Navigating COVID-19 Testing
Authors: Palak Shah, Christopher R. deFilippi, Kristin E. Mullins
Journal: Circulation (2020)

oronaviruses are a family of viruses pathogenic to humans.Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), a single-stranded, positive-sense RNA virus.The virus can be translated directly into protein, and the synthesized negative RNA strand serves as a template for viral replication.The SARS-CoV2 genome encodes for structural proteins (spike, membrane, envelope, nucleocapsid) and regions necessary for viral replication (the open-reading frame 1a or 1b, RNA-dependent RNA polymerase, hemagglutinin-esterase). Laboratory diagnostics typically target 2 distinct genetic regions in the development of their assays to improve test accuracy and to reduce false-positive results through cross-reactivity with other Coronaviridae.Most commonly, the diagnosis is made using a nasopharyngeal swab, but nasal swabs and saliva samples are becoming more common.Samples are preserved and transported in a viral stabilizing medium.The major laboratory-based methods currently available for the detection of SARS-CoV2 include real-time reverse-transcription polymerase chain reaction (RT-PCR), loop or isothermal nucleic acid amplification testing, next-generation sequencing, antigen-based testing, and clustered regularly interspaced short palindromic repeat-based technologies (Figure).RT-PCR is the most widely used technique for diagnosis of COVID-19.It is a specialized, quantitative molecular technique available in most major laboratories and the current gold standard for SARS-CoV2 infection diagnosis.RT-PCR involves 2 major steps: viral extraction and amplification.Initially, no SARS-CoV2-specific primers existed.Once the nucleotide sequence of SARS-CoV2 was determined, then primers were designed that bind target regions of the genome (eg, nucleocapsid and envelope regions).In RT-PCR, viral RNA is first extracted from the medium and then reverse transcribed to cDNA.Using SARS-CoV2-specific primers, a DNA polymerase amplifies cDNA through rapid heating and cooling (ie, thermocycling).In RT-PCR, a fluorescent nucleotide probe can then bind its target sequence region, and a fluorophore is emitted.The process is repeated ≈35 to 45 times, and once the accumulation of fluorescence signal passes a specified level, the cycle threshold, the test is considered positive.The highly manual and technical aspects of RT-PCR limit testing batches to ≈100 to 400 samples, which require 6 to 8 hours to complete.Recent technological innovations in RT-PCR, including the elimination of a dedicated extraction step, have reduced testing time to as little as 45 minutes.Loop-mediated isothermal amplification or standard isothermal nucleic acid amplification testing is an alternative to RT-PCR that uses multiple primers that bind to complementary regions of the SARS-CoV2 genome and synthesize cDNA at a fixed temperature.Because no temperature cycling is required, the test can provide results within 15 to 20 minutes. 1 The sensitivity of isothermal nucleic acid amplification testing came under scrutiny recently with the ID Now assay (Abbott Laboratories, Abbott Park IL), which approximates 75% 2 and is more prone to