Infectious Diseases - Research Publications
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ItemNo Preview AvailableRapid detection of monkeypox virus using a CRISPR-Cas12a mediated assay: a laboratory validation and evaluation(Elsevier, 2023-10)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.
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ItemNo Preview AvailableIntra- and interhost genomic diversity of monkeypox virus(WILEY, 2023-08)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.
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ItemCorrelation between monkeypox viral load and infectious virus in clinical specimens(ELSEVIER, 2023-04)BACKGROUND: In the 2022 mpox outbreak, several studies have explored longitudinal DNA shedding of mpox virus (MPXV) using PCR. However, there are fewer studies assessing infectivity in cell culture, and, by inference, MPXV transmissibility. Such information could help inform infection control and public health guidelines. AIMS AND METHODS: The aim of this study was to correlate cell culture infectivity of clinical samples with viral loads in clinical samples. Between May to October 2022, clinical samples from different body sites sent to the Victorian Infectious Diseases Reference Laboratory in Melbourne, Australia for MPXV PCR detection were cultured in Vero cells as a surrogate for infectivity. RESULTS: In the study period, 144 samples from 70 patients were tested by MPXV PCR. Viral loads in skin lesions were significantly higher than those in throat or nasopharyngeal samples (median Ct 22.0 vs 29.0, p = 0.0013 and median Ct 22.0 vs 36.5, p = 0.0001, respectively). Similarly, viral loads were significantly higher in anal samples compared to throat or nasopharyngeal samples (median Ct 20.0 vs. 29.0, p=<0.0001 and median Ct 20.0 vs. 36.5, p=<0.0001, respectively). Viral culture was successfully performed in 80/94 samples. Using logistic regression analysis, 50% of the samples were positive in viral culture at Ct 34.1 (95% confidence intervals 32.1-37.4). CONCLUSIONS: Our data further validate recent findings showing that samples with a higher MPXV viral load are more likely to demonstrate infectivity in cell culture. Although the presence of infectious virus in cell culture may not directly translate with clinical transmission risk, our data may be used as an adjunct help inform guidelines on testing and isolation policies in individuals with mpox.