Parallel RT-qPCR Testing

Samples were taken simultaneously as nasopharyngeal swabs for PCR testing. In the clinical setting, all PCRs were locally or nationally approved tests. All samples were analyzed at the same state-approved laboratory.

Nucleic acid extraction and real-time RT-qPCR for virus detection were performed to allow the identification of SARS-CoV-2. The extraction of total nucleic acid (DNA and RNA) from collected samples was performed using the BioGene Extraction kit (Bioclin, K204-4, Brazil), following the manufacturer’s instructions. Specimens were handled under the laboratory biosafety guidance required for the novel coronavirus (2019-nCoV) designated by WHO at the Central Laboratory of the Espírito Santo state (LACEN-ES). A combination of four tests was employed to detect viral RNA. The first was using the IDT (Integrated DNA Technologies; Coralville, IA) kit, which is developed in association with the CDC and employs primers and probes for the N1, N2, and RP genes. The second was Maccura (designed by Maccura Biotechnology Co., Hi-tech Zone, Chengdu, China), which is a single-well triple target assay and identifies three genes from SARS-CoV-2 (E, N, and ORF1ab) and provides a separate positive internal control (IC). The third was the Molecular SARS-CoV-2 (E/RP genes) kit (Instituto de Tecnologia em Imunobiológicos, Bio-Manguinhos, FioCruz, RJ, Brazil), which uses primers and probes, as reported by Corman et al.²² The detection of viral RNA was carried out on an ABI 7500 real-time PCR machine (Applied Biosystems, Weiterstadt, Germany) using the published protocol and the sequence of primers and probe for E gene and RNAse P. Lastly, the IBMP (Instituto de Biologia Molecular do Paraná—FioCRUZ) kit was employed, which is a single-well test and detects N and ORF1ab genes, and uses the RP gene as an internal control.

All assays were performed using manufacturers’ recommendations. First, all samples were tested in a single-well assay (IBMP or Maccura) for the qPCR run, and interpretations of all results were added to a spreadsheet, together with the values of Cts obtained. Samples with inconclusive results, either by nonamplification in the internal control or by nonamplification of another gene, were tested with the other two qPCR kits (IDT or Bio-manguinhos gene E). If the PCR result remained inconclusive, the result and Ct values were added to the spreadsheet as a negative.