Defining the phylogenetics and resistome of the major Clostridioides difficile ribotypes circulating in Australia.

Clostridioides difficile infection (CDI) remains a significant public health threat globally. New interventions to treat CDI rely on an understanding of the evolution and epidemiology of circulating strains. Here we provide longitudinal genomic data on strain diversity, transmission dynamics and antimicrobial resistance (AMR) of C. difficile ribotypes (RTs) 014/020 (n=169), 002 (n=77) and 056 (n=36), the three most prominent C. difficile strains causing CDI in Australia. Genome scrutiny showed that AMR was uncommon in these lineages, with resistance-conferring alleles present in only 15/169 RT014/020 strains (8.9 %), 1/36 RT056 strains (2.78 %) and none of 77 RT002 strains. Notably, ~90 % of strains were resistant to MLSB agents in vitro, but only ~5.9 % harboured known resistance alleles, highlighting an incongruence between AMR genotype and phenotype. Core genome analyses revealed all three RTs contained genetically heterogeneous strain populations with limited evidence of clonal transmission between CDI cases. The average number of pairwise core genome SNP (cgSNP) differences within each RT group ranged from 23.3 (RT056, ST34, n=36) to 115.6 (RT002, ST8, n=77) and 315.9 (RT014/020, STs 2, 13, 14, 49, n=169). Just 19 clonal groups (encompassing 40 isolates), defined as isolates differing by ≤2 cgSNPs, were identified across all three RTs (RT014/020, n=14; RT002, n=3; RT056, n=2). Of these clonal groups, 63 % (12/19) comprised isolates from the same Australian State and 37 % (7/19) comprised isolates from different States. The low number of plausible transmission events found for these major RTs (and previously documented populations in animal and environmental sources/reservoirs) points to widespread and persistent community sources of diverse C. difficile strains as opposed to ongoing nationwide healthcare outbreaks dominated by a single clone. Together, these data provide new insights into the evolution of major lineages causing CDI in Australia and highlight the urgent need for enhanced surveillance, and for public health interventions to move beyond the healthcare setting and into a One Health paradigm to effectively combat this complex pathogen.

Metatranscriptomic Sequencing of Medically Important Mosquitoes Reveals Extensive Diversity of RNA Viruses and Other Microbial Communities in Western Australia.

Mosquitoes harbor a wide diversity of microorganisms, including viruses that are human pathogens, or that are insect specific. We used metatranscriptomics, an unbiased high-throughput molecular approach, to describe the composition of viral and other microbial communities in six medically important mosquito species from across Western Australia: Aedes vigilax, Culex annulirostris, Cx. australicus, Cx. globocoxitus, Cx. pipiens biotype molestus, and Cx. quinquefasciatus. We identified 42 viral species, including 13 novel viruses, from 19 families. Culex mosquitoes exhibited a significantly higher diversity of viruses than Aedes mosquitoes, and no virus was shared between the two genera. Comparison of mosquito populations revealed a heterogenous distribution of viruses between geographical regions and between closely related species, suggesting that geography and host species may play a role in shaping virome composition. We also detected bacterial and parasitic microorganisms, among which Wolbachia bacteria were detected in three members of the Cx. pipiens complex, Cx. australicus, Cx. pipiens biotype molestus, and Cx. quinquefasciatus. In summary, our unbiased metatranscriptomics approach provides important insights into viral and other microbial diversity in Western Australian mosquitoes that vector medically important viruses.

Respiratory Syncytial Virus Reinfections in Children in Western Australia.

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.

Circulation of influenza and other respiratory viruses during the COVID-19 pandemic in Australia and New Zealand, 2020-2021.

Objective: Circulation patterns of influenza and other respiratory viruses have been globally disrupted since the emergence of coronavirus disease (COVID-19) and the introduction of public health and social measures (PHSMs) aimed at reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Methods: We reviewed respiratory virus laboratory data, Google mobility data and PHSMs in five geographically diverse regions in Australia and New Zealand. We also described respiratory virus activity from January 2017 to August 2021. Results: We observed a change in the prevalence of circulating respiratory viruses following the emergence of SARS-CoV-2 in early 2020. Influenza activity levels were very low in all regions, lower than those recorded in 2017-2019, with less than 1% of laboratory samples testing positive for influenza virus. In contrast, rates of human rhinovirus infection were increased. Respiratory syncytial virus (RSV) activity was delayed; however, once it returned, most regions experienced activity levels well above those seen in 2017-2019. The timing of the resurgence in the circulation of both rhinovirus and RSV differed within and between the two countries. Discussion: The findings of this study suggest that as domestic and international borders are opened up and other COVID-19 PHSMs are lifted, clinicians and public health professionals should be prepared for resurgences in influenza and other respiratory viruses. Recent patterns in RSV activity suggest that these resurgences in non-COVID-19 viruses have the potential to occur out of season and with increased impact.

Factors Predicting Secondary Respiratory Morbidity Following Early-Life Respiratory Syncytial Virus Infections: Population-Based Cohort Study.

Background: The association between early-life respiratory syncytial virus (RSV) infections and later respiratory morbidity is well established. However, there is limited evidence on factors that influence this risk. We examined sociodemographic and perinatal factors associated with later childhood respiratory morbidity requiring secondary care following exposure to a laboratory-confirmed RSV episode in the first 2 years. Methods: We used a probabilistically linked whole-of-population-based birth cohort including 252 287 children born in Western Australia between 2000 and 2009 with follow-up to the end of 2012. Cox proportional hazards models estimated adjusted hazard ratios (aHRs) of the association of various risk factors with the first respiratory episode for asthma, wheezing, and unspecified acute lower respiratory infection beyond the age of 2 years. Results: The analytic cohort included 4151 children with a confirmed RSV test before age 2 years. The incidence of subsequent respiratory morbidity following early-life RSV infection decreased with child age at outcome (highest incidence in 2-<4-year-olds: 41.8 per 1000 child-years; 95% CI, 37.5-46.6), increased with age at RSV infection (6-<12-month-olds: 23.6/1000 child-years; 95% CI, 19.9-27.8; 12-<24-month-olds: 22.4/1000 child-years; 95% CI, 18.2-22.7) and decreasing gestational age (50.8/1000 child-years; 95% CI, 33.5-77.2 for children born extremely preterm, <28 weeks gestation). Risk factors included age at first RSV episode (6-<12 months: aHR, 1.42; 95% CI, 1.06-1.90), extreme prematurity (<28 weeks: aHR, 2.22; 95% CI, 1.40-3.53), maternal history of asthma (aHR, 1.33; 95% CI, 1.04-1.70), and low socioeconomic index (aHR, 1.76; 95% CI, 1.03-3.00). Conclusions: Our results suggest that in addition to preterm and young infants, children aged 12-<24 months could also be potential target groups for RSV prevention to reduce the burden of later respiratory morbidities associated with RSV.

Determining the true incidence of seasonal respiratory syncytial virus-confirmed hospitalizations in preterm and term infants in Western Australia.

Respiratory syncytial virus contributes to significant global infant morbidity and mortality. We applied a previously developed statistical prediction model incorporating pre-pandemic RSV testing data and hospital admission data to estimate infant RSV-hospitalizations by birth month and prematurity, focused on infants aged <1 year. The overall predicted RSV-hospitalization incidence rates in infants <6 months were 32.7/1,000 child-years (95 % CI: 31.8, 33.5) and 3.1/1,000 child-years (95 % CI: 3.0, 3.1) in infants aged 6-<12 months. Predicted RSV-hospitalization rates for infants aged <6 months were highest for infants born in April/May. Predicted rates for preterm infants born 29-32 weeks gestation were highest in March-May, whereas infants born >33 weeks had peak RSV-hospitalization rates from May-June, similar to late preterm or term births. RSV-hospitalization rates in the pre-pandemic era were highly seasonal, and seasonality varied with degree of prematurity. Accurate estimates of RSV-hospitalization in high-risk sub-groups are essential to understand preventable burden of RSV especially given the current prevention landscape.

A surge in human metapneumovirus paediatric respiratory admissions in Western Australia following the reduction of SARS-CoV-2 non-pharmaceutical interventions.

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.

The seasonality of respiratory syncytial virus in Western Australia prior to implementation of SARS-CoV-2 non-pharmaceutical interventions.

Background: Respiratory syncytial virus (RSV) seasonality is dependent on the local climate. We assessed the stability of RSV seasonality prior to the SARS-CoV-2 pandemic in Western Australia (WA), a state spanning temperate and tropical regions. Method: RSV laboratory testing data were collected from January 2012 to December 2019. WA was divided into three regions determined by population density and climate: Metropolitan, Northern and Southern. Season threshold was calculated per region at 1.2% annual cases, with onset the first of ≥2 weeks above this threshold and offset as the last week before ≥2 weeks below. Results: The detection rate of RSV in WA was 6.3/10,000. The Northern region had the highest detection rate (15/10,000), more than 2.5 times the Metropolitan region (detection rate ratio 2.7; 95% CI, 2.6-2.9). Test percentage positive was similar in the Metropolitan (8.6%) and Southern (8.7%) regions, with the lowest in the Northern region (8.1%). RSV seasons in the Metropolitan and Southern regions occurred annually, with a single peak and had consistent timing and intensity. The Northern tropical region did not experience a distinct season. Proportion of RSV A to RSV B in the Northern region differed from the Metropolitan region in 5 of the 8 years studied. Conclusions: Detection rate of RSV in WA is high, especially in the Northern region, where climate, an expanded at-risk population and increased testing may have contributed to greater numbers. Before the SARS-CoV-2 pandemic, RSV seasonality in WA was consistent in timing and intensity for the Metropolitan and Southern regions.

Molecular detection and characterisation of the first Japanese encephalitis virus belonging to genotype IV acquired in Australia.

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_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.

An Unusual Resurgence of Human Metapneumovirus in Western Australia Following the Reduction of Non-Pharmaceutical Interventions to Prevent SARS-CoV-2 Transmission.

Non-pharmaceutical interventions (NPIs) to reduce SARS-CoV-2 transmission disrupted respiratory virus seasonality. We examined the unusual return of human metapneumovirus (hMPV) in Western Australia following a period of absence in 2020. We analysed hMPV laboratory testing data from 1 January 2017 to 31 December 2021. Whole-genome sequencing of selected hMPV-positive samples was performed using a tiled-amplicon approach. Following an absence in spring 2020, an unusual hMPV surge was observed during the wet summer season in the tropical Northern region in late 2020. Following a six-month delay, an intense winter season occurred in the subtropical/temperate Southern and Metropolitan regions. Compared to 2017-2019, hMPV incidence in 2021 increased by 3-fold, with a greater than 4-fold increase in children aged 1-4 years. There was a collapse in hMPV diversity in 2020, with the emergence of a single subtype. NPIs contributed to an absent 2020 season and a clonal hMPV resurgence. The summer surge and delayed winter season suggest that prevailing temperature and humidity are keys determinant of hMPV transmission. The increased incidence in 2021 was linked to an expanded cohort of hMPV-naïve 1-4-year-old children and waning population immunity. Further intense and unusual respiratory virus seasons are expected as COVID-19 associated NPIs are removed.

Whole genome sequencing of SARS-CoV-2 from wastewater links to individual cases in catchments.

After a limited first wave of community transmission in March 2020 and until 2022, Western Australia was largely free of COVID-19, with cases restricted to hotel quarantine, commercial vessels, and small, infrequent community clusters. Despite the low case load setting, sequencing of wastewater samples from large municipal treatment plants produced SARS-CoV-2 genomes with coverage up to 99.7 % and depth to 4000×, which was sufficient to link wastewater sequences to those of active cases in the catchment at the time. This study demonstrates that ≤5 positive individuals can be enough to produce high genomic coverage (>90 %) assemblies even in catchments of up to a quarter of a million people. Genomic analysis of wastewater contemporaneous with clinical cases can also be used to rule out transmission between cases in different catchments, when their SARS-CoV-2 genomes have distinguishing nucleotide polymorphisms. These findings reveal a greater potential of wastewater WGS to inform outbreak management and disease surveillance than previously recognized.

Travel-associated SARS-CoV-2 transmission documented with whole genome sequencing following a long-haul international flight.

BACKGROUND: Multiple instances of flight-associated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission during long-haul flights have been reported during the COVID-19 pandemic. However, comprehensive investigations of passenger risk behaviours, before, during and after the flight, are scarce. METHODS: To investigate suspected SARS-CoV-2 transmission during a flight from United Arab Emirates to Australia in July 2020, systematic, repeated polymerase chain reaction (PCR) testing of passengers in hotel quarantine was linked to whole genome sequencing. Epidemiological analyses of in-depth interviews covering behaviours during the flight and activities pre- and post-boarding were used to identify risk factors for infection. RESULTS: Seventeen of the 95 passengers from four different travel origins had PCR-confirmed infection yielding indistinguishable genomic sequences. Two of the 17 passengers were symptomatic within 2 days of the flight, and classified as co-primary cases. Seven secondary cases were seated within two rows of the co-primary cases, but five economy passengers seated further away and three business class passengers were also infected (attack rate = 16% [15/93]). In multivariable analysis, being seated within two rows of a primary case [odds ratio (OR) 7.16; 95% confidence interval (CI) 1.66-30.85] and spending more than an hour in the arrival airport (OR 4.96; 95% CI 1.04-23.60) were independent predictors of secondary infection, suggesting travel-associated SARS-CoV-2 transmission likely occurred both during and after the flight. Self-reported increased hand hygiene, frequent aisle walking and using the bathroom on the plane did not independently affect the risk of SARS-CoV-2 acquisition. CONCLUSIONS: This investigation identified substantial in-flight transmission among passengers seated both within and beyond two rows of the primary cases. Infection of passengers in separate cabin classes also suggests transmission occurred outside the cabin environment, likely at the arrival airport. Recognizing that transmission may occur pre- and post-boarding may inform contact tracing advice and improve efforts to prevent future travel-associated outbreaks.

Off-season RSV epidemics in Australia after easing of COVID-19 restrictions.

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.

Examining the interseasonal resurgence of respiratory syncytial virus in Western Australia.

BACKGROUND: Following a relative absence in winter 2020, a large resurgence of respiratory syncytial virus (RSV) detections occurred during the 2020/2021 summer in Western Australia. This seasonal shift was linked to SARS-CoV-2 public health measures. We examine the epidemiology and RSV testing of respiratory-coded admissions, and compare clinical phenotype of RSV-positive admissions between 2019 and 2020. METHOD: At a single tertiary paediatric centre, International Classification of Diseases, 10th edition Australian Modification-coded respiratory admissions longer than 12 hours were combined with laboratory data from 1 January 2019 to 31 December 2020. Data were grouped into bronchiolitis, other acute lower respiratory infection (OALRI) and wheeze, to assess RSV testing practices. For RSV-positive admissions, demographics and clinical features were compared between 2019 and 2020. RESULTS: RSV-positive admissions peaked in early summer 2020, following an absent winter season. Testing was higher in 2020: bronchiolitis, 94.8% vs 89.2% (p=0.01); OALRI, 88.6% vs 82.6% (p=0.02); and wheeze, 62.8% vs 25.5% (p<0.001). The 2020 peak month, December, contributed almost 75% of RSV-positive admissions, 2.5 times the 2019 peak. The median age in 2020 was twice that observed in 2019 (16.4 vs 8.1 months, p<0.001). The proportion of RSV-positive OALRI admissions was greater in 2020 (32.6% vs 24.9%, p=0.01). There were no clinically meaningful differences in length of stay or disease severity. INTERPRETATION: The 2020 RSV season was in summer, with a larger than expected peak. There was an increase in RSV-positive non-bronchiolitis admissions, consistent with infection in older RSV-naïve children. This resurgence raises concern for regions experiencing longer and more stringent SARS-CoV-2 public health measures.

Single-Point Mutations in the N Gene of SARS-CoV-2 Adversely Impact Detection by a Commercial Dual Target Diagnostic Assay.

Accurate and rapid diagnostic tests are a critical component for the early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of the overall control strategy for the current pandemic. Nucleic acid amplification tests are the gold standard for diagnosis of acute SARS-CoV-2 infection, and many real-time PCR diagnostic assays have been developed. Mutations that occur within the primer/probe binding regions of the SARS-CoV-2 genome can negatively impact the performance of diagnostic assays. Here, we report two single-point mutations in the N gene of SARS-CoV-2 associated with N gene target detection failures in the Cepheid Xpert Xpress SARS-CoV-2 assay, the first a C to T mutation at position 29197, found in five patients, and the second a C to T mutation at position 29200, found in eight patients. By sequencing the Xpert amplicons, we showed both mutations to be located within the amplified region of the Xpert N gene target. This report highlights the necessity for multiple genetic targets and the continual monitoring and evaluation of diagnostic assay performance. IMPORTANCE This paper reports the identification of single-point mutations in the N gene of SARS-CoV-2 associated with a gene target failure by the Cepheid Xpert commercial system. In order to determine the mutation(s) responsible for the N gene detection failures, the genomic products from the Cepheid Xpert system were sequenced and compared to whole genomes of SARS-CoV-2 from clinical cases. This report is the first to our knowledge which characterizes the amplified PCR products of the Xpert system, confirming the mutations associated with the gene target failure. The mutations identified have previously been reported.

The spatial-temporal dynamics of respiratory syncytial virus infections across the east-west coasts of Australia during 2016-17.

Respiratory syncytial virus (RSV) is an important human respiratory pathogen. In temperate regions, a distinct seasonality is observed, where peaks of infections typically occur in early winter, often preceding the annual influenza season. Infections are associated with high rates of morbidity and mortality and in some populations exceed that of influenza. Two subtypes, RSV-A and RSV-B, have been described, and molecular epidemiological studies have shown that both viruses mostly co-circulate. This trend also appears to be the case for Australia; however, previous genomic studies have been limited to cases from one Eastern state-New South Wales. As such, the broader spatial patterns and viral traffic networks across the continent are not known. Here, we conducted a whole-genome study of RSV comparing strains across eastern and Western Australia during the period January 2016 to June 2017. In total, 96 new RSV genomes were sequenced, compiled with previously generated data, and examined using a phylodynamic approach. This analysis revealed that both RSV-A and RSV-B strains were circulating, and each subtype was dominated by a single genotype, RSV-A ON1-like and RSV-B BA10-like viruses. Some geographical clustering was evident in strains from both states with multiple distinct sub-lineages observed and relatively low mixing across jurisdictions, suggesting that endemic transmission was likely seeded from imported, unsampled locations. Overall, the RSV phylogenies reflected a complex pattern of interactions across multiple epidemiological scales from fluid virus traffic across global and regional networks to fine-scale local transmission events.

Impact of Coronavirus Disease 2019 Public Health Measures on Detections of Influenza and Respiratory Syncytial Virus in Children During the 2020 Australian Winter.

Public health measures targeting coronavirus disease 2019 have potential to impact transmission of other respiratory viruses. We found 98.0% and 99.4% reductions in respiratory syncytial virus and influenza detections, respectively, in Western Australian children through winter 2020 despite schools reopening. Border closures have likely been important in limiting external introductions.