PMC:7170415 / 13840-17305 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7170415","sourcedb":"PMC","sourceid":"7170415","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7170415","text":"Laboratory-Based Molecular Testing\nThe current diagnostic strategy recommended by the CDC to identify patients with COVID-19 is to test samples taken from the respiratory tract to assess for the presence of 1 or several nucleic acid targets specific to SARS–CoV-2 (25). A nasopharyngeal specimen is the preferred choice for swab-based SARS–CoV-2 testing, but oropharyngeal, mid-turbinate, or anterior nares samples also are acceptable (31, 32). Samples should be obtained by using a flocked swab, if available, to enhance the collection and release of cellular material. Swabs with an aluminum or plastic shaft are preferred. Swabs that contain calcium alginate, wood, or cotton should be avoided, because they may contain substances that inhibit PCR testing. Ideally, swabs should be transferred into universal transport medium immediately after sample collection to preserve viral nucleic acid. Samples taken from sputum, endotracheal aspirates, and bronchoalveolar lavage also may be sent directly to the microbiology laboratory for processing, and may have greater sensitivity than upper respiratory tract specimens (33). Inadequate sample collection may result in a false-negative test. After specimen collection, samples undergo RNA extraction followed by qualitative RT-PCR for target detection.\nIn the United States, the CDC has developed the most widely used SARS–CoV-2 assay. The kit contains PCR primer–probe sets for 2 regions of the viral nucleocapsid gene (N1 and N2), and for the human RNase P gene to ensure the RNA extraction was successful. This assay differs from the World Health Organization primer–probe sets, which target the SARS–CoV-2 RNA-dependent RNA polymerase (RdRP) and envelope (E) genes (25). Both assays have high analytic sensitivity and specificity for SARS–CoV-2, with minimal cross-reactivity with other circulating strains of coronaviruses, and both use a cycle threshold of less than 40 as the criterion for positivity. The CDC kit may be used by state public health laboratories, other laboratories determined by the state to be qualified, and clinical laboratories that meet the regulatory requirements of the Clinical Laboratories Improvement Amendment (CLIA) to perform high-complexity testing (27). Dozens of laboratories have applied for Emergency Use Authorization (EUA) from the FDA for their own laboratory-developed assays (34). The FDA also has granted an EUA for several commercial assays (35), further expanding the ability of clinical laboratories to use these platforms (Table).\nTable. The 28 Commercial SARS–CoV-2 in Vitro Diagnostic Assays Given an EUA From the FDA as of 4 April 2020 The lack of an established reference standard, use of differing sample collection and preparation methods, and an incomplete understanding of viral dynamics across the time course of infection hamper rigorous assessment of the diagnostic accuracy of the many newly introduced SARS–CoV-2 assays (36). Serum and urine are usually negative for the presence of viral nucleic acid, regardless of illness severity (33). Of importance, the ability of RT-PCR assays to rule out COVID-19 on the basis of upper respiratory tract samples obtained at a single time point remains unclear. Conversely, after a patient has had a positive test result, several authorities have recommended obtaining at least 2 negative upper respiratory tract samples, collected at intervals of 24 hours or longer, to document SARS–CoV-2 clearance (37, 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