Genetic Study of SARS-CoV-2 Non Structural Protein 12 in COVID-19 Patients Non Responders to Remdesivir.

Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (n = 36/111, 32.4%) or prolonged (5 to 20 days) (n = 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the nsp12 gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the de novo E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication. IMPORTANCE This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the nsp12 pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.

Viral Culture Confirmed SARS-CoV-2 Subgenomic RNA Value as a Good Surrogate Marker of Infectivity.

Determining SARS-CoV-2 viral infectivity is crucial for patient clinical assessment and isolation decisions. We assessed subgenomic RNA (sgRNA) as a surrogate marker of SARS-CoV-2 infectivity in SARS-CoV-2-positive reverse transcription PCR (RT-PCR) respiratory samples (n = 105) in comparison with viral culture as the reference standard for virus replication. sgRNA and viral isolation results were concordant in 99/105 cases (94%), indicating highly significant agreement between the two techniques (Cohen's kappa coefficient 0.88, 95% confidence interval [CI] 0.78 to 0.97, P < 0.001). sgRNA RT-PCR showed a sensitivity of 97% and a positive predictive value of 94% to detect replication-competent virus, further supporting sgRNA as a surrogate marker of SARS-CoV-2 infectivity. sgRNA RT-PCR is an accurate, rapid, and affordable technique that can overcome culture and cycle threshold (CT) value limitations and be routinely implemented in hospital laboratories to detect viral infectivity, which is essential for optimizing patient monitoring, the efficacy of treatments/vaccines, and work reincorporation policies, as well as for safely shortening isolation precautions.

Severe Acute Respiratory Syndrome Coronavirus 2 Normalized Viral Loads and Subgenomic RNA Detection as Tools for Improving Clinical Decision Making and Work Reincorporation.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse-transcription polymerase chain reaction (RT-PCR) provides a highly variable cycle threshold (Ct) value that cannot distinguish viral infectivity. Subgenomic ribonucleic acid (sgRNA) has been used to monitor active replication. Given the importance of long RT-PCR positivity and the need for work reincorporation and discontinuing isolation, we studied the functionality of normalized viral loads (NVLs) for patient monitoring and sgRNA for viral infectivity detection. METHODS: The NVLs measured through the Nucleocapsid and RNA-dependent-RNA-polymerase genes and sgRNA RT-PCRs were performed in 2 consecutive swabs from 84 healthcare workers. RESULTS: The NVLs provided similar and accurate quantities of both genes of SARS-CoV-2 at 2 different timepoints of infection, overcoming Ct-value and swab collection variability. Among SARS-CoV-2-positive samples, 51.19% were sgRNA-positive in the 1st RT-PCR and 5.95% in the 2nd RT-PCR. All sgRNA-positive samples had >4 log10 RNA copies/1000 cells, whereas samples with ≤1 log10 NVLs were sgRNA-negative. Although NVLs were positive until 29 days after symptom onset, 84.1% of sgRNA-positive samples were from the first 7 days, which correlated with viral culture viability. Multivariate analyses showed that sgRNA, NVLs, and days of symptoms were significantly associated (P < .001). CONCLUSIONS: The NVLs and sgRNA are 2 rapid accessible techniques that could be easily implemented in routine hospital practice providing a useful proxy for viral infectivity and coronavirus disease 2019 patient follow-up.