Molecular diagnosis for the novel coronavirus sars-cov-2: The experience of the san giuliano hospital (Asl Napoli 2 Nord)

ABSTRACT

The current COVID-19 pandemic, caused by SARSCoV-2 virus, has made it necessary to identify positive, symptomatic and asymptomatic patients, in a timely manner to activate appropriate containment measures, in order to limiting the spread of infection. The standard test for the diagnosis of COVID-19 is based on the detection of the viral RNA in respiratory specimens using the Real Time reverse-transcription PCR (rtRT-qPCR). In our Molecular Medicine Laboratory, from November 2020 to May 2021, we tested 98.540 respiratory samples collected in UTM swabs (Copan Diagnostics). Viral RNA were extracted with Nextractor magnetic automated system NX48S (Genolution) and they were tested for the detection of SARS-CoV-2 with two different rtRTqPCR kits i) NeoPlex kit COVID-19 Detection Kit (GeneMatrix, Eurospital), whose gene targets are RdRp and N (N=68.951), and ii) KHB Diagnostic kit for Sars-CoV-2 (KHB), whose gene targets are ORF1ab region, N and E (N=29.589). About 30% of the total analyzed samples, in the mentioned period, were found to be positive to SARS-CoV-2 RNA. However, 1.538 samples found to be positive to only ORF1ab region with KHB Diagnostic kit for Sars-CoV-2, resulting then negative to both N and RdRP genes after being re-tested, within the same working day, with NeoPlex kit COVID-19 Detection Kit, as confirmed by further clinical/diagnostic and followup insights. This indicated a false positive rate of about 5% by using ORF1ab region as target gene in SARS-CoV-2 detection. In conclusion, our preliminary data strongly suggest to use a kit for RdRp gene, whose detection has a greater clinical specificity, particularly, in the late phase of infection. From May 2021, we introduced in the diagnosis of SARS-CoV-2 routine a new kit, 1copy COVID-19 qPCR 4plex (1drop, Eurospital), which not only amplifies three genes in a single tube (i.e., RdRp, N and E), but allows to obtain results after 50 minutes from the extraction, thereby improving the TAT. These results suggest the need of use more than one diagnostic kit, with different targets, in order to obtain more reliable diagnostic data.