Multiplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assays for Diagnostic Testing of Severe Acute Respiratory Syndrome Coronavirus 2 and Seasonal Influenza Viruses: A Challenge of the Phase 3 Pandemic Setting

Abstract

Background: Pandemic coronavirus disease 2019 (COVID-19) disease represents a challenge for healthcare structures. The molecular confirmation of samples from infected individuals is crucial and therefore guides public health decision making. Clusters and possibly increased diffuse transmission could occur in the context of the next influenza season. For this reason, a diagnostic test able to discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza viruses is urgently needed.

Methods: A multiplex real-time reverse-transcription polymerase chain reaction (PCR) assay was assessed using 1 laboratory protocol with different real-time PCR instruments. Overall, 1000 clinical samples (600 from samples SARS-CoV-2-infected patients, 200 samples from influenza-infected patients, and 200 negative samples) were analyzed.

Results: The assay developed was able to detect and discriminate each virus target and to intercept coinfections. The limit of quantification of each assay ranged between 5 and 10 genomic copy numbers, with a cutoff value of 37.7 and 37.8 for influenza and SARS-CoV-2 viruses, respectively. Only 2 influenza coinfections were detected in COVID-19 samples.

Conclusions: This study suggests that multiplex assay is a rapid, valid, and accurate method for the detection of SARS-CoV-2 and influenza viruses in clinical samples. The test may be an important diagnostic tool for both diagnostic and surveillance purposes during the seasonal influenza activity period.

Keywords: COVID-19; SARS-CoV-2; differential diagnosis; influenza viruses; multiplex real-time PCR.

Figure 1.

LC480II plot amplifications and standard curves of the singleplex and multiplex assays. Each assay was tested using related standard ranging from 10⁷ to 10 copies/μL. Polymerase chain reaction curve data are represented by relative fluorescence plotted against cycle threshold (Ct) values. Standard curves were generated from the Ct values obtained against known concentrations. Slope of the regression curve, the coefficient of determination (R²), and efficiency (E) are indicated for each assay.

Figure 2.

Receiver operating characteristic (ROC) curve graphs for each gene target in multiplex assay. The optimal cycle threshold (Ct) cutoff values and the area under the ROC curve (AUC) are indicated.