Novel Assays for Molecular Detection of Severe Acute Respiratory Syndrome Coronavirus 2.

From the onset of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/COVID-19 pandemic, there has been a major emphasis on molecular laboratory tests for the virus. Shortages in various testing supplies, the desire to increase testing capacity, and a push to make point-of-care or home-based testing available have fostered considerable innovation for SARS-CoV-2 molecular diagnostics, advancements likely to be applicable to other diagnostic uses. The authors attempt to cover some of the most compelling novel types of molecular assays or novel approaches in adapting established molecular methodologies for SARS-CoV-2 detection or characterization.

The Limit of Detection Matters: The Case for Benchmarking Severe Acute Respiratory Syndrome Coronavirus 2 Testing.

BACKGROUND: Resolving the coronavirus disease 2019 (COVID-19) pandemic requires diagnostic testing to determine which individuals are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard is to perform reverse-transcription polymerase chain reaction (PCR) on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of approximately 100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss infected patients. However, the relative clinical sensitivity of these assays remains unknown. METHODS: Here we model the clinical sensitivities of assays based on their LoD. Cycle threshold (Ct) values were obtained from 4700 first-time positive patients using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization test. We derived viral loads from Ct based on PCR principles and empiric analysis. A sliding scale relationship for predicting clinical sensitivity was developed from analysis of viral load distribution relative to assay LoD. RESULTS: Ct values were reliably repeatable over short time testing windows, providing support for use as a tool to estimate viral load. Viral load was found to be relatively evenly distributed across log10 bins of incremental viral load. Based on these data, each 10-fold increase in LoD is expected to lower assay sensitivity by approximately 13%. CONCLUSIONS: The assay LoD meaningfully impacts clinical performance of SARS-CoV-2 tests. The highest LoDs on the market will miss a majority of infected patients. Assays should therefore be benchmarked against a universal standard to allow cross-comparison of SARS-CoV-2 detection methods.

Large-Scale, In-House Production of Viral Transport Media To Support SARS-CoV-2 PCR Testing in a Multihospital Health Care Network during the COVID-19 Pandemic.

The COVID-19 pandemic has severely disrupted worldwide supplies of viral transport media (VTM) due to widespread demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription-PCR (RT-PCR) testing. In response to this ongoing shortage, we began production of VTM in-house in support of diagnostic testing in our hospital network. As our diagnostic laboratory was not equipped for reagent production, we took advantage of space and personnel that became available due to closure of the research division of our medical center. We utilized a formulation of VTM described by the CDC that was simple to produce, did not require filtration for sterilization, and used reagents that were available from commercial suppliers. Performance of VTM was evaluated by several quality assurance measures. Based on cycle threshold (CT ) values of spiking experiments, we found that our VTM supported highly consistent amplification of the SARS-CoV-2 target (coefficient of variation = 2.95%) using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization (EUA) assay on the Abbott m2000 platform. VTM was also found to be compatible with multiple swab types and, based on accelerated stability studies, able to maintain functionality for at least 4 months at room temperature. We further discuss how we met logistical challenges associated with large-scale VTM production in a crisis setting, including use of a staged assembly line for VTM transport tube production.

Open Development and Clinical Validation of Multiple 3D-Printed Nasopharyngeal Collection Swabs: Rapid Resolution of a Critical COVID-19 Testing Bottleneck.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a severe international shortage of the nasopharyngeal swabs that are required for collection of optimal specimens, creating a critical bottleneck blocking clinical laboratories' ability to perform high-sensitivity virological testing for SARS-CoV-2. To address this crisis, we designed and executed an innovative, cooperative, rapid-response translational-research program that brought together health care workers, manufacturers, and scientists to emergently develop and clinically validate new swabs for immediate mass production by 3D printing. We performed a multistep preclinical evaluation of 160 swab designs and 48 materials from 24 companies, laboratories, and individuals, and we shared results and other feedback via a public data repository (http://github.com/rarnaout/Covidswab/). We validated four prototypes through an institutional review board (IRB)-approved clinical trial that involved 276 outpatient volunteers who presented to our hospital's drive-through testing center with symptoms suspicious for COVID-19. Each participant was swabbed with a reference swab (the control) and a prototype, and SARS-CoV-2 reverse transcriptase PCR (RT-PCR) results were compared. All prototypes displayed excellent concordance with the control (κ = 0.85 to 0.89). Cycle threshold (CT ) values were not significantly different between each prototype and the control, supporting the new swabs' noninferiority (Mann-Whitney U [MWU] test, P > 0.05). Study staff preferred one of the prototypes over the others and preferred the control swab overall. The total time elapsed between identification of the problem and validation of the first prototype was 22 days. Contact information for ordering can be found at http://printedswabs.org Our experience holds lessons for the rapid development, validation, and deployment of new technology for this pandemic and beyond.