Emergence of transmissible SARS-CoV-2 variants with decreased sensitivity to antivirals in immunocompromised patients with persistent infections
Mohammed Nooruzzaman, Katherine E E Johnson, Ruchi Rani, Eli J Finkelsztein, Leonardo C Caserta, Rosy P Kodiyanplakkal, Wei Wang, Jingmei Hsu, Maria T Salpietro, Stephanie Banakis, Joshua Albert, Lars F Westblade, Claudio Zanettini, Luigi Marchionni, Rosemary Soave, Elodie Ghedin, Diego G Diel, Mirella Salvatore
Nature Communications, doi:10.1038/s41467-024-51924-3
We investigated the impact of antiviral treatment on the emergence of SARS-CoV-2 resistance during persistent infections in immunocompromised patients (n = 15). All patients received remdesivir and some also received nirmatrelvir-ritonavir (n = 3) or therapeutic monoclonal antibodies (n = 4). Sequence analysis showed that nine patients carried viruses with mutations in the nsp12 (RNA dependent RNA polymerase), while four had viruses with nsp5 (3C protease) mutations. Infectious SARS-CoV-2 with a double mutation in nsp5 (T169I) and nsp12 (V792I) was recovered from respiratory secretions 77 days after initial COVID-19 diagnosis from a patient sequentially treated with nirmatrelvir-ritonavir and remdesivir. In vitro characterization confirmed its decreased sensitivity to remdesivir and nirmatrelvir, which was overcome by combined antiviral treatment. Studies in golden Syrian hamsters demonstrated efficient transmission to contact animals. This study documents the isolation of SARS-CoV-2 carrying resistance mutations to both nirmatrelvir and remdesivir from a patient and demonstrates its transmissibility in vivo. Infection with SARS-CoV-2 in immunocompromised patients poses major clinical, therapeutic, and public health challenges. These patients often experience more severe infection outcome(s) than the general population with disease progression being influenced by the treatment of the underlying condition 1 . Moreover, while most people with a competent immune system successfully clear SARS-CoV-2 infection within days, immunocompromised patients may become persistently infected and present prolonged virus replication and shedding. Long-term viral replication contributes to intra-host evolution leading to the emergence of variants with mutations in the virus
Nucleic acid isolation and real-time reverse transcriptase PCR (rRT-PCR) Nucleic acid was extracted from oropharyngeal swabs and tissues collected at necropsy. A 10% (w/v) homogenate was prepared in DMEM from tissues (nasal turbinate, trachea, and lungs) using a stomacher (one speed cycle of 60 s, Stomacher® 80 Biomaster). The tissue homogenate was clarified by centrifuging at 2000×g for 10 min. In total, 200 µL of oropharyngeal swabs and clarified tissue homogenate was used for RNA extraction using the MagMax Core extraction kit (Thermo Fisher, Waltham, MA, USA) and the automated KingFisher Flex nucleic acid extractor (Thermo Fisher, Waltham, MA, USA). The rRT-PCR for total viral RNA detection was performed using the EZ-SARS-CoV-2 Real-Time RT-PCR assay (Tetracore Inc., Rockville, MD, USA), which detects both genomic and subgenomic viral RNA targeting the viral nucleoprotein gene. An internal inhibition control was included in all reactions. Positive and negative amplification controls were run side-by-side with test samples. Relative viral genome copy numbers were calculated based on the standard curve and determined using GraphPad Prism 9 (GraphPad, La Jolla, CA, USA). The amount of viral RNA detected in samples was expressed as log (genome copy number) per mL.
Virus isolation and titration All oropharyngeal swabs and tissue homogenates were subjected to virus isolation under Biosafety Level 3 (BSL-3) conditions at the Animal Health Diagnostic Center (ADHC) Research..
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