory data. The incidence of reinfection in the first five years of life was estimated using the total birth population from 2012 to 2017. Clinical data on a subset of reinfection episodes were obtained from two metropolitan pediatric centers. A total of 466 children with hospital-attended reinfections were identified. The median interval between RSV detections was 460 days (interquartile range: 324, 812), with a reinfection rate of 95 per 100,000 individuals (95% confidence interval: 82, 109). Reinfection was most common in children who experienced their first RSV detection <6 months of age. Predisposing factors were identified in 56% of children; children with predisposing factors were older at first and second detections, were more likely to be admitted, and had a longer length of stay. This study highlights the significant burden of hospital-attended RSV reinfections in children with and without predisposing factors. Expanded surveillance with in-depth clinical data is required to further characterize the impact of RSV reinfection.

The COVID-19 pandemic has necessitated the rapid development and implementation of whole-genome sequencing (WGS) and bioinformatic methods for managing the pandemic. However, variability in methods and capabilities between laboratories has posed challenges in ensuring data accuracy. A national working group comprising 18 laboratory scientists and bioinformaticians from Australia and New Zealand was formed to improve data concordance across public health laboratories (PHLs). One effort, presented in this study, sought to understand the impact of the methodology on consensus genome concordance and interpretation. SARS-CoV-2 WGS proficiency testing programme (PTP) data were retrospectively obtained from the 2021 Royal College of Pathologists of Australasia Quality Assurance Programmes (RCPAQAP), which included 11 participating Australian laboratories. The submitted consensus genomes and reads from eight contrived specimens were investigated, focusing on discordant sequence data and findings were presented to the working group to inform best practices. Despite using a variety of laboratory and bioinformatic methods for SARS-CoV-2 WGS, participants largely produced concordant genomes. Two participants returned five discordant sites in a high-Cτ replicate, which could be resolved with reasonable bioinformatic quality thresholds. We noted ten discrepancies in genome assessment that arose from nucleotide heterogeneity at three different sites in three cell-culture-derived control specimens. While these sites were ultimately accurate after considering the participants' bioinformatic parameters, it presented an interesting challenge for developing standards to account for intrahost single nucleotide variation (iSNV). Observed differences had little to no impact on key surveillance metrics, lineage assignment and phylogenetic clustering, while genome coverage <90 % affected both. We recommend PHLs bioinformatically generate two consensus genomes with and without ambiguity thresholds for quality control and downstream analysis, respectively, and adhere to a minimum 90 % genome coverage threshold for inclusion in surveillance interpretations. We also suggest additional PTP assessment criteria, including primer efficiency, detection of iSNVs and minimum genome coverage of 90 %. This study underscores the importance of multidisciplinary national working groups in informing guidelines in real time for bioinformatic quality acceptance criteria. It demonstrates the potential for enhancing public health responses through improved data concordance and quality control in SARS-CoV-2 genomic analysis during pandemic surveillance.

Aim: Western Australian laboratory data demonstrated a decrease in human metapneumovirus (hMPV) detections through 2020 associated with SARS-CoV-2-related non-pharmaceutical interventions (NPIs), followed by a subsequent surge in metropolitan region in mid-2021. We aimed to assess the impact of the surge in hMPV on paediatric hospital admissions and the contribution of changes in testing., Methods: All respiratory-coded admissions of children aged <16 years at a tertiary paediatric centre between 2017 and 2021 were matched with respiratory virus testing data. Patients were grouped by age at presentation and by ICD-10 AM codes into bronchiolitis, other acute lower respiratory infection (OALRI), wheeze and upper respiratory tract infection (URTI). For analysis, 2017-2019 was utilised as a baseline period., Results: hMPV-positive admissions in 2021 were more than 2.8 times baseline. The largest increase in incidence was observed in the 1-4 years group (incidence rate ratio (IRR) 3.8; 95% confidence interval (CI): 2.5-5.9) and in OALRI clinical phenotype (IRR 2.8; 95% CI: 1.8-4.2). The proportion of respiratory-coded admissions tested for hMPV in 2021 doubled (32-66.2%, P < 0.001), with the greatest increase in wheeze (12-75% in 2021, P < 0.001). hMPV test percentage positivity in 2021 was higher than in the baseline period (7.6% vs. 10.1% in 2021, P = 0.004)., Conclusion: The absence and subsequent surge underline the susceptibility of hMPV to NPIs. Increased hMPV-positive admissions in 2021 can be partially attributable to testing, but test-positivity remained high, consistent with a genuine increase. Continued comprehensive testing will help ascertain true burden of hMPV respiratory diseases., (© 2023 The Authors. Journal of Paediatrics and Child Health published by John Wiley & Sons Australia, Ltd on behalf of Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

Emerging infectious disease threats require rapid response tools to inform diagnostics, treatment, and outbreak control. RNA-based metagenomics offers this; however, most approaches are time-consuming and laborious. Here, we present a simple and fast protocol, the RAPID prep assay, with the aim of providing a cause-agnostic laboratory diagnosis of infection within 24 h of sample collection by sequencing ribosomal RNA-depleted total RNA. The method is based on the synthesis and amplification of double-stranded cDNA followed by short-read sequencing, with minimal handling and clean-up steps to improve processing time. The approach was optimized and applied to a range of clinical respiratory samples to demonstrate diagnostic and quantitative performance. Our results showed robust depletion of both human and microbial rRNA, and library amplification across different sample types, qualities, and extraction kits using a single workflow without input nucleic-acid quantification or quality assessment. Furthermore, we demonstrated the genomic yield of both known and undiagnosed pathogens with complete genomes recovered in most cases to inform molecular epidemiological investigations and vaccine design. The RAPID prep assay is a simple and effective tool, and representative of an important shift toward the integration of modern genomic techniques with infectious disease investigations.

Background: A fatal case of Japanese encephalitis (JE) occurred in a resident of the Tiwi Islands, in the Northern Territory of Australia in February 2021, preceding the large JE outbreak in south-eastern Australia in 2022. This study reports the detection, whole genome sequencing and analysis of the virus responsible (designated JEV/Australia/NT&#95;Tiwi Islands/2021)., Methods: Reverse transcription quantitative PCR (RT-qPCR) testing was performed on post-mortem brain specimens using a range of JE virus (JEV)-specific assays. Virus isolation from brain specimens was attempted by inoculation of mosquito and mammalian cells or embryonated chicken eggs. Whole genome sequencing was undertaken using a combination of Illumina next generation sequencing methodologies, including a tiling amplicon approach. Phylogenetic and selection analyses were performed using alignments of the Tiwi Islands JEV genome and envelope (E) protein gene sequences and publicly available JEV sequences., Results: Virus isolation was unsuccessful and JEV RNA was detected only by RT-qPCR assays capable of detecting all JEV genotypes. Phylogenetic analysis revealed that the Tiwi Islands strain is a divergent member of genotype IV (GIV) and is closely related to the 2022 Australian outbreak virus (99.8% nucleotide identity). The Australian strains share highest levels of nucleotide identity with Indonesian viruses from 2017 and 2019 (96.7-96.8%). The most recent common ancestor of this Australian-Indonesian clade was estimated to have emerged in 2007 (95% HPD range: 1998-2014). Positive selection was detected using two methods (MEME and FEL) at several sites in the E and non-structural protein genes, including a single site in the E protein (S194N) unique to the Australian GIV strains., Conclusion: This case represents the first detection of GIV JEV acquired in Australia, and only the second confirmed fatal human infection with a GIV JEV strain. The close phylogenetic relationship between the Tiwi Islands strain and recent Indonesian viruses is indicative of the origin of this novel GIV lineage, which we estimate has circulated in the region for several years prior to the Tiwi Islands case., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Sikazwe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human respiratory syncytial virus (RSV) is an important cause of acute respiratory infection with the most severe disease in the young and elderly. Non-pharmaceutical interventions and travel restrictions for controlling COVID-19 have impacted the circulation of most respiratory viruses including RSV globally, particularly in Australia, where during 2020 the normal winter epidemics were notably absent. However, in late 2020, unprecedented widespread RSV outbreaks occurred, beginning in spring, and extending into summer across two widely separated regions of the Australian continent, New South Wales (NSW) and Australian Capital Territory (ACT) in the east, and Western Australia. Through genomic sequencing we reveal a major reduction in RSV genetic diversity following COVID-19 emergence with two genetically distinct RSV-A clades circulating cryptically, likely localised for several months prior to an epidemic surge in cases upon relaxation of COVID-19 control measures. The NSW/ACT clade subsequently spread to the neighbouring state of Victoria and to cause extensive outbreaks and hospitalisations in early 2021. These findings highlight the need for continued surveillance and sequencing of RSV and other respiratory viruses during and after the COVID-19 pandemic, as mitigation measures may disrupt seasonal patterns, causing larger or more severe outbreaks., (© 2022. The Author(s).)

To investigate potential transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during a domestic flight within Australia, we performed epidemiologic analyses with whole-genome sequencing. Eleven passengers with PCR-confirmed SARS-CoV-2 infection and symptom onset within 48 hours of the flight were considered infectious during travel; 9 had recently disembarked from a cruise ship with a retrospectively identified SARS-CoV-2 outbreak. The virus strain of those on the cruise and the flight was linked (A2-RP) and had not been previously identified in Australia. For 11 passengers, none of whom had traveled on the cruise ship, PCR-confirmed SARS-CoV-2 illness developed between 48 hours and 14 days after the flight. Eight cases were considered flight associated with the distinct SARS-CoV-2 A2-RP strain; the remaining 3 cases (1 with A2-RP) were possibly flight associated. All 11 passengers had been in the same cabin with symptomatic persons who had culture-positive A2-RP virus strain. This investigation provides evidence of flight-associated SARS-CoV-2 transmission.

Background: Detection of pneumonia-causing respiratory viruses in the nasopharynx of asymptomatic children has made their actual contribution to pneumonia unclear. We compared nasopharyngeal viral density between children with and without pneumonia to understand if viral density could be used to diagnose pneumonia., Methods: Nasopharyngeal swabs (NPS) were collected from hospitalised pneumonia cases at Princess Margaret Hospital (PMH) and contemporaneous age-matched controls at PMH outpatient clinics and a local immunisation clinic in Perth, Australia. The density (copies/mL) of respiratory syncytial virus (RSV), influenza A virus (InfA), human metapneumovirus (HMPV) and rhinovirus in NPS was determined using quantitative PCR. Linear regression analysis was done to assess the trend between viral density and age in months. The association between viral density and disease status was examined using logistic regression. Area under receiver operating characteristic (AUROC) curves were assessed to determine optimal discriminatory viral density cut-offs., Results: Through May 2015 to October 2017, 230 pneumonia cases and 230 controls were enrolled. Median nasopharyngeal density for any respiratory virus was not substantially higher in cases than controls (p>0.05 for each). A decreasing density trend with increasing age was observed-the trend was statistically significant for RSV (regression coefficient -0.04, p=0.004) but not for other viruses. After adjusting for demographics and other viral densities, for every log 10 copies/mL density increase, the odds of being a case increased by six times for RSV, three times for HMPV and two times for InfA. The AUROC curves were <0.70 for each virus, suggesting poor case-control discrimination based on viral density., Conclusion: The nasopharyngeal density of respiratory viruses was not significantly higher in children with pneumonia than those without; however, the odds of being a case increased with increased density for some viruses. The utility of viral density, alone, in defining pneumonia was limited., Competing Interests: Competing interests: PR receives grants from GlaxoSmithKline, Novavax, Medimmune and Janssen outside the submitted work;, (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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

Introduction: Pneumonia is the leading cause of childhood morbidity and mortality globally. Introduction of the conjugate Haemophilus influenzae B and multivalent pneumococcal vaccines in developed countries including Australia has significantly reduced the overall burden of bacterial pneumonia. With the availability of molecular diagnostics, viruses are frequently detected in children with pneumonia either as primary pathogens or predispose to secondary bacterial infection. Many respiratory pathogens that are known to cause pneumonia are also identified in asymptomatic children, so the true contribution of these pathogens to childhood community-acquired pneumonia (CAP) remains unclear. Since the introduction of pneumococcal vaccines, very few comprehensive studies from developed countries have attempted to determine the bacterial and viral aetiology of pneumonia. We aim to determine the contribution of bacteria and viruses to childhood CAP to inform further development of effective diagnosis, treatment and preventive strategies., Methods and Analysis: We are conducting a prospective case-control study (PneumoWA) where cases are children with radiologically confirmed pneumonia admitted to Princess Margaret Hospital for Children (PMH) and controls are healthy children identified from PMH outpatient clinics and from local community immunisation clinics. The case-control ratio is 1:1 with 250 children to be recruited in each arm. Nasopharyngeal swabs are collected from both cases and controls to detect the presence of viruses and bacteria by PCR; pathogen load will be assessed by quantitative PCR. The prevalence of pathogens detected in cases and controls will be compared, the OR of detection and population attributable fraction to CAP for each pathogen will be determined; relationships between pathogen load and disease status and severity will be explored., Ethics and Dissemination: This study has been approved by the human research ethics committees of PMH, Perth, Australia (PMH HREC REF 2014117EP). Findings will be disseminated at research conferences and in peer-reviewed journals., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Recurrent acute otitis media (rAOM, recurrent ear infection) is a common childhood disease caused by bacteria termed otopathogens, for which current treatments have limited effectiveness. Generic probiotic therapies have shown promise, but seem to lack specificity. We hypothesised that healthy children with no history of AOM carry protective commensal bacteria that could be translated into a specific probiotic therapy to break the cycle of re-infection. We characterised the nasopharyngeal microbiome of these children (controls) in comparison to children with rAOM (cases) to identify potentially protective bacteria. As some children with rAOM do not appear to carry any of the known otopathogens, we also hypothesised that characterisation of the middle ear microbiome could identify novel otopathogens, which may also guide the development of more effective therapies., Results: Middle ear fluids, middle ear rinses and ear canal swabs from the cases and nasopharyngeal swabs from both groups underwent 16S rRNA gene sequencing. The nasopharyngeal microbiomes of cases and controls were distinct. We observed a significantly higher abundance of Corynebacterium and Dolosigranulum in the nasopharynx of controls. Alloiococcus, Staphylococcus and Turicella were abundant in the middle ear and ear canal of cases, but were uncommon in the nasopharynx of both groups. Gemella and Neisseria were characteristic of the case nasopharynx, but were not prevalent in the middle ear., Conclusions: Corynebacterium and Dolosigranulum are characteristic of a healthy nasopharyngeal microbiome. Alloiococcus, Staphylococcus and Turicella are possible novel otopathogens, though their rarity in the nasopharynx and prevalence in the ear canal means that their role as normal aural flora cannot be ruled out. Gemella and Neisseria are unlikely to be novel otopathogens as they do not appear to colonise the middle ear in children with rAOM.

Background: In experimental studies viral infections have been shown to induce type 2 inflammation in asthmatics, but whether this is a feature of naturally occurring virus-induced asthma exacerbations is unknown. Thymic stromal lymphopoietin (TSLP) released from the airway epithelium in response to damage, has been suggested as a link between viral infection and type 2 inflammation, but the role of TSLP in asthma exacerbations is unknown., Objective: To assess whether type 2 inflammation, as measured by sputum eosinophils and fractional exhaled nitric oxide (FeNO), is a feature of naturally occurring virus-induced exacerbations of asthma and whether TSLP is associated with this type 2 inflammation., Methods: Patients presenting to hospital with acute asthma were examined during the exacerbation, and after 4 weeks recovery. The assessments included spirometry, FeNO and induced sputum for differential counts and TSLP mRNA levels. Nasal swabs were collected for viral detection., Results: Sputum eosinophils and FeNO were similar between virus-positive (n = 44) and negative patients (n = 44). In virus-positive patients, TSLP expression was lower at exacerbation than follow-up (p = 0.03). High TSLP at exacerbation was associated with lower sputum eosinophils (p = 0.01) and higher FEV1 (p = 0.03). In virus-positive patients, %-predicted FEV1 negatively correlated with both FeNO and sputum eosinophils (p = 0.02 and p = 0.05, respectively)., Conclusion: Our findings support that type 2 inflammation is present in patients during virus-induced asthma exacerbations, to the same degree as non-viral exacerbations, and correlate negatively with FEV1. However, in virus-positive patients, high TSLP expression during exacerbation was associated with low sputum eosinophils, suggesting that the effect of TSLP in vivo, in the setting of an asthma exacerbation, might be different than the type 2 inducing effects observed in experimental studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background and Objective: Airway eosinophilia is associated with an increased risk of asthma exacerbations; however, the impact on the severity of exacerbations is largely unknown. We describe the sputum inflammatory phenotype during asthma exacerbation and correlate it with severity and treatment response., Methods: Patients presenting to hospital with an asthma exacerbation were recruited during a 12-month period and followed up after 4 weeks. Induced sputum was collected at both visits. Patients underwent spirometry, arterial blood gas analysis, fractional exhaled nitric oxide analysis, white blood cell counts and a screening for common respiratory viruses and bacteria. An eosinophilic exacerbation (EE) was defined as having sputum eosinophils ≥ 3% and a non-eosinophilic exacerbation as < 3% (NEE)., Results: A total of 47 patients were enrolled; 37 (79%) had successful sputum induction at baseline, of whom 43% had sputum eosinophils ≥3% (EE). Patients with EE had a significantly lower forced expiratory volume in 1 s (FEV 1 ) % predicted (70.8%, P = 0.03) than patients with NEE (83.6%). Furthermore, EE patients were more likely to require supplemental oxygen during admission (63% vs 14%, P = 0.002). The prevalence of respiratory viruses was the same in EE and NEE patients (44% vs 52%, P = 0.60), as was bacterial infection (6% vs 14%, P = 0.44). Fractional expiratory nitric oxide (FeNO) correlated with sputum %-eosinophils (ρ = 0.57, P < 0.001), and predicted airway eosinophilia with a sensitivity of 86% and a specificity of 70%., Conclusion: Our findings suggest that eosinophilic asthma exacerbations may be clinically more severe than NEEs, supporting the identification of these higher risk patients for specific interventions., (© 2016 Asian Pacific Society of Respirology.)

Background: Human rhinovirus (RV) is the most common respiratory virus and has been associated with frequent and severe acute lower respiratory infections (ALRI). The prevalence of RV species among HIV-infected children in South Africa is unknown., Objectives: To describe the prevalence of respiratory viruses, including RV species, associated with HIV status and other clinical symptoms in children less than two years of age with and without ALRI in Pretoria, South Africa., Study Design: Nasopharyngeal aspirates were collected from 105 hospitalized ALRI cases and 53 non-ALRI controls less than two years of age. HIV status was determined. Common respiratory viruses were identified by PCR, and RV species and genotypes were identified by semi-nested PCR, sequencing and phylogenetic tree analyses., Results: Respiratory viruses were more common among ALRI cases than controls (83.8% vs. 69.2%; p=0.041). RV was the most commonly identified virus in cases with pneumonia (45.6%) or bronchiolitis (52.1%), regardless of HIV status, as well as in controls (39.6%). RV-A was identified in 26.7% of cases and 15.1% of controls while RV-C was identified in 21.0% of cases and 18.9% of controls. HIV-infected children were more likely to be diagnosed with pneumonia than bronchiolitis (p<0.01). RSV was not identified in any HIV-infected cases (n=15) compared with 30.6% of HIV-uninfected cases (n=85, p=0.013), and was identified more frequently in bronchiolitis than in pneumonia cases (43.8% vs. 12.3%; p<0.01)., Conclusions: RV-A and RV-C are endemic in South African children and HIV infection may be protective against RSV and bronchiolitis., (Copyright © 2016 Elsevier B.V. All rights reserved.)