A Comparison of Five SARS-CoV-2 Molecular Assays With Clinical Correlations

Gary W. Procop, MD, MS; Jay E. Brock, PhD; Edmunds Z. Reineks, MD; Nabin K. Shrestha, MD, MPH; Ryan Demkowicz, MD; Eleanor Cook, MD; Emad Ababneh, MD; Susan M. Harrington, PhD

Disclosures

Am J Clin Pathol. 2021;155(1):69-78. 

In This Article

Abstract and Introduction

Abstract

Objectives: Comparative assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular assays that have been operationalized through the US Food and Drug Administration's Emergency Use Authorization process are warranted to assess real-world performance. Characteristics such as sensitivity, specificity, and false-negative rate are important to inform clinical use.

Methods: We compared five SARS-CoV-2 assays using nasopharyngeal and nasal swab specimens submitted in transport media; we enriched this cohort for positive specimens, since we were particularly interested in the sensitivity and false-negative rate. Performance of each test was compared with a composite standard.

Results: The sensitivities and false-negative rates of the 239 specimens that met inclusion criteria were, respectively, as follows: Centers for Disease Control and Prevention 2019 nCoV Real-Time RT-PCR Diagnostic Panel, 100% and 0%; TIB MOLBIOL/Roche z 480 Assay, 96.5% and 3.5%; Xpert Xpress SARS-CoV-2 (Cepheid), 97.6% and 2.4%; Simplexa COVID-19 Direct Kit (DiaSorin), 88.1% and 11.9%; and ID Now COVID-19 (Abbott), 83.3% and 16.7%.

Conclusions: The assays that included a nucleic acid extraction followed by reverse transcription polymerase chain reaction were more sensitive than assays that lacked a full extraction. Most false negatives were seen in patients with low viral loads, as extrapolated from crossing threshold values.

Introduction

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and the subsequent pandemic has resulted in the need to rapidly deploy molecular diagnostic assays for the detection of infected individuals. Molecular diagnostic assays targeting the SARS-CoV-2 virus have been provided by commercial manufacturers and/or designed in individual laboratories and implemented through the authority of the US Food and Drug Administration's (FDA) Emergency Use Authorization (EUA) Act (https://www.fda.gov/emergency-preparedness-and-response/mcm-legal-regulatory-and-policy-framework/emergency-use-authorization). Assays that achieve EUA clearance have been assessed through a variety of experiments, including but not limited to an assessment of the lower limit of detection (LoD) and in silico assessment of primer and probe sequences for potential cross-reactivity.[1] Although these and the other FDA EUA requirements are excellent initial means of assessing an assay, there is not a defined LoD that is necessary for an assay to achieve EUA status. In addition, analytical and clinical sensitivity and specificity determinations are not required, which is understandable when responding to an emergency.

Laboratories that have had a research interest in severe acute respiratory syndrome coronaviruses have had the opportunity to more thoroughly study test performance characteristics through various primer/probe combinations and concentrations and reaction conditions. Most clinical laboratories, which by necessity have been thrust into SARS-CoV-2 testing, have not had this opportunity, however. Therefore, comparative studies of FDA EUA–cleared molecular tests for SARS-CoV-2 are warranted to more fully understand test performance characteristics.

We, therefore, compared five SARS-CoV-2 molecular assays to one another to determine the sensitivity and specificity of these assays. This comparison was enriched for positive specimens, as we were particularly interested in determining the false-negative rates for these assays, since mischaracterizing an infected patient as coronavirus disease 2019 (COVID-19) negative could have considerable infection prevention implications in the hospital. This comparison also afforded our group the opportunity to compare test characteristics, such as crossing thresholds (Cts), with alternate test results, as well as with the clinical and demographic findings of infected patients.

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