Clinical evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test.

The GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test (PLUS assay) received Health Canada approval in January 2022. The PLUS assay is similar to the SARS-CoV-2/Flu/RSV combination test, with modifications to improve assay robustness against circulating and emerging variants. The performance characteristics of the PLUS assay were assessed at the Lakeridge Health Oshawa Hospital Centre and the National Microbiology Laboratory of Canada. The PLUS assay was directly compared to the SARS-CoV-2/Flu/RSV combination test using SARS-CoV-2 culture from five variants and remnant clinical specimens collected across the coronavirus disease 2019 pandemic. This included 50 clinical specimens negative for all pathogens, 110 clinical specimens positive for SARS-CoV-2, influenza A, influenza B, RSVA, and(or) RSVB and an additional 11 mixed samples to screen for target interactions. The PLUS assay showed a high % agreement with the widely used SARS-CoV-2/Flu/RSV combination test. Based on these findings, the PLUS assay and the Xpert SARS-CoV-2/Flu/RSV combination test results are largely consistent with no observed difference in sensitivity, specificity, or time to result when challenged with various SARS-CoV-2 variants. The reported cycle threshold (Ct) values provided by the new PLUS assay were also unchanged, with the exception of a possible 1-2 decrease reported in Ct for RSVA across a limited sample size.

A Sensitive and Rapid Wastewater Test for SARS-COV-2 and Its Use for the Early Detection of a Cluster of Cases in a Remote Community.

Throughout the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has been used to monitor trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence in the community. A major challenge in establishing wastewater surveillance programs, especially in remote areas, is the need for a well-equipped laboratory for sample analysis. Currently, no options exist for rapid, sensitive, mobile, and easy-to-use wastewater tests for SARS-CoV-2. The performance of the GeneXpert system, which offers cartridge-based, rapid molecular clinical testing for SARS-CoV-2 in a portable platform, was evaluated using wastewater as the input. The GeneXpert demonstrated a SARS-CoV-2 limit of detection in wastewater below 32 copies/mL with a sample processing time of less than an hour. Using wastewater samples collected from multiple sites across Canada during February and March 2021, a high overall agreement (97.8%) was observed between the GeneXpert assay and laboratory-developed tests regarding the presence or absence of SARS-CoV-2. Additionally, with the use of centrifugal filters, the detection threshold of the GeneXpert system was improved to <10 copies/mL in wastewater. Finally, to support on-site wastewater surveillance, GeneXpert testing was implemented in Yellowknife, a remote community in Northern Canada, where its use successfully alerted public health authorities to undetected transmission of COVID-19. The identification of SARS-CoV-2 in wastewater triggered clinical testing of recent travelers and identification of new COVID-19 cases/clusters. Taken together, these results suggest that GeneXpert is a viable option for surveillance of SARS-CoV-2 in wastewater in locations that do not have access to established testing laboratories. IMPORTANCE Wastewater-based surveillance is a powerful tool that provides an unbiased measure of COVID-19 prevalence in a community. This work describes a sensitive wastewater rapid test for SARS-CoV-2 based on a widely distributed technology, the GeneXpert. The advantages of an easy-to-use wastewater test for SARS-CoV-2 are clear: it supports surveillance in remote communities, improves access to testing, and provides faster results allowing for an immediate public health response. The application of wastewater rapid testing in a remote community facilitated the detection of a COVID-19 cluster and triggered public health action, clearly demonstrating the utility of this technology. Wastewater surveillance will become increasingly important in the postvaccination pandemic landscape as individuals with asymptomatic/mild infections continue transmitting SARS-CoV-2 but are unlikely to be tested.

Generation of False-Positive SARS-CoV-2 Antigen Results with Testing Conditions outside Manufacturer Recommendations: A Scientific Approach to Pandemic Misinformation.

Antigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. However, misleading demonstrations of the Abbott Panbio coronavirus disease 2019 (COVID-19) Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside manufacturer recommendations. Using Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and Tween 20) as well as the impact of temperature (4°C, 20°C, and 45°C) and humidity (90%) on assay performance. Direct sample testing (without the kit buffer) resulted in false-positive signals resembling those obtained with SARS-CoV-2 positive controls tested under proper conditions. The likely explanation of these artifacts is nonspecific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside manufacturer claims. Our data support strict adherence to manufacturer instructions to avoid false-positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation. IMPORTANCE With the Panbio severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test being deployed in over 120 countries worldwide, understanding conditions required for its ideal performance is critical. Recently on social media, this kit was shown to generate false positives when manufacturer recommendations were not followed. While erroneous results from improper use of a test may not be surprising to some health care professionals, understanding why false positives occur can help reduce the propagation of misinformation and provide a scientific rebuttal for these aberrant findings. This study demonstrated that the kit buffer's pH, ionic strength, and buffering capacity were critical components to ensure proper kit function and avoid generation of false-positive results. Typically, false positives arise from cross-reacting or interfering substances; however, this study demonstrated a mechanism where false positives were generated under conditions favoring nonspecific interactions between the two antibodies designed for SARS-CoV-2 antigen detection. Following the manufacturer instructions is critical for accurate test results.

Clinical evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV combination test.

The Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV combination test received emergency use authorization approval by the United States Food and Drug Administration in December 2020, and Health Canada approval in January 2021. The performance characteristics of the GeneXpert Xpert Xpress SARS-CoV-2/Flu/RSV combination test were assessed at Lakeridge Health Oshawa and the National Microbiology Laboratory of Canada. The combination test was compared to the Xpert SARS-CoV-2 and Xpert Flu/RSV assays, and the BioFire FilmArray Respiratory Panel 2.1 (RP2.1) test kit. Materials evaluated were serial dilutions of chemically-inactivated SARS-CoV-2 and remnant clinical specimens (nasal or nasopharyngeal swabs) collected from patients. The limit of detection (LOD) for the SARS-CoV-2 component of the Xpert SARS-CoV-2/Flu/RSV combination test was determined to be <100 viral copies/mL when using chemically-inactivated SARS-CoV-2. In total, 86 clinical positive and 51 clinical negative samples were used for this study, with mixtures of clinical positives being used to mimic coinfection and screen for competitive inhibition. The combination test showed a high percent agreement with the Xpert SARS-CoV-2 and Xpert Flu/RSV tests, as well as the BioFire FilmArray RP2.1. Based on the findings from this study and a growing body of research, the Xpert SARS-CoV-2/Flu/RSV combination test will serve as an effective replacement for the Xpert SARS-CoV-2 and Xpert Flu/RSV assays.

Recommendations for sample pooling on the Cepheid GeneXpert® system using the Cepheid Xpert® Xpress SARS-CoV-2 assay.

The coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. In pooled sample testing, multiple samples are combined (or pooled) together and tested as a single unit. If the pool is positive, the individual samples can then be individually tested to identify the positive case(s). Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert® Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the limit of detection of this assay was comparable to laboratory-developed reverse-transcription quantitative PCR SARS-CoV-2 tests, with observed detection below 100 copies/mL. The Xpert® Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were pooled in groups of six. Based on these data, we recommend the adoption of pooled testing with the Xpert® Xpress SARS-CoV-2 assay where warranted based on public health needs. The suggested number of samples per pool, or the pooling depth, is unique for each point-of-care testing site and can be determined by the positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.