key: cord-1047717-4ej0pajc authors: Isles, Nicole; Badman, Steven G.; Ballard, Susan; Zhang, Bowen; Howden, Benjamin P.; Guy, Rebecca; Williamson, Deborah A. title: Analytical sensitivity and specificity of the Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV assay date: 2021-10-22 journal: Pathology DOI: 10.1016/j.pathol.2021.09.002 sha: 657f12e3010bf62945722d20e07b044c60dda4ee doc_id: 1047717 cord_uid: 4ej0pajc nan Testing was conducted at the Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL), The University of Melbourne at the Doherty Institute, Melbourne, Australia. In brief, SARS-CoV-2-positive nasopharyngeal or deep nasal swabs were obtained from routine clinical testing at MDU PHL, and stored SARS-CoV-2-negative nasopharyngeal or deep nasal swabs were obtained from the Department of Microbiology, Royal Melbourne Hospital (RMH), Melbourne, Australia. All clinical samples were previously tested for SARS-CoV-2 using the AusDiagnostics Coronavirus Typing (8-well) panel (AusDiagnostics, Australia) at RMH, as previously described, 8 and using the Aptima SARS-CoV-2 assay (Hologic, USA) at MDU PHL, according to the manufacturer's instructions. Analytical sensitivity for SARS-CoV-2 detection was assessed using two approaches. First, a 50 µL volume of quantified inactivated whole virus (SARS-CoV-2 Analytical Q Panel; Qnostics, UK) supplied as a standardised dilution series (6.0 to 1.7 log10 digital copies/mL) was spiked into universal transport media (UTM). Subsequently, 300 µL of spiked UTM was used in the Xpert Xpress SARS-CoV-2/Flu/RSV assay and also in the Xpert Xpress SARS-CoV-2 assay for comparison. The limit of detection (LOD) was determined, and all testing was performed in triplicate. Second, analytical sensitivity was also determined using heat-killed SARS-CoV-2 virus stock quantified at 1.04 × 10 5 TCID50/mL, obtained from previously isolated SARS-CoV-2 in Melbourne. 9 Virus was diluted in saline, and 50 µL was spiked into universal transport media (obtained from the Media Preparation Unit, University of Melbourne). Three replicates at 10×, 1× and 0.1× LOD (LOD determined by Xpert Xpress SARS-CoV-2) were tested. Clinical sensitivity was assessed by testing 46 RT-PCR confirmed positive samples (previously tested on the Panther Fusion SARS-CoV-2 assay; Hologic), spanning a range of cycle Ct values between 18.2 and 36.1 (Table 1) . Cross-reactivity was assessed using a commercial panel of respiratory control organisms (NATRPC2-BIO; Zeptometrix, USA) comprising purified, intact virus particles and bacterial cells suspended in a matrix that mimics the composition of a clinical specimen. Cross-reactivity was also examined using gamma-irradiated influenza virus (A/Victoria/31/2020 and B/Darwin/58/2019), respiratory syncytial virus (RSV) A 16144363 and RSV B 15136810 and two seasonal human coronavirus strains OC43 and 229E obtained from the Victorian Infectious Diseases Reference Laboratory (VIDRL), Melbourne, Australia, and spiked into pooled nasopharyngeal swab samples that tested negative to SARS-CoV-2. Clinical specificity was assessed by testing 50 SARS-CoV-2 RT-PCR-negative samples obtained from patients with respiratory symptoms attending RMH. Using the Qnostics SARS-CoV-2 panel, the LoD for SARS-CoV-2 with the Xpert Xpress SARS-CoV-2/Flu/RSV assay was 8.3 copies/mL, and for the Xpert Xpress SARS-CoV-2, assay was 8.3 copies/mL. Using dilutions of heat inactivated SARS-CoV-2, the LoD for the Xpert Xpress SARS-CoV-2/Flu/RSV assay was 0.002 TCID50/mL, and for the Xpert Xpress, SARS-CoV-2 assay was 0.002 TCID50/mL. Agreement of the Xpert Xpress SARS-CoV-2/Flu/RSV assay with the Panther Fusion SARS-CoV-2 assay was 100%, across a range of Ct values (Table 1) . Inactivated influenza A B and RSV were serially diluted in UTM; 10 µL, 25 µL and 50 µL of diluted virus were spiked into pooled negative swab matrix at approx 1×, 2× and 5× LOD (previously determined by MDU). Further limiting dilutions were prepared from the above spike preparations and diluted in negative swab matrix to determine LOD and were tested in triplicate. The LoD values for influenza and RSV strains are described in Table 2 . Using the Zeptometrix respiratory panel, all expected targets were detected using the Xpert Xpress SARS-CoV-2/Flu/RSV assay. In addition, no cross-reactivity was observed across any of the four assay channels in the Xpert Xpress SARS-CoV-2/Flu/RSV assay (SARS-CoV-2; influenza A; influenza B; RSV). Further, the Xpert Xpress SARS-CoV-2 assay did not display any cross-reactivity with the Zeptometrix panels. Using heat-inactivated virus spiked into negative swab samples, no cross-reactivity was observed, and all expected positive samples were detected (Table 3) . Moreover, all 50 clinical samples that tested negative for SARS-CoV-2 at RMH also tested negative using the Xpert Xpress SARS-CoV-2/Flu/RSV assay, giving a negative percentage agreement of 100%. Here, we provide a comprehensive evaluation of the performance of the new Xpert Xpress SARS-CoV-2/Flu/RSV assay. We demonstrate that the performance characteristics of this assay are comparable to the existing Xpert Xpress SARS-CoV-2 assay for the detection of SARS-CoV-2, with similar LoD and specificity for both assays. Further, our results are in keeping with a recent study by Mostafa et al. that demonstrated an overall positive percentage agreement for the SARS-CoV-2 target of 98.7% when compared to a range of other RT-PCR platforms (including the Xpert Xpress SARS-CoV-2 and Hologic Panther Fusion SARS-CoV-2 assays), and a negative percentage agreement of 100% with other targets (influenza A/B and RSV) showing 100% total agreement. 6 More recently, Lueng et al. also reported high concordance between the Xpert Xpress SARS-CoV-2 and Xpert Xpress SARS-CoV-2/Flu/RSV assay. 10 For end-users, one notable difference exists between the two Xpert assay types. The single Xpert Xpress SARS-CoV-2 assay reports N2 and E cycle threshold values independently within the analyte results for each sample. The test also calls out three result types: positive, presumptive positive and not detected. In contrast, the Xpert Xpress SARS-CoV-2/Flu/RSV assay reports a single Ct value for each of the four pathogens and each result is reported as detected or undetected. While some purchase cost differential is likely between the single Xpert SARS-CoV-2 assay and its 4plex cousin, commercial pricing had not been finalised at the time of writing. More broadly, rapid and reliable testing for SARS-CoV-2 has been critical to the COVID-19 response. Given the overlap in clinical symptoms between COVID-19 and other respiratory illnesses such as influenza, rapid differentiation of causative pathogens is essential in ensuring appropriate clinical and public health control measures. This is particularly important for responses to COVID-19, i.e., rapid isolation, treatment intervention and contact tracing. Moreover, as COVID-19 vaccination is gradually implemented globally, and national and international movement resumes over the next few years, inevitably, the circulation of other respiratory viruses such as influenza and RSV will increase. There is both an immediate and ongoing need for rapid multiplex testing, particularly testing that can be performed near or at the point of care, such as the Xpert Xpress SARS-CoV-2/Flu/RSV assay which requires minimal sample preparation steps. Use of this assay in combination with a validated viral inactivating transport medium 11 also reduces pathogenic exposure risk for test operators at that point of care or in laboratory settings. Possible uses include testing in aged care and emergency hospital settings, where rapid diagnosis will enable J o u r n a l P r e -p r o o f triage of patients to appropriate treatment, isolation rooms, and prompt implementation of infection control measures. Remote locations would also benefit from this molecular point of care test given the Xpert Xpress 4plex assay covers four major respiratory pathogens and may reduce the need for laboratory reflex testing and shorten clinical decision making time. The presence of such a test could also avert unnecessary and costly evacuations of individuals to larger health facilities. J o u r n a l P r e -p r o o f World Health Organization. WHO Coronavirus Disease (COVID-19) Dashboard. Cited 10 Impact of delays on effectiveness of contact tracing strategies for COVID-19: a modelling study Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China Multicenter evaluation of the Cepheid Xpert Xpress SARS-CoV-2 test Multi-center evaluation of cepheid xpert® Xpress SARS-CoV-2 point-of-care test during the SARS-CoV-2 pandemic A decentralised point-of-care testing model to address inequities in the COVID-19 response Multi-center evaluation of the Cepheid Xpert® Xpress SARS-CoV-2/Flu/RSV Test Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia Implementation and evaluation of a novel real-time multiplex assay for SARS-CoV-2: in-field learnings from a clinical microbiology laboratory Evaluation of the Xpert Xpress SARS-CoV-2/Flu/RSV assay for simultaneous detection of SARS-CoV-2, Influenza A/B and respiratory syncytial viruses in nasopharyngeal specimens Evaluation of commercially available viral transport medium (VTM) for SARS-CoV-2 inactivation and use in point-of-care (POC) testing Acknowledgements: Our sincere thanks to Tanya Applegate, Socheata Chea and Eloise Williams for their support with this evaluation.Conflicts of interest and sources of funding: Laboratory work was supported by a grant from the NHMRC Medical Research Future Fund (APP2002317). We acknowledge in-kind support from Cepheid who supplied the SARS-CoV-2/Flu A-B/RSV cartridges free of charge for this validation study.