Prolonged Detection of SARS CoV2 RNA in Extracellular Vesicles in Nasal Swab RT-PCR Negative Patients

ABSTRACT Background There is a prolonged RT-PCR positivity seen in COVID-19 infected patients up to 2-3 months. We aim to investigate the presence of viral particles inside Extracellular vesicles (EV) and its role in underlying liver disease patients. Methods SARS CoV2 nasal RT-PCR positive n=78 {n=24(66.6%) chronic liver disease (CLD); n=52 (81.3%) non-liver disease} were studied. SARS CoV2 patients were also followed up for day (d) 7, 14 and 28. Extracellular vesicles were isolated using differential ultracentrifugation. SARS CoV2 RNA was measured using qRT-PCR by Altona Real Star kit. Results In baseline RT-PCR positive patients, SARS-CoV2 RNA inside the EV was present in 64/74 (82%) patients with comparable viral load between VTM and EV (mean 1CT – 0.033±0.005 vs. 1CT – 0.029±0.014, p=ns). On follow-up at day 7, of the 24 patients negative for COVID19, 10 (41%) had persistence of virus in the EV (1CT – 0.028±0.004) and on day 14, 14 of 40 (35%) negative RT-PCR had EVs with SARS CoV2 RNA (1CT – 0.028±0.06). The mean viral load decreased at day7 and day14 in nasal swab from baseline (p=0.001) but not in EV. SARS-CoV2 RNA otherwise undetectable in plasma, was found to be positive in EV in 12.5% of COVID19 positive patients. Interestingly, significantly prolonged and high viral load was found in EV at day 14 in CLD-COVID19 patients compared to COVID19 alone (p=0.002). The high cellular injury was seen in CLD-COVID19 infected patients with significant high levels of EV associated with endothelial cells and hepatocytes than COVID19 alone (p=0.004; 0.001). Conclusion Identification of SARS-CoV2 RNA in EV, in RT-PCR negative patients indicates persistence of infection for and likely recurrence of the infection. EV associated RNA may determine the clinical course of subjects with undetectable SARS-CoV2 virus and this may also have relevance in management of chronic liver disease patients.