Inference of Active Viral Replication in Cases with Sustained Positive Reverse Transcription-PCR Results for SARS-CoV-2

Abstract

Objectives: The purpose of this study was to detect COVID-19 cases with persistent positive RT-PCR results for SARS-CoV-2, for which viable virus can be inferred, due to the presence of subgenomic (SG) viral RNA, which is expressed only in replicating viruses. Methods: RNA remnants, purified from diagnostic nasopharyngeal specimens, were used as templates for RT-PCR specific detection of SG E gene RNA. As controls, we also detected viral genomic RNA for the E gene and/or a human housekeeping gene (RNase P). Results: We assessed the samples of 60 RT-PCR-positive cases with a prolonged viral SARS-CoV-2 shedding (24-101 days) since the first diagnostic RT-PCR. SG viral RNA was detected in 12/60 (20%) of the persistent cases, 28-79 days after the onset of symptoms. The age range of the cases with prolonged viral shedding and presence of SG RNA was quite wide (40-100 years), and they were equally distributed between males (42%) and females (58%). None was HIV positive, although seven were immunosuppressed. According to the severity of the COVID-19 episodes they were mild (40%), intermediate (20%), and severe (40%) Conclusion: In a percentage of persistent positive SARS-CoV-2 PCR positive cases the presence of actively replicating virus may be inferred, far beyond diagnosis. We should not assume a universal lack of infectiousness for COVID-19 cases with prolonged viral shedding.

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