Global surveillance of potential antiviral drug resistance in SARS-CoV-2: proof of concept focussing on the RNA-dependent RNA polymerase (preprint)

Antiviral treatments for COVID-19 have involved many repurposed drugs. Currently, SARS-CoV-2 RNA-dependent RNA polymerase (RdRp, encoded by nsp12-nsp7-nsp8) has been targeted by numerous inhibitors with debated clinical impact. Among these, remdesivir has been conditionally approved for the treatment of COVID-19 patients. Although the emergence of antiviral resistance, an indirect proxy for antiviral efficacy, poses a considerable healthcare threat, an evolutionary perspective on emerging resistant mutants is still lacking. Here we show that SARS-CoV-2 RdRp is under purifying selection, that potential escape mutations are rare, and unlikely to lead to viral fitness loss. In more than 56,000 viral genomes from 105 countries dating from December 2019 to July 2020 we found negative selective pressure affecting nsp12 (Tajimas D = -2.62), with potential antiviral escape mutations in only 0.3% of sequenced genomes. Those affected known key residues, such as Nsp12:Val473 and Nsp12:Arg555. Of the potential escape mutations found globally, in silico structural models show that this rarely implies loss of stability in RdRp. No potential escape mutation were found in our local cohort of remdesivir treated patients from the first wave (n=8). Our results indicate that RdRp is a suitable drug target, and that remdesivir does not seem to exert high selective pressure. Our study could be the starting point of a larger monitoring effort of drug resistance throughout the COVID-19 pandemic. We recommend the application of repetitive genome sequencing of SARS-CoV-2 from patients treated with antivirals to provide early insights into the evolution or antiviral resistance.

Effect of Systemic Inflammatory Response to SARS-CoV-2 on Lopinavir and Hydroxychloroquine Plasma Concentrations (preprint)

Background: Coronavirus disease 2019 (COVID-19) leads to inflammatory cytokine release, which can downregulate the expression of metabolizing enzymes. This cascade affects drug concentrations in the plasma. We investigated the association between lopinavir (LPV) and hydroxychloroquine (HCQ) plasma concentrations and the values of acute phase inflammation marker C-reactive protein (CRP). Methods: LPV plasma concentrations were prospectively collected in 92 patients hospitalized at our institution. Lopinavir/ritonavir was administered 12 hourly, 800/200 mg on day 1, and 400/100 mg on day 2 until day 5 or 7. HCQ was given at 800 mg, followed by 400 mg after 6, 24 and 48 hours. Hematological, liver, kidney, and inflammation laboratory values were analyzed on the day of drug level determination. Results: The median age of study participants was 59 (range 24 up to 85) years, and 71% were male. The median duration from symptom onset to hospitalization and treatment initiation was 7 days (IQR 4;10) and 8 days (IQR 5;10), respectively. The median LPV trough concentration on day 3 of treatment was 26.5 ug/mL (IQR 18.9;31.5). LPV plasma concentrations positively correlated with CRP values (r=0.37, p<0.001), and were significantly lower when tocilizumab was preadministrated. No correlation was found between HCQ concentrations and CRP values. Conclusions: High LPV plasma concentrations were observed in COVID-19 patients. The ratio of calculated unbound drug fraction to published SARS-CoV2 EC50 values indicated insufficient LPV concentrations in the lung. CRP values significantly correlated with LPV but not HCQ plasma concentrations, implying inhibition of cytochrome P450 3A4 (CYP3A4) metabolism by inflammation.