Influenza epidemiology spanning pre-COVID-19 pandemic to post-COVID-19 pandemic periods in Australia is insufficiently described. This study reviewed influenza epidemiology in two metropolitan New South Wales (NSW) health districts between 2018 and 2023 and investigated influenza virus (IFV) co-infections with other respiratory viruses (ORVs). A retrospective analysis of diagnostic polymerase chain reaction data from patients requiring testing for IFV and/or ORVs was conducted. Influenza detections were exceptionally low (n=57, <0.2% positivity) between April 2020 and 2021 when compared to those in 2019 (n=3,312, 14.4% positivity). Subsequent relaxation of public health measures corresponded with increased positivity rates: from 0.1% (33) in 2021 to 2.1% (4,028) in 2022 and 3.8% (4,362) in 2023. Influenza A virus (IAV) activity peaked earlier in 2022 and 2023 compared to most prepandemic years. Influenza B virus (IBV) detections were notably higher in 2019 and 2023. Co-infections were identified in 17.2% (346/2010) of IFV-positive samples, with rhinovirus being the most frequent co-infecting virus (7.4%). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was only detected in 1.3% of IFV infections. Logistic regression revealed significantly higher odds of IFV co-infections in children aged under 5 years [odds ratio (OR) 8.18; 95% confidence interval (CI) 5.44-12.29; p<0.01] and in those aged 5-17 years (OR 2.45; 95% CI 1.59-3.77; p<0.01). A significant increase in the likelihood of IFV co-infection was also observed in 2022 (OR 2.42; 95% CI 1.23-4.75; p<0.05). This study described influenza epidemiology across pre-COVID-19 pandemic, during-COVID-19 pandemic, and post-COVID-19 pandemic periods in NSW. Key findings include the earlier IAV peak activity in 2022-2023 and a rapid increase in IBV detection rate from 2022 to 2023, underscoring the need for sustained influenza surveillance to monitor the persistence of these trends. The surge in influenza detections in 2022-2023, accompanied with increased testing volumes, suggests that future surveillance efforts should account for changes in rates of testing when assessing severity of influenza seasons. The higher IFV co-infection frequency was observed in children and adolescents. 'Flurona' cases remain infrequent and exclusively associated with IAVs. These insights also inform the future application of multiplex diagnostic methods.

BACKGROUND:Potential kidney donors with active hepatitis B virus (HBV: positive hepatitis B surface antigen [HBsAg] and/or nucleic acid test [NAT]) are usually declined for HBsAg-negative recipients. Safety may be improved by recipient vaccination and/or antivirals, thereby increasing transplantation opportunities. We quantified HBV transmission risk in this setting to inform donation decisions. METHODS:Systematic review and meta-analysis (MEDLINE, to November 2024) of cohorts comprised of kidney donors with active HBV intentionally used for HBsAg-negative recipients. Transmission was defined as new HBsAg or NAT positivity posttransplant. Transmission proportions and exact 95% confidence intervals (CIs) were pooled using generalized linear mixed models with logistic transformation and random effects. RESULTS:We included 20 cohorts involving 600 HBsAg-negative recipients from donors with active HBV. Most donors were living (52%), with negative NAT (60%). Most recipients had positive surface antibody (>10 IU/L, 86%); many were core antibody positive (47%). Antiviral prophylaxis was given to 49% recipients, varying in type, duration, and strategy. There were 29 of 600 HBV transmissions, mostly transient low-level viremia only (62%). The pooled transmission rate was 4.0% (95% CI, 1.8%-8.3%) with low heterogeneity ( I2  = 0%) but some between-study variance (Tau 2  = 1.21). Transmission rates were higher where all donors had positive NAT (16.0%; 95% CI, 10.2%-24.3%), and lower where all recipients were living (0.8%; 95% CI, 0.1%-6.4%) or had positive surface antibody (1.4%; 95% CI, 0.2%-8.8%). Three deaths because of HBV transmissions occurred, all among recipients not taking posttransplant antiviral prophylaxis. CONCLUSIONS:Given low transmission rates and mitigating strategies, kidney transplantation may be considered from donors with active HBV, with donor/recipient risk stratification, consent, and monitoring/prophylaxis.

BACKGROUND:During the 2019 severe influenza season, New South Wales (NSW) experienced the highest number of cases in Australia. This study retrospectively investigated the genetic characteristics of influenza viruses circulating in NSW in 2019 and identified genetic markers related to antiviral resistance and potential virulence. METHODS:The complete genomes of influenza A and B viruses were amplified using reverse transcription-polymerase chain reaction (PCR) and sequenced with an Illumina MiSeq platform. RESULTS:When comparing the sequencing data with the vaccine strains and reference sequences, the phylogenetic analysis revealed that most NSW A/H3N2 viruses (n = 68; 94%) belonged to 3C.2a1b and a minority (n = 4; 6%) belonged to 3C.3a. These viruses all diverged from the vaccine strain A/Switzerland/8060/2017. All A/H1N1pdm09 viruses (n = 20) showed genetic dissimilarity from vaccine strain A/Michigan/45/2015, with subclades 6B.1A.5 and 6B.1A.2 identified. All B/Victoria-lineage viruses (n = 21) aligned with clade V1A.3, presenting triple amino acid deletions at positions 162-164 in the hemagglutinin protein, significantly diverging from the vaccine strain B/Colorado/06/2017. Multiple amino acid substitutions were also found in the internal proteins of influenza viruses, some of which have been previously reported in hospitalized influenza patients in Thailand. Notably, the oseltamivir-resistant marker H275Y was present in one immunocompromised patient infected with A/H1N1pdm09 and the resistance-related mutation I222V was detected in another A/H3N2-infected patient. CONCLUSIONS:Considering antigenic drift and the constant evolution of circulating A and B strains, we believe continuous monitoring of influenza viruses in NSW via the high-throughput sequencing approach provides timely and pivotal information for both public health surveillance and clinical treatment.

OBJECTIVES: The 2022 seasonal respiratory syncytial virus (RSV) epidemic in Sydney, Australia saw an unprecedented number of RSV detections. We aimed to characterize genomic and immunologic factors associated with the surge in RSV cases. METHODS: Whole genome sequences of RSV were generated from 264 RSV-infected infants and linked to case-matched clinical data from the 2022 southern hemisphere RSV season. We then performed an immunologic analysis of baseline RSV-specific humoral immunity in women of childbearing age before and throughout the coronavirus disease 2019 pandemic. RESULTS: Clinical analysis revealed a high burden of disease across patients of all health backgrounds. More than one-half of RSV-related health care visits by infants resulted in hospitalization, and one-quarter required high-flow respiratory support or a higher level of care. Viral phylogenetic analyses revealed that 2022 Sydney RSV sequences were closely related to viruses that had been circulating globally since 2017, including those detected in recent US outbreaks. Nonsynonymous mutations within the palivizumab and nirsevimab binding sites were detected at low frequencies. There was no difference in baseline RSV-neutralizing antibody titers between 2020 and 2022. CONCLUSIONS: Collectively, these findings suggest that neither the emergence of a novel RSV genotype nor hypothesized immune debt was associated with the surge of RSV cases and hospitalizations in 2022. Continued genomic and immunologic surveillance is required to further understand the factors driving outbreaks of RSV globally, and to inform guidelines for the rollout and ongoing use of recently developed immunotherapeutics and vaccines.

IntroductionQuery (Q) fever is a zoonosis caused by the bacterium Coxiella burnetii typically presenting as an influenza-like illness (ILI) with or without hepatitis. The infection may be missed by clinicians in settings of low endemicity, as the presentation is clinically not specific, and there are many more common differential diagnoses for ILI including SARS-CoV-2 infection.MethodsResidual serum samples were retrospectively tested for Phase 1 and 2 Q fever-specific IgM, IgG, IgA antibodies by indirect immunofluorescence and C. burnetii DNA by polymerase chain reaction. They had not been previously tested for Q fever, originating from undiagnosed patients with probable ILI, aged 10-70 years and living in regional New South Wales, Australia. The results were compared with contemperaneous data on acute Q fever diagnostic tests which had been performed based on clinicians requests from a geographically similar population.ResultsOnly one (0.2%) instance of missed acute Q fever was identified after testing samples from 542 eligible patients who had probable ILI between 2016-2023. Laboratory data showed that during the same period, 731 samples were tested for acute Q fever for clinician-initiated requests and of those 70 (9.6%) were positive. Probability of being diagnosed with Q fever after a clinician initiated request was similar regardless of the patients sex, age and the calendar year of sampling.ConclusionIn this sample, Q fever was most likely to be diagnosed via clinician requested testing rather than by testing of undiagnosed patients with an influenza like illness.

This study retrospectively analyzed the genetic characteristics of influenza A H3N2 (A/H3N2) viruses circulating in New South Wales (NSW), the Australian state with the highest number of influenza cases in 2022, and explored the phylodynamics of A/H3N2 transmission within Australia during this period. Sequencing was performed on 217 archived specimens, and A/H3N2 evolution and spread within Australia were analyzed using phylogenetic and phylodynamic methods. Hemagglutinin genes of all analyzed NSW viruses belonged to subclade 3C.2a1b.2a.2 and clustered together with the 2022 vaccine strain. Complete genome analysis of NSW viruses revealed highly frequent interclade reassortments between subclades 3C.2a1b.2a.2 and 3C.2a1b.1a. The estimated earliest introduction time of the dominant subgroup 3C.2a1b.2a.2a.1 in Australia was February 22, 2022 (95% highest posterior density: December 19, 2021-March 13, 2022), following the easing of Australian travel restrictions, suggesting a possible international source. Phylogeographic analysis revealed that Victoria drove the transmission of A/H3N2 viruses across the country during this season, while NSW did not have a dominant role in viral dissemination to other regions. This study highlights the importance of continuous surveillance and genomic characterization of influenza viruses in the postpandemic era, which can inform public health decision-making and enable early detection of novel strains with pandemic potential.

Background: Seasonal epidemics of respiratory syncytial virus (RSV) in the Southern Hemisphere typically occur in late autumn and winter. However, implementation of public health measures for the Coronavirus Disease (COVID-19) pandemic resulted in an almost complete absence of RSV detections during the 2020 season. This was followed by unusual summer outbreaks worldwide throughout 2021, and in 2022 the seasonal RSV epidemic in Sydney (Australia) saw an unprecedented number of RSV detections.Methods: Whole genome sequences of RSV were generated from 264 RSV-infected infants and linked to case-matched clinical data from the 2022 Southern Hemisphere RSV season. We then performed immunological analysis of baseline RSV-specific humoral immunity in women of childbearing age before and throughout the COVID-19 pandemic.Findings: Clinical analysis found a high burden of disease across patients of all health backgrounds. Over half of RSV-related healthcare visits by infants resulted in hospitalization, and one quarter required high-flow respiratory support or a higher level of care. Viral phylogenetic analyses found that 2022 Sydney RSV sequences were closely related to viruses that were circulating globally since 2017, including those detected in recent USA outbreaks. Non-synonymous mutations within the palivizumab and nirsevimab binding sites were detected at low frequencies. There was no difference in baseline RSV-neutralizing antibody titres between 2020 and 2022.Interpretation: Collectively, these findings suggest an interplay of factors unrelated to changes in local host immunity to RSV contributed to the surge of RSV cases and hospitalisations in 2022. That is, we did not demonstrate the emergence of a novel RSV genotype or hypothesized immune debt associated with the increase in cases. Continued genomic and immunological surveillance is required to further understand factors driving outbreaks of RSV globally, and to inform guidelines for the rollout and ongoing use of recently developed immunotherapeutics and vaccines.Funding: Laboratory consumable costs were supported by the Thrasher Research Fund (Early Career Award, GW). This study was partly supported by MRFF (#2023323, PW, AB) and NHMRC (#2006755, AK, WR) grants.Declaration of Interest: The authors have no conflicts of interest to declare.Ethical Approval: This study was approved by the Sydney Children's Hospitals Network Human Research Ethics Committee (2020/ETH00718).

Aim: To observe upper respiratory tract infection (URTI) symptoms, rhinovirus levels and compliance with daily carrageenan nasal spray. Methods: 102 adults were randomized to carrageenan or saline placebo three times daily for 8 weeks and URTI symptoms were recorded. A control group (n = 42) only recorded URTI symptoms. Participants collected nasal swabs when symptomatic. Results: Regular daily carrageenan prophylaxis resulted in consistent but nonsignificant reductions in URTI symptoms versus the placebo group. Saline placebo decreased and increased some cold symptoms compared with no treatment. Conclusion: Daily prophylactic administration of antiviral carrageenan may not significantly reduce URTI symptoms. Due to low compliance, use in a population with specific reasons to avoid URTIs may be more appropriate. Disease-specific outcomes may be more useful than symptom reporting.

Plasmonic nanoparticles are finding applications within the single molecule sensing field in a "dimer" format, where interaction of the target with hairpin DNA causes a decrease in the interparticle distance, leading to a localized surface plasmon resonance shift. While this shift may be detected using spectroscopy, achieving statistical relevance requires the measurement of thousands of nanoparticle dimers and the timescales required for spectroscopic analysis are incompatible with point-of-care devices. However, using dark-field imaging of the dimer structures, simultaneous digital analysis of the plasmonic resonance shift after target interaction of thousands of dimer structures may be achieved in minutes. The main challenge of this digital analysis on the single-molecule scale was the occurrence of false signals caused by non-specifically bound clusters of nanoparticles. This effect may be reduced by digitally separating dimers from other nanoconjugate types. Variation in image intensity was observed to have a discernible impact on the color analysis of the nanoconjugate constructs and thus the accuracy of the digital separation. Color spaces wherein intensity may be uncoupled from the color information (hue, saturation, and value (HSV) and luminance, a* vector, and b* vector (LAB) were contrasted to a color space which cannot uncouple intensity (RGB) to train a classifier algorithm. Each classifier algorithm was validated to determine which color space produced the most accurate digital separation of the nanoconjugate types. The LAB-based learning classifier demonstrated the highest accuracy for digitally separating nanoparticles. Using this classifier, nanoparticle conjugates were monitored for their plasmonic color shift after interaction with a synthetic RNA target, resulting in a platform with a highly accurate yes/no response with a true positive rate of 88% and a true negative rate of 100%. The sensor response of tested single stranded RNA (ssRNA) samples was well above control responses for target concentrations in the range of 10 aM-1 pM.

Acute respiratory infection is the third most frequent cause of mortality worldwide, causing over 4.25 million deaths annually. Although most diagnosed acute respiratory infections are thought to be of viral origin, the aetiology often remains unclear. The advent of next-generation sequencing (NGS) has revolutionised the field of virus discovery and identification, particularly in the detection of unknown respiratory viruses. We systematically reviewed the application of NGS technologies for detecting respiratory viruses from clinical samples and outline potential barriers to the routine clinical introduction of NGS. The five databases searched for studies published in English from 01 January 2010 to 01 February 2021, which led to the inclusion of 52 studies. A total of 14 different models of NGS platforms were summarised from included studies. Among these models, second-generation sequencing platforms (e.g., Illumina sequencers) were used in the majority of studies (41/52, 79%). Moreover, NGS platforms have proven successful in detecting a variety of respiratory viruses, including influenza A/B viruses (9/52, 17%), SARS-CoV-2 (21/52, 40%), parainfluenza virus (3/52, 6%), respiratory syncytial virus (1/52, 2%), human metapneumovirus (2/52, 4%), or a viral panel including other respiratory viruses (16/52, 31%). The review of NGS technologies used in previous studies indicates the advantages of NGS technologies in novel virus detection, virus typing, mutation identification, and infection cluster assessment. Although there remain some technical and ethical challenges associated with NGS use in clinical laboratories, NGS is a promising future tool to improve understanding of respiratory viruses and provide a more accurate diagnosis with simultaneous virus characterisation.

Acute respiratory infections (ARIs) are a major cause of morbidity among children. Respiratory viruses are commonly detected in both symptomatic and asymptomatic periods. The rates of infection and community epidemiology of respiratory viruses in healthy children needs further definition to assist interpretation of molecular diagnostic assays in this population. Children otherwise healthy aged 1 to 8 years were prospectively enrolled in the study during two consecutive winters, when ARIs peak in New Zealand. Parents completed a daily symptom diary for 8 weeks, during which time they collected a nasal swab from the child for each clinical ARI episode. A further nasal swab was collected by research staff during a clinic visit at the conclusion of the study. All samples were tested for 15 respiratory viruses commonly causing ARI using molecular multiplex polymerase chain reaction assays. There were 575 ARIs identified from 301 children completing the study, at a rate of 1.04 per child-month. Swabs collected during an ARI were positive for a respiratory virus in 76.8% (307 of 400), compared with 37.3% (79 of 212) of swabs collected during asymptomatic periods. The most common viruses detected were human rhinovirus, coronavirus, parainfluenza viruses, influenzavirus, respiratory syncytial virus, and human metapneumovirus. All of these were significantly more likely to be detected during ARIs than asymptomatic periods. Parent-administered surveillance is a useful mechanism for understanding infectious disease in healthy children in the community. Interpretation of molecular diagnostic assays for viruses must be informed by understanding of local rates of asymptomatic infection by such viruses.

Journal of Medical VirologyVolume 94, Issue 9 p. 4043-4046 LETTER TO THE EDITOR Persistent high-level shedding of cultivable SARS-CoV-2 Delta virus 33 days after onset of COVID-19 in a hospitalized patient with pneumonia Ki Wook Kim, Corresponding Author Ki Wook Kim [email protected] orcid.org/0000-0001-9579-6408 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Correspondence Ki Wook Kim, Virology Research Laboratory, Serology and Virology Division (SAViD), Level 3 Clinical Sciences Bldg, Prince of Wales Hospital, Randwick, Sydney, NSW 2031, Australia. Email: [email protected]Search for more papers by this authorXinye Wang, Xinye Wang Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorAnurag Adhikari, Anurag Adhikari Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMalinna Yeang, Malinna Yeang Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorFrances Jenkins, Frances Jenkins Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorZin Naing, Zin Naing Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorGregory J. Walker, Gregory J. Walker Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorCharles S. P. Foster, Charles S. P. Foster Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorSacha Stelzer-Braid, Sacha Stelzer-Braid orcid.org/0000-0001-6037-9305 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorIra Deveson, Ira Deveson Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, New South Wales, Australia Faculty of Medicine, St Vincent's Clinical School, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMaria E. Craig, Maria E. Craig orcid.org/0000-0001-6004-576X Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Sydney, New South Wales, AustraliaSearch for more papers by this authorNicodemus Tedla, Nicodemus Tedla Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorRowena A. Bull, Rowena A. Bull Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMarianne Martinello, Marianne Martinello Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorAngie N. Pinto, Angie N. Pinto Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorRaymond Chan, Raymond Chan Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorStuart Turville, Stuart Turville Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorWilliam D. Rawlinson, William D. Rawlinson orcid.org/0000-0003-0988-7827 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia Faculty of Science, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorSebastiaan van Hal, Sebastiaan van Hal Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, AustraliaSearch for more papers by this author Ki Wook Kim, Corresponding Author Ki Wook Kim [email protected] orcid.org/0000-0001-9579-6408 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Correspondence Ki Wook Kim, Virology Research Laboratory, Serology and Virology Division (SAViD), Level 3 Clinical Sciences Bldg, Prince of Wales Hospital, Randwick, Sydney, NSW 2031, Australia. Email: [email protected]Search for more papers by this authorXinye Wang, Xinye Wang Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorAnurag Adhikari, Anurag Adhikari Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMalinna Yeang, Malinna Yeang Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorFrances Jenkins, Frances Jenkins Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorZin Naing, Zin Naing Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorGregory J. Walker, Gregory J. Walker Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorCharles S. P. Foster, Charles S. P. Foster Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorSacha Stelzer-Braid, Sacha Stelzer-Braid orcid.org/0000-0001-6037-9305 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorIra Deveson, Ira Deveson Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, New South Wales, Australia Faculty of Medicine, St Vincent's Clinical School, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMaria E. Craig, Maria E. Craig orcid.org/0000-0001-6004-576X Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Sydney, New South Wales, AustraliaSearch for more papers by this authorNicodemus Tedla, Nicodemus Tedla Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorRowena A. Bull, Rowena A. Bull Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMarianne Martinello, Marianne Martinello Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorAngie N. Pinto, Angie N. Pinto Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorRaymond Chan, Raymond Chan Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorStuart Turville, Stuart Turville Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorWilliam D. Rawlinson, William D. Rawlinson orcid.org/0000-0003-0988-7827 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia Faculty of Science, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorSebastiaan van Hal, Sebastiaan van Hal Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, AustraliaSearch for more papers by this author First published: 04 May 2022 https://doi.org/10.1002/jmv.27832Citations: 1 William D. Rawlinson and Sebastiaan van Hal are Joint Senior authors. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Supporting Information Filename Description jmv27832-sup-0001-JMV_Supplementary_Material_final.docx17.9 KB Supplementary information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. 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Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infection hospitalisations in Aboriginal infants specifically those aged <6 months. Maternally derived RSV antibody (Ab) can protect against severe RSV disease in infancy. However, the efficiency of transplacental transfer of maternal anti‐RSV Ab remains unknown in Aboriginal infants. We characterised RSV Ab in Australian First Nations mother‐infant pairs (n = 78). We investigated impact of covariates including low birthweight, gestational age (GA), sex of the baby, maternal age and multiparity of the mother on cord to maternal anti‐RSV Ab titre ratio (CMTR) using multivariable logistic regression model. All (n = 78) but one infant was born full term (median GA: 39 weeks, interquartile range: 38–40 weeks) and 56% were males. The mean log2 RSV Ab titre was 10.7 (SD± 1.3) in maternal serum and 11.0 (SD ± 1.3) in cord serum at birth; a ratio of 1.02 (SD ± 0.06). One‐third of the pairs had a CMTR of <1 indicating impaired transfer. Almost 9% (7/78) of the term infants had cord RSV Ab levels below

Whole-genome sequencing of viral isolates is critical for informing transmission patterns and for the ongoing evolution of pathogens, especially during a pandemic. However, when genomes have low variability in the early stages of a pandemic, the impact of technical and/or sequencing errors increases. We quantitatively assessed inter-laboratory differences in consensus genome assemblies of 72 matched SARS-CoV-2-positive specimens sequenced at different laboratories in Sydney, Australia. Raw sequence data were assembled using two different bioinformatics pipelines in parallel, and resulting consensus genomes were compared to detect laboratory-specific differences. Matched genome sequences were predominantly concordant, with a median pairwise identity of 99.997%. Identified differences were predominantly driven by ambiguous site content. Ignoring these produced differences in only 2.3% (5/216) of pairwise comparisons, each differing by a single nucleotide. Matched samples were assigned the same Pango lineage in 98.2% (212/216) of pairwise comparisons, and were mostly assigned to the same phylogenetic clade. However, epidemiological inference based only on single nucleotide variant distances may lead to significant differences in the number of defined clusters if variant allele frequency thresholds for consensus genome generation differ between laboratories. These results underscore the need for a unified, best-practices approach to bioinformatics between laboratories working on a common outbreak problem.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

The incidence of enterovirus D68 (EV-D68) in New South Wales, Australia, is unknown. As part of a state-wide surveillance program, enterovirus positive diagnostic specimens were assessed from patients presenting to hospitals with respiratory and meningitis syndromes from August 2018 to November 2019. Diagnostic enterovirus positive samples were collected from 339 patients and re-extracted followed by targeted PCR across the whole EV-D68 genome (7.4 kb). Obtained amplicons (n=208) were sequenced using Illumina sequencing technology and the phylogenetic relationships analysed relative to EV-D68 Fermon strain. We identified EV-D68 in 31 patients, both children (n=27) and adults (n=4). Phylogenetically, the majority (n=30) were from subclade B3, the same as that causing outbreaks of EV-D68 across the USA and Europe during 2018. These data strengthen the importance of having an active enterovirus surveillance network.

Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n = 200), and negative control sera collected prior to the COVID-19 pandemic (n = 100), were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. Neutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. These results suggest the marker used (total Ab vs. IgG vs. IgA) and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrates their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.

Accumulating evidence supports the high prevalence of co-infections among Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) patients, and their potential to worsen the clinical outcome of COVID-19. However, there are few data on Southern Hemisphere populations, and most studies to date have investigated a narrow spectrum of viruses using targeted qRT-PCR. Here we assessed respiratory viral co-infections among SARS-CoV-2 patients in Australia, through respiratory virome characterization. Nasopharyngeal swabs of 92 SARS-CoV-2-positive cases were sequenced using pan-viral hybrid-capture and the Twist Respiratory Virus Panel. In total, 8% of cases were co-infected, with rhinovirus (6%) or influenzavirus (2%). Twist capture also achieved near-complete sequencing (> 90% coverage, > tenfold depth) of the SARS-CoV-2 genome in 95% of specimens with Ct < 30. Our results highlight the importance of assessing all pathogens in symptomatic patients, and the dual-functionality of Twist hybrid-capture, for SARS-CoV-2 whole-genome sequencing without amplicon generation and the simultaneous identification of viral co-infections with ease.