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. 2021 Jan 21;59(2):e02277-20.
doi: 10.1128/JCM.02277-20. Print 2021 Jan 21.

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

Cristina Rodríguez-Grande  1   2 , Javier Adán-Jiménez  1   2 , Pilar Catalán  1   2   3 , Luis Alcalá  1   2   3 , Agustín Estévez  1   2 , Patricia Muñoz  1   2   3   4 , Laura Pérez-Lago  1   2 , Darío García de Viedma  5   2   3
Affiliations

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

Cristina Rodríguez-Grande et al. J Clin Microbiol. .

Abstract

The purpose of this study was to detect coronavirus disease 2019 (COVID-19) cases with persistent positive reverse transcription-PCR (RT-PCR) results for severe acute respiratory syndrome coronavirus 2 (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. RNA remnants purified from diagnostic nasopharyngeal specimens were used as the 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). We assessed the samples of 60 RT-PCR-positive cases with prolonged viral SARS-CoV-2 shedding (24 to 101 days) since the first diagnostic RT-PCR. SG viral RNA was detected in 12/60 (20%) of the persistent cases, 28 to 79 days after the onset of symptoms. The age range of the cases with prolonged viral shedding and the presence of SG RNA was quite wide (40 to 100 years), and the cases were equally distributed between males (42%) and females (58%). No case was HIV positive, although seven were immunosuppressed. According to the severities of the COVID-19 episodes, they were mild (40%), intermediate (20%), and severe (40%). In a percentage of persistent 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.

Keywords: COVID-19; SARS-CoV-2; persistence; subgenomic RNA.

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Figures

FIG 1
FIG 1
Agarose gel electrophoresis indicating the proper amplicon size (171 bp) expected for E gene subgenomic RNA RT-PCR from positive specimens taken close to symptom onset (diagnostic specimens), when active viral replication is expected. A selection of RT-PCR genomic SARS-CoV-2-positive specimens with negative E gene subgenomic RNA RT-PCR results among those taken far beyond diagnosis (persistence specimens) is also shown. Finally, an RT-PCR genomic SARS-CoV-2-negative specimen is included.
See this image and copyright information in PMC

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