Identifying Inconclusive Data in the SARS-CoV-2 Molecular Diagnostic Using Nucleocapsid Phosphoprotein Gene as Target

ABSTRACT Context: The gold standard test to identify the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in coronavirus disease 2019 (COVID-19) patients is the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR), but the inconclusive data and presence of false positive diagnosis remain the major problem of this approach. Objective: To compare the fitness of two primers sets to the SARS-CoV-2 nucleocapsid phosphoprotein (NP) gene in the molecular diagnosis of COVID-19, we verify the inconclusive data and confidence of high cycle threshold (Ct) values in the SARS-CoV-2 detection. Design: The 970 patient samples were tested using United States Centers for Disease Control and Prevention protocol. We compared the fitness of two primers sets to two different regions of NP gene. In addition, we check the consistency of positive samples with high Ct values by retesting extracted SARS-CoV-2 RNA or by second testing of patients. Results: The N1 and N2 displayed similar fitness during testing with no differences between Ct values. Then, we verified security range Cts related to positive diagnostic with Ct above 34 failing in 21/32 (65.6%) after retesting of samples. The samples patients with Ct above 34.89 that were doubly positive revealed a low sensitivity (52.4%) and specificity (63.6%) of the test in samples with Ct above 34. Conclusions: It is secure to use one primer set to the NP gene to identify SARS-CoV-2 in samples. However, samples with high Ct values may be considered inconclusive and retested to avoid false positive diagnosis.