Trends in SARS-CoV-2 cycle threshold (Ct) values from nucleic acid testing predict the trajectory of COVID-19 waves.

Forecasting COVID-19 waves helps with public health planning and resource allocation. Cycle threshold (Ct) values obtained from positive SARS-CoV-2 nucleic acid amplification test (NAAT) results offer limited value for individual patient management, but real-time analysis of temporal trends of aggregated Ct values may provide helpful information to predict the trajectories of COVID-19 waves in the community. Ct value trends on 59,609 SARS-CoV-2 NAAT-positive results from 574,403 tests using a single testing assay system, between September 2021 and January 2023, were examined to monitor the trend of the proportion of positive NAAT with lower Ct values (≤28) in relation to changing COVID-19 case numbers over time. We applied regression with autoregressive integrated moving average errors modelling approach to study the relation between Ct values and case counts. We also developed an insight product to monitor the temporal trends with Ct values obtained from SARS-CoV-2 NAAT-positive results. In this study, the proportion of lower Ct values preceded by a range of 7-32 days the rising population COVID-19 testing rate reflecting onset of a COVID-19 wave. Monitoring population Ct values may assist in predicting increased disease activity.

Seroprevalence of Japanese encephalitis virus-specific antibodies in Australia following novel epidemic spread: protocol for a national cross-sectional study.

INTRODUCTION: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes encephalitis and other morbidity in Southeast Asia. Since February 2022, geographically dispersed JEV human, animal and vector detections occurred on the Australian mainland for the first time. This study will determine the prevalence of JEV-specific antibodies in human blood with a focus on populations at high risk of JEV exposure and determine risk factors associated with JEV seropositivity by location, age, occupation and other factors. METHOD: Samples are collected using two approaches: from routine blood donors (4153 samples), and active collections targeting high-risk populations (convenience sampling). Consent-based sampling for the latter includes a participant questionnaire on demographic, vaccination and exposure data. Samples are tested for JEV-specific total antibody using a defined epitope-blocking ELISA, and total antibody to Australian endemic flaviviruses Murray Valley encephalitis and Kunjin viruses. ANALYSIS: Two analytic approaches will occur: descriptive estimates of seroprevalence and multivariable logistic regression using Bayesian hierarchical models. Descriptive analyses will include unadjusted analysis of raw data with exclusions for JEV-endemic country of birth, travel to JEV-endemic countries, prior JEV-vaccination, and sex-standardised and age-standardised analyses. Multivariable logistic regression will determine which risk factors are associated with JEV seropositivity likely due to recent transmission within Australia and the relative contribution of each factor when accounting for effects within the model. ETHICS: National Mutual Acceptance ethical approval was obtained from the Sydney Children's Hospitals Network Human Research Ethics Committee (HREC). Local approvals were sought in each jurisdiction. Ethical approval was also obtained from the Australian Red Cross Lifeblood HREC. DISSEMINATION: Findings will be communicated to participants and their communities, and human and animal health stakeholders and policy-makers iteratively and after final analyses. Understanding human infection rates will inform procurement and targeted allocation of limited JEV vaccine, and public health strategies and communication campaigns, to at-risk populations.

A laboratory framework for ongoing optimization of amplification-based genomic surveillance programs.

IMPORTANCE: This study provides a laboratory framework to ensure ongoing relevance and performance of amplification-based whole genome sequencing to strengthen public health surveillance during extended outbreaks or pandemics. The framework integrates regular reviews of the performance of a genomic surveillance system and highlights the importance of ongoing monitoring and the identification and implementation of improvements to whole genome sequencing methods to enhance public health responses to pathogen outbreaks.

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.

The Question of the Origins of COVID-19 and the Ends of Science.

Intense public interest in scientific claims about COVID-19, concerning its origins, modes of spread, evolution, and preventive and therapeutic strategies, has focused attention on the values to which scientists are assumed to be committed and the relationship between science and other public discourses. A much discussed claim, which has stimulated several inquiries and generated far-reaching political and economic consequences, has been that SARS-CoV-2 was deliberately engineered at the Wuhan Institute of Virology and then, either inadvertently or otherwise, released to the public by a laboratory worker. This has been pursued despite a clear refutation, through comprehensive genomic analysis, of the hypothesis that the virus was deliberately engineered and the failure of detailed investigations to identify any evidence in support of a laboratory leak. At the same time a substantial, established body of knowledge about the many factors underlying the emergence of novel zoonotic diseases has been largely ignored-including climate change and other mechanisms of environmental destruction, tourism, patterns of trade, and cultural influences. The existence and conduct of these debates have raised questions about the vulnerability of science to manipulation for political purposes. Scientific discourses are vulnerable because: (i) claims can be made with no more than probabilistic force; (ii) alleged "facts" are always subject to interpretation, which depends on social, ethical, and epistemological assumptions; and (iii) science and scientists are not inherently committed to any single set of values and historically have served diverse, and sometimes perverse, social and political interests. In the face of this complexity, the COVID-19 experience highlights the need for processes of ethical scrutiny of the scientific enterprise and its strategic deployment. To ensure reliability of truth claims and protection from corrupting influences robust ethical discourses are required that are independent of, and at times even contrary to, those of science itself.

Understanding the treatment benefit of hyperimmune anti-influenza intravenous immunoglobulin (Flu-IVIG) for severe human influenza.

BACKGROUNDAntibody-based therapies for respiratory viruses are of increasing importance. The INSIGHT 006 trial administered anti-influenza hyperimmune intravenous immunoglobulin (Flu-IVIG) to patients hospitalized with influenza. Flu-IVIG treatment improved outcomes in patients with influenza B but showed no benefit for influenza A.METHODSTo probe potential mechanisms of Flu-IVIG utility, sera collected from patients hospitalized with influenza A or B viruses (IAV or IBV) were analyzed for antibody isotype/subclass and Fcγ receptor (FcγR) binding by ELISA, bead-based multiplex, and NK cell activation assays.RESULTSInfluenza-specific FcγR-binding antibodies were elevated in Flu-IVIG-infused IBV- and IAV-infected patients. In IBV-infected participants (n = 62), increased IgG3 and FcγR binding were associated with more favorable outcomes. Flu-IVIG therapy also improved the odds of a more favorable outcome in patients with low levels of anti-IBV Fc-functional antibody. Higher FcγR-binding antibody was associated with less favorable outcomes in IAV-infected patients (n = 50), and Flu-IVIG worsened the odds of a favorable outcome in participants with low levels of anti-IAV Fc-functional antibody.CONCLUSIONThese detailed serological analyses provide insights into antibody features and mechanisms required for a successful humoral response against influenza, suggesting that IBV-specific, but not IAV-specific, antibodies with Fc-mediated functions may assist in improving influenza outcome. This work will inform development of improved influenza immunotherapies.TRIAL REGISTRATIONClinicalTrials.gov NCT02287467.FUNDINGFunding for this research was provided by subcontract 13XS134 under Leidos Biomedical Research Prime Contract HHSN261200800001E and HHSN261201500003I, NCI/NIAID.

Risk of a first clinical diagnosis of central nervous system demyelination in relation to human herpesviruses in the context of Epstein-Barr virus.

BACKGROUND AND PURPOSE: Epstein-Barr virus (EBV) is implicated in multiple sclerosis (MS) risk; evidence for other herpesviruses is inconsistent. Here, we test blood markers of infection with human herpesvirus 6 (HHV-6), varicella zoster virus (VZV), and cytomegalovirus (CMV) as risk factors for a first clinical diagnosis of central nervous system demyelination (FCD) in the context of markers of EBV infection. METHODS: In the Ausimmune case-control study, cases had an FCD, and population controls were matched on age, sex, and study region. We quantified HHV-6- and VZV-DNA load in whole blood and HHV-6, VZV, and CMV antibodies in serum. Conditional logistic regression tested associations with FCD risk, adjusting for Epstein-Barr nuclear antigen (EBNA) IgG, EBV-DNA load, and other covariates. RESULTS: In 204 FCD cases and 215 matched controls, only HHV-6-DNA load (positive vs. negative) was associated with FCD risk (adjusted odds ratio = 2.20, 95% confidence interval = 1.08-4.46, p = 0.03). Only EBNA IgG and HHV-6-DNA positivity were retained in a predictive model of FCD risk; the combination had a stronger association than either alone. CMV-specific IgG concentration modified the association between an MS risk-related human leucocyte antigen gene and FCD risk. Six cases and one control had very high HHV-6-DNA load (>1.0 × 106 copies/mL). CONCLUSIONS: HHV-6-DNA positivity and high load (possibly due to inherited HHV-6 chromosomal integration) were associated with increased FCD risk, particularly in association with markers of EBV infection. With growing interest in prevention/management of MS through EBV-related pathways, there should be additional consideration of the role of HHV-6 infection.

RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples.

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 RAPIDprep 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 RAPIDprep assay is a simple and effective tool, and representative of an important shift toward the integration of modern genomic techniques with infectious disease investigations.

Arthropod-Borne Flaviviruses in Pregnancy.

Flaviviruses are a diverse group of enveloped RNA viruses that cause significant clinical manifestations in the pregnancy and postpartum periods. This review highlights the epidemiology, pathophysiology, clinical features, diagnosis, and prevention of the key arthropod-borne flaviviruses of concern in pregnancy and the neonatal period-Zika, Dengue, Japanese encephalitis, West Nile, and Yellow fever viruses. Increased disease severity during pregnancy, risk of congenital malformations, and manifestations of postnatal infection vary widely amongst this virus family and may be quite marked. Laboratory confirmation of infection is complex, especially due to the reliance on serology for which flavivirus cross-reactivity challenges diagnostic specificity. As such, a thorough clinical history including relevant geographic exposures and prior vaccinations is paramount for accurate diagnosis. Novel vaccines are eagerly anticipated to ameliorate the impact of these flaviviruses, particularly neuroinvasive disease manifestations and congenital infection, with consideration of vaccine safety in pregnant women and children pivotal. Moving forward, the geographical spread of flaviviruses, as for other zoonoses, will be heavily influenced by climate change due to the potential expansion of vector and reservoir host habitats. Ongoing 'One Health' engagement across the human-animal-environment interface is critical to detect and responding to emergent flavivirus epidemics.

Emergent Omicron BR.2.1 sublineage of SARS-CoV-2 in New South Wales, Australia: a subvariant with high fitness but without increased disease severity.

OBJECTIVES: To describe the epidemiology and impact of Omicron BR.2.1, an emergent SARS-CoV-2 Omicron BA.2.75 sublineage displaying high fitness compared to other cocirculating subvariants in New South Wales, Australia. METHODS: From September 01 to November 26, 2022, 4971 SARS-CoV-2 consensus genomes from unique patients were generated, and correlated with international travel and reinfection history, and admission to the intensive care unit. RESULTS: BR.2.1 became the predominant variant by late November, and was responsible for a significantly higher proportion of community-acquired cases during the study period (55.1% vs 38.4%, P < 0.001). Reinfections (defined as occurring between 6 and 24 weeks after a prior diagnosis of COVID-19) were significantly higher among BR.2.1 compared to non-BR.2.1 infected persons (17.0% vs 6.0%, P < 0.001). BR.2.1 cases were also significantly younger compared to non-BR.2.1 (median age 48 years (interquartile range [IQR] 32) vs 53 years (IQR 32), P = 0.004). The proportion of patients admitted to the intensive care unit with BR.2.1 was not significantly higher than other subvariants (2.3% vs 2.0%, P = 0.717). CONCLUSION: Having emerged locally within New South Wales, BR.2.1 caused a significant number of SARS-CoV-2 reinfections, but with disease severity comparable with other currently circulating lineages. Given its rapid rise in prevalence, BR.2.1 has the potential to become established internationally.

New Detection of Locally Acquired Japanese Encephalitis Virus Using Clinical Metagenomics, New South Wales, Australia.

In the context of an emerging Japanese encephalitis outbreak within Australia, we describe a novel locally acquired case in New South Wales. A man in his 70s had rapidly progressive, fatal meningoencephalitis, diagnosed as caused by Japanese encephalitis virus by RNA-based metagenomic next-generation sequencing performed on postmortem brain tissue.

The Origins of Severe Acute Respiratory Syndrome-Coronavirus-2.

An outbreak of severe pneumonia of unknown cause was identified in Wuhan, China in December 2019: the causative agent was a novel betacoronavirus, severe acute respiratory syndrome-cotonavirus-2 (SARS-CoV-2), a virus that joins a list of coronaviruses causing severe (e.g., SARS and Middle East respiratory syndrome) or milder (e.g., 229E, OC43, NL63, and HKU1) respiratory tract infection. The World Health Organization (WHO) classified the spreading outbreak as a pandemic on March 11, 2020. Many SARS-related coronaviruses (SARSr-CoVs) have been identified in bats, particularly in Rhinolophus horseshoe bats, animals that are common in southern China and Southeast Asia. Many of the features of SARS-CoV-2 that facilitate human infection-the furin cleavage site, the receptor binding domain that binds to the human ACE2 receptor-can be identified in SARSr-CoVs. Related coronaviruses can be detected in pangolins and other animals, and human SARS-CoV-2 itself can infect various animals, some of which can transmit SARS-CoV-2 back to humans. Investigation by the WHO and others pointed to the initial outbreak being centered on the Huanan wet market in Wuhan where wild and farmed animals were sold, and where environmental testing revealed widespread SARS-CoV-2 contamination. This supports the hypothesis that bats, probably via an intermediate animal, are the origin of SARS-CoV-2. Other possible origins have been postulated, such as an accidental or deliberate laboratory leak, or virus present in frozen foods, but evidence for these ideas has not surfaced. Study of the origins of SARS-CoV-2 have been complicated by intense media and political commentary, features that may slow the studies required to understand the viral origins. Such studies are complex and may be slow: international openness and co-operation is vital. Origins explanations are needed to predict or prevent future pandemics and support the "One Health" approach to disease.

SARS-CoV-2 Seroprevalence in a Cohort of International Travellers Returning to Rural Australia: Enablers and Barriers to Containment of COVID-19.

OBJECTIVE: To describe the effectiveness of the public health response to COVID-19 in our local region by documenting detection of SARS-CoV-2 infection by nucleic acid testing (NAT) positivity and seroprevalence. METHODS: In this prospective study (ACTRN12620000487910), symptomatic adult international travellers returning to regional Australia in March 2020 underwent SARS-CoV-2 NAT and SARS-CoV-2-specific serology. RESULTS: Ninety-nine eligible participants were included. Nine participants had laboratory confirmed SARS-CoV-2, all returning between 16-20 March 2020. Eight (89%) had a positive NAT and seven (78%) had a positive serology test. The majority returned from New Zealand. Participants most frequently presented with cough (100%), headache (66.7%) and sore throat (44.4%). No community cases were detected from 1 March to 30 June 2020. CONCLUSIONS: The study cohort of international travellers returning to regional Australia in March 2020 returned eight positive SARS-CoV-2 NAT results over a five-day window. Serology identified one additional case and was negative in two cases who were PCR positive. Longitudinal data confirmed an absence of local community transmission to 30 June 2020. IMPLICATIONS FOR PUBLIC HEALTH: A combination of local, national and environmental factors were necessary to prevent the establishment of community transmission in our local region.

Rapid on-site molecular Point of Care Testing during influenza outbreaks in aged care facilities improves antiviral use and reduces hospitalisation.

OBJECTIVE: Western Sydney Local Health District (WSLHD) measured the utility and validity of rapid molecular point-of-care testing (POCT) in aged care facilities (ACFs) experiencing influenza-like illness (ILI) outbreaks against routine laboratory testing. METHODS: A descriptive epidemiological study into 82 respiratory outbreaks reported across 63 ACFs within WSLHD supporting approximately 6,500 residents aged ≥65 years and staffed by ∼6,500 employees, from 1 August 2018 to 31 December 2019. RESULTS: WSLHD Public Health Unit performed on-site testing at 27 ACF outbreaks (34%), while 53(66%) ACFs conducted only routine laboratory testing. The Xpert®Xpress Flu/RSV molecular PCR provided a sensitivity and specificity of 100%. Those with on-site testing, antiviral prophylaxis was prescribed at 75% of facilities within 24 hours of testing, as opposed to 32% of those using laboratory testing (p<0.01). There were 24 of 181 ACF residents hospitalised in the POCT group compared to 76 of 357 in the laboratory-only group (OR=0.57; p=0.02). CONCLUSIONS: On-site ACF testing is reliable and practical for early identification of influenza, enabling timely use of antiviral treatment and prophylaxis, and was associated with decreased hospitalisation. PUBLIC HEALTH IMPLICATIONS: Enhanced respiratory surveillance and on-site testing should be strongly considered as part of routine management of respiratory outbreaks in ACFs and may reduce outbreak severity.

Guiding the design of SARS-CoV-2 genomic surveillance by estimating the resolution of outbreak detection.

Genomic surveillance of SARS-CoV-2 has been essential to inform public health response to outbreaks. The high incidence of infection has resulted in a smaller proportion of cases undergoing whole genome sequencing due to finite resources. We present a framework for estimating the impact of reduced depths of genomic surveillance on the resolution of outbreaks, based on a clustering approach using pairwise genetic and temporal distances. We apply the framework to simulated outbreak data to show that outbreaks are detected less frequently when fewer cases are subjected to whole genome sequencing. The impact of sequencing fewer cases depends on the size of the outbreaks, and on the genetic and temporal similarity of the index cases of the outbreaks. We also apply the framework to an outbreak of the SARS-CoV-2 Delta variant in New South Wales, Australia. We find that the detection of clusters in the outbreak would have been delayed if fewer cases had been sequenced. Existing recommendations for genomic surveillance estimate the minimum number of cases to sequence in order to detect and monitor new virus variants, assuming representative sampling of cases. Our method instead measures the resolution of clustering, which is important for genomic epidemiology, and accommodates sampling biases.

Emerging Genotype IV Japanese Encephalitis Virus Outbreak in New South Wales, Australia.

The detection of a new and unexpected Japanese encephalitis virus (JEV) outbreak in March 2022 in Australia, where JEV is not endemic, demanded the rapid development of a robust diagnostic framework to facilitate the testing of suspected patients across the state of New South Wales (NSW). This nascent but comprehensive JEV diagnostic service encompassed serological, molecular and metagenomics testing within a centralised reference laboratory. Over the first three months of the outbreak (4 March 2022 to 31 May 2022), 1,061 prospective samples were received from 878 NSW residents for JEV testing. Twelve confirmed cases of Japanese encephalitis (JE) were identified, including ten cases diagnosed by serology alone, one case by metagenomic next generation sequencing and real-time polymerase chain reaction (RT-PCR) of brain tissue and serology, and one case by RT-PCR of cerebrospinal fluid, providing an incidence of JE over this period of 0.15/100,000 persons in NSW. As encephalitis manifests in <1% of cases of JEV infection, the population-wide prevalence of JEV infection is likely to be substantially higher. Close collaboration with referring laboratories and clinicians was pivotal to establishing successful JEV case ascertainment for this new outbreak. Sustained and coordinated animal, human and environmental surveillance within a OneHealth framework is critical to monitor the evolution of the current outbreak, understand its origins and optimise preparedness for future JEV and arbovirus outbreaks.

Improved Neutralisation of the SARS-CoV-2 Omicron Variant following a Booster Dose of Pfizer-BioNTech (BNT162b2) COVID-19 Vaccine.

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired over 30 mutations in the spike protein (with 15 in the receptor-binding domain), raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three, and six months post-two doses of Pfizer-BioNTech BNT162b2 had a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres were boosted. Despite this increase, neutralising antibody titres were reduced fourfold for Omicron compared to lineage A.2.2 SARS-CoV-2.

Development and Validation of an In-House Real-Time Reverse-Transcriptase Polymerase Chain Reaction Assay for SARS-CoV-2 Omicron Lineage Subtyping between BA.1 and BA.2.

In order to rapidly differentiate sublineages BA.1 and BA.2 of the SARS-CoV-2 variant of concern Omicron, we developed a real-time reverse-transcriptase polymerase chain reaction to target the discriminatory spike protein deletion at amino acid position 69-70 (S:del69-70). Compared to the gold standard of whole genome sequencing, the candidate assay was 100% sensitive and 99.4% specific. Sublineage typing by RT-PCR can provide a rapid, high throughput and cost-effective method to enhance surveillance as well as potentially guiding treatment and infection control decisions.

Emergence of Japanese encephalitis in Australia: a diagnostic perspective.

The unprecedented emergence of Japanese encephalitis (JE) in mainland Australia represents an outbreak of high clinical and public health significance. JE is a zoonosis spread by mosquitoes and is one of the most important causes of endemic viral encephalitis in South-East Asia and the Indian subcontinent. While occasional cases of human Japanese encephalitis virus (JEV) infection have occurred in far north Australia, its detection in pigs and the substantial number of locally acquired human cases across multiple jurisdictions in early 2022 prompted the declaration of this outbreak as a Communicable Disease Incident of National Significance. Laboratory testing for JEV is complex, and most cases are diagnosed by serology, for which interpretation is difficult. This review provides a comprehensive outline of currently available methods for JEV diagnosis including serology, nucleic acid amplification testing, virus isolation, sequencing and metagenomics. The relative advantages and disadvantages of the diagnostic tests are presented, as well as their value in clinical and public health contexts. This review also explores the role of mosquito, veterinary and human surveillance as part of the laboratory response to JEV. As JEV may become endemic in Australia, a collaborative and coordinated One Health approach involving animal, human and environmental health is required for optimal disease response and control.