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Contents

        Preface: A Tale of Trial and Triumph: Molecular Diagnostics for Severe Acute Respiratory Coronavirus 2 Over the First Two Years of the Coronavirus Disease 2019 Pandemic xiii

        Sanjat Kanjilal and Yi-Wei Tang

        Analytic and Clinical Performance of Major Commercial Severe Acute Respiratory Syndrome Coronavirus 2 Molecular Assays in the United States 129

        Michelle R. Campbell and Matthew J. Binnicker
        The rapid development of commercially available molecular assays in response to the COVID-19 pandemic has been essential in identifying positive cases and guiding state and national response plans. With over 200 SARS-CoV-2 molecular tests having received emergency use authorization by the US Food and Drug Administration, numerous studies have been conducted to evaluate these methods and compare their analytical and clinical performance. By applying the lessons learned from the rapid development of molecular assays in response to the COVID-19 pandemic, the diagnostic industry will be better prepared to respond to future outbreaks of novel infectious diseases.

        The Successes and Challenges of SARS-CoV-2 Molecular Testing in the United States 147

        Jennifer Dien Bard and N. Esther Babady
        SARS-CoV-2 was identified and diagnostic methods developed at an impressive speed due in great part to the wider use of molecular methods in 2019 compared with 2002 during the SARS pandemic. The development of rapid and novel molecular diagnostic assays, leveraging of the high adaptability of molecular tests, and the integration of SARS-CoV-2 genotyping into public health, clinical, and research laboratories have been some of the successes in SARS-CoV-2 molecular testing. The main challenges are related to regulatory hurdles, supply chain constraints, and laboratory preparation.

        An Overview of SARS-CoV-2 Molecular Diagnostics in Europe 161

        Emma Davies, Hamzah Z. Farooq, Benjamin Brown, Peter Tilston, Ashley McEwan, Andrew Birtles, Robert William O’Hara, Shazaad Ahmad, Nicholas Machin, Louise Hesketh, and Malcolm Guiver
        The COVID-19 pandemic has led to the rapid development of a plethora of molecular diagnostic assays with real-time polymerase chain reaction (RT-PCR) at the forefront. In this review, we will discuss the history and utility of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) molecular diagnostics and the associated current and future regulatory process in Europe. We will assess the performance characteristics of a range of the most common SARS-CoV-2 molecular tests currently used in Europe with a focus on as rapid molecular platforms, stand-alone RT-PCR kits, the role of low-throughput and high-throughput end-to-end testing platforms, and the rapidly evolving field of SARS-CoV-2 variant of concern identification.

        SARS-CoV-2 Molecular Diagnostics in China 193

        Yanjun Lu and Ziyong Sun
        The COVID-19 pandemic remains a significant problem involving health systems worldwide. Several diagnostic methods are reported for detecting the coronavirus in clinical, research, and public health laboratories. rRT-PCR is considered the gold standard; however, as it required skilled personnel and special equipment, rapid antigen tests have been developed and used as first-line screening. The serologic testing of antibodies can also be used to enhance the detection sensitivity and accuracy, which are used to assess the overall infection rate. This review summarizes the molecular techniques and serologic assays widely used in China and discusses the advantages and disadvantages of these techniques.

        Rapid Antigen Assays for SARS-CoV-2: Promise and Peril 203

        Thao T. Truong, Jennifer Dien Bard, and Susan M. Butler-Wu
        Though rapid antigen tests have historically problematic performance characteristics for the diagnosis of respiratory viral infections such as influenza, they have attained an unprecedented level of use in the context of the COVID-19 pandemic. Ease of use and scalability of rapid antigen tests has facilitated a democratization and scale of testing beyond anything reasonably achievable by traditional laboratory-based testing. In this chapter, we discuss the performance characteristics of rapid antigen testing for SARS-CoV-2 detection and their application to non-traditional uses beyond clinical diagnostic testing.

        Clinical Diagnostic Point-of-Care Molecular Assays for SARS-CoV-2 223

        Nicole V. Tolan and Gary L. Horowitz
        Laboratories faced many challenges throughout the COVID-19 pandemic. Point-of-care (POC) SARS-CoV-2 nucleic acid amplification tests (NAATs) provided a key solution to the need for rapid turnaround time in select patient populations and were implemented at the POC but also within laboratories to supplement traditional molecular assays. Clinical Laboratory Improvement Amendments–waived rapid POC SARS-CoV-2 NAATs offer the benefit of reduced educational requirements for operators and can be performed by non–laboratory-trained individuals. However, these methods must be validated to ensure the manufacturer’s performance specifications are met and they are found to be fit-for-purpose in the clinical workflows they are implemented.

        Cycle Threshold Values from Severe Acute Respiratory Syndrome Coronavirus-2 Reverse Transcription-Polymerase Chain Reaction Assays: Interpretation and Potential Use Cases 237

        Alexander J. McAdam
        Reverse transcription-polymerase chain reaction (RT-PCR) tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), are approved for qualitative use. The cycle threshold (Ct) value reflects the concentration of viral RNA in the sample, with lower Ct values indicating higher levels of RNA. Caregivers may wish to use the Ct value to determine the progression of infection, how severe the infection will be, and whether the patient can transmit the virus. Variability of Ct values and the data supporting these uses should be considered when deciding whether and how to use Ct values in clinical care.

        Performance of Non-nasopharyngeal Sample Types for Molecular Detection of SARS-CoV-2 249

        Benjamin Kukull, Salika M. Shakir, and Kimberly E. Hanson
        Nasopharyngeal swabs have historically been considered the preferred specimen type for the detection of respiratory viruses, including SARS-CoV-2. However, in response to a global pandemic with shortages of swabs and specimen transport media, limited access to qualified health care personnel, and needs for large-scale testing in nonmedical settings, alternative sample types have been validated for COVID-19 diagnosis. The purpose of this review is to highlight the diagnostic accuracy and clinical utility of non-nasopharyngeal respiratory samples for SARS-CoV-2 molecular diagnostic testing.

        Strategies for Scaling up SARS-CoV-2 Molecular Testing Capacity 261

        Sanchita Das and Karen M. Frank
        Scaling up SARS-CoV-2 testing during the COVID-19 pandemic was critical to maintaining clinical operations and an open society. Pooled testing and automation were two critical strategies used by laboratories to meet the unprecedented demand. Here, we review these and other cutting-edge strategies that sought to expand SARS-CoV-2 testing capacity while maintaining high individual test performance.

        Approaches to Deployment of Molecular Testing for SARS-CoV-2 in Resource-Limited Settings 283

        Gama Bandawe, Moses Chitenje, Joseph Bitiliyu-Bangoh, and Elizabeth Kampira
        Deployment of molecular testing for SARS-CoV-2 in resource-limited settings is challenging. Scale-up of molecular had to be conducted with a laboratory system strengthening approach that emphasize laboratory integration. National reference laboratories play a central role. In Malawi the molecular testing was underpinned by existing pathogen control programs for human immunodeficiency virus and tuberculosis that use Abbott and GeneXpert machines and reagents. Despite this, the impact on these programs was well managed. Antigen testing increased access to testing. Pooled testing and direct-to-polymerase chain reaction methods have the potential to save costs and further increase access to molecular tests.

        Novel Assays for Molecular Detection of Severe Acute Respiratory Syndrome Coronavirus 2 299

        Kyle G. Rodino, Kenneth P. Smith, and Matthew A. Pettengill
        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.