Non-SARS-CoV-2 respiratory viral detection and whole genome sequencing from COVID-19 rapid antigen test devices: a laboratory evaluation study.

BACKGROUND: There has been high uptake of rapid antigen test device use for point-of-care COVID-19 diagnosis. Individuals who are symptomatic but test negative on COVID-19 rapid antigen test devices might have a different respiratory viral infection. We aimed to detect and sequence non-SARS-CoV-2 respiratory viruses from rapid antigen test devices, which could assist in the characterisation and surveillance of circulating respiratory viruses in the community. METHODS: We applied archival clinical nose and throat swabs collected between Jan 1, 2015, and Dec 31, 2022, that previously tested positive for a common respiratory virus (adenovirus, influenza, metapneumovirus, parainfluenza, rhinovirus, respiratory syncytial virus [RSV], or seasonal coronavirus; 132 swabs and 140 viral targets) on PCR to two commercially available COVID-19 rapid antigen test devices, the Panbio COVID-19 Ag Rapid Test Device and Roche SARS-CoV-2 Antigen Self-Test. In addition, we collected 31 COVID-19 rapid antigen test devices used to test patients who were symptomatic at The Royal Melbourne Hospital emergency department in Melbourne, Australia. We extracted total nucleic acid from the device paper test strips and assessed viral recovery using multiplex real-time PCR (rtPCR) and capture-based whole genome sequencing. Sequence and genome data were analysed through custom computational pipelines, including subtyping. FINDINGS: Of the 140 respiratory viral targets from archival samples, 89 (64%) and 88 (63%) were positive on rtPCR for the relevant taxa following extraction from Panbio or Roche rapid antigen test devices, respectively. Recovery was variable across taxa: we detected influenza A in nine of 18 samples from Panbio and seven of 18 from Roche devices; parainfluenza in 11 of 20 samples from Panbio and 12 of 20 from Roche devices; human metapneumovirus in 11 of 16 from Panbio and 14 of 16 from Roche devices; seasonal coronavirus in eight of 19 from Panbio and two of 19 from Roche devices; rhinovirus in 24 of 28 from Panbio and 27 of 28 from Roche devices; influenza B in four of 15 in both devices; and RSV in 16 of 18 in both devices. Of the 31 COVID-19 devices collected from The Royal Melbourne Hospital emergency department, 11 tested positive for a respiratory virus on rtPCR, including one device positive for influenza A virus, one positive for RSV, four positive for rhinovirus, and five positive for SARS-CoV-2. Sequences of target respiratory viruses from archival samples were detected in 55 (98·2%) of 56 samples from Panbio and 48 (85·7%) of 56 from Roche rapid antigen test devices. 98 (87·5%) of 112 viral genomes were completely assembled from these data, enabling subtyping for RSV and influenza viruses. All 11 samples collected from the emergency department had viral sequences detected, with near-complete genomes assembled for influenza A and RSV. INTERPRETATION: Non-SARS-CoV-2 respiratory viruses can be detected and sequenced from COVID-19 rapid antigen devices. Recovery of near full-length viral sequences from these devices provides a valuable opportunity to expand genomic surveillance programmes for public health monitoring of circulating respiratory viruses. FUNDING: Australian Government Medical Research Future Fund and Australian National Health and Medical Research Council.

Prevention and post-exposure management of occupational exposure to Ebola virus.

There have been significant advances in the prevention and management of Ebola virus disease (EVD) caused by Zaire Ebola virus (ZEBOV), including the development of two effective vaccines, rVSV-ZEBOV and Ad26.ZEBOV/MVA-BN-Filo. In addition, ZEBOV monoclonal antibodies have become first-line therapy for EVD. However, the 2022-23 outbreak of Sudan Ebola virus (SUDV) in Uganda has highlighted the gap in current therapies and vaccines, whose efficacy is uncertain against non-ZEBOV species. Health-care and laboratory staff working in EVD treatment centres or Ebola virus diagnostic and research laboratories face unique risks relating to potential occupational exposure to Ebola viruses. Given the substantial morbidity and mortality associated with EVD, facilities should have strategies in place to manage occupational exposures, including consideration of post-exposure therapies. In this Review, we discuss currently available evidence for prevention and post-exposure prophylaxis of EVD, including therapies currently under evaluation for SUDV.

Assessment of hepatitis B virus infection and interhost cellular responses using intrahepatic cholangiocyte organoids.

The intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma-derived HBV (genotype B and C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT-PCR (RNA, covalently closed circular DNA [cccDNA], extracellular DNA) and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon-stimulated genes [ISG]) to interferon-α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well-differentiated ICOs using spinoculation, with time and donor-dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg and HBsAg. Donor-dependent drug responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon-α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor-dependent variation in viral dynamics and cellular responses. These features can be utilized for the development of personalized drug testing platform for antivirals.

An integrated public health response to an outbreak of Murray Valley encephalitis virus infection during the 2022-2023 mosquito season in Victoria.

Introduction: Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus known to cause infrequent yet substantial human outbreaks around the Murray Valley region of south-eastern Australia, resulting in significant mortality. Methods: The public health response to MVEV in Victoria in 2022-2023 included a climate informed pre-season risk assessment, and vector surveillance with mosquito trapping and laboratory testing for MVEV. Human cases were investigated to collect enhanced surveillance data, and human clinical samples were subject to serological and molecular testing algorithms to assess for co-circulating flaviviruses. Equine surveillance was carried out via enhanced investigation of cases of encephalitic illness. Integrated mosquito management and active health promotion were implemented throughout the season and in response to surveillance signals. Findings: Mosquito surveillance included a total of 3,186 individual trapping events between 1 July 2022 and 20 June 2023. MVEV was detected in mosquitoes on 48 occasions. From 2 January 2023 to 23 April 2023, 580 samples (sera and CSF) were tested for flaviviruses. Human surveillance detected 6 confirmed cases of MVEV infection and 2 cases of "flavivirus-unspecified." From 1 September 2022 to 30 May 2023, 88 horses with clinical signs consistent with flavivirus infection were tested, finding one probable and no confirmed cases of MVE. Discussion: The expanded, climate-informed vector surveillance system in Victoria detected MVEV in mosquitoes in advance of human cases, acting as an effective early warning system. This informed a one-health oriented public health response including enhanced human, vector and animal surveillance, integrated mosquito management, and health promotion.

Rapid detection of monkeypox virus using a CRISPR-Cas12a mediated assay: a laboratory validation and evaluation study.

BACKGROUND: The 2022 outbreak of mpox (formerly known as monkeypox) led to the spread of monkeypox virus (MPXV) in over 110 countries, demanding effective disease management and surveillance. As current diagnostics rely largely on centralised laboratory testing, our objective was to develop a simple rapid point-of-care assay to detect MPXV in clinical samples using isothermal amplification coupled with CRISPR and CRISPR-associated protein (Cas) technology. METHODS: In this proof-of-concept study, we developed a portable isothermal amplification CRISPR-Cas12a-based assay for the detection of MPXV. We designed a panel of 22 primer-guide RNA sets using pangenome and gene-agnostic approaches, and subsequently shortlisted the three sets producing the strongest signals for evaluation of analytical sensitivity and specificity using a fluorescence-based readout. The set displaying 100% specificity and the lowest limit of detection (LOD) was selected for further assay validation using both a fluorescence-based and lateral-flow readout. Assay specificity was confirmed using a panel of viral and bacterial pathogens. Finally, we did a blind concordance study on genomic DNA extracted from 185 clinical samples, comparing assay results with a gold-standard quantitative PCR (qPCR) assay. We identified the optimal time to detection and analysed the performance of the assay relative to qPCR using receiver operating characteristic (ROC) curves. We also assessed the compatibility with lateral-flow strips, both visually and computationally, where strips were interpreted blinded to the fluorescence results on the basis of the presence or absence of test bands. FINDINGS: With an optimal run duration of approximately 45 min from isothermal amplification to CRISPR-assay readout, the MPXV recombinase polymerase amplification CRISPR-Cas12a-based assay with the selected primer-guide set had an LOD of 1 copy per µL and 100% specificity against tested viral pathogens. Blinded concordance testing of 185 clinical samples resulted in 100% sensitivity (95% CI 89·3-100) and 99·3% specificity (95% CI 95·7-100) using the fluorescence readout. For optimal time to detection by fluorescence readout, we estimated the areas under the ROC curve to be 0·98 at 2 min and 0·99 at 4 min. Lateral-flow strips had 100% sensitivity (89·3-100) and 98·6% specificity (94·7-100) with both visual and computational assessment. Overall, lateral-flow results were highly concordant with fluorescence-based readouts (179 of 185 tests, 96·8% concordant), with discrepancies associated with low viral load samples. INTERPRETATION: Our assay for the diagnosis of mpox displayed good performance characteristics compared with qPCR. Although optimisation of the assay will be required before deployment, its usability and versatility present a potential solution to MPXV detection in low-resource and remote settings, as well as a means of community-based, on-site testing. FUNDING: Victorian Medical Research Accelerator Fund and the Australian Government Department of Health.

Intra- and interhost genomic diversity of monkeypox virus.

The impact and frequency of infectious disease outbreaks demonstrate the need for timely genomic surveillance to inform public health responses. In the largest known outbreak of mpox, genomic surveillance efforts have primarily focused on high-incidence nations in Europe and the Americas, with a paucity of data from South-East Asia and the Western Pacific. Here we analyzed 102 monkeypox virus (MPXV) genomes sampled from 56 individuals in Melbourne, Australia. All genomes fell within the 2022 MPXV outbreak lineage (B.1), with likely onward local transmission detected. We observed within-host diversity and instances of co-infection, and highlight further examples of structural variation and apolipoprotein B editing complex-driven micro-evolution in the current MPXV outbreak. Updating our understanding of MPXV emergence and diversification will inform public health measures and enable monitoring of the virus' evolutionary trajectory throughout the mpox outbreak.

Whole-Genome Sequencing of SARS-CoV-2 from Quarantine Hotel Outbreak.

Hotel quarantine for international travelers has been used to prevent coronavirus disease spread into Australia. A quarantine hotel-associated community outbreak was detected in South Australia. Real-time genomic sequencing enabled rapid confirmation tracking the outbreak to a recently returned traveler and linked 2 cases of infection in travelers at the same facility.