ABSTRACT
Background: There is a prolonged RT-PCR positivity seen in COVID-19 infected patients up to 2-3 months.It is assumed that this virus is usually non-infective but there are hardly any study on the reactivation of this virus within the respiratory tract. 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 and throat swab RT-PCR positive n=64 {n=12(18.7%) chronic liver disease (CLD);n=52 (81.3%) non-liver disease} n=5 RT PCR negative subjects (HC) were studied. SARS CoV2 patients were also followed up for day(d) 7 and 14. Nasal swab [collected in viral transport media (VTM)] and plasma samples were investigated at each time point. Extracellular vesicles were isolated using differential ultracentrifugation. SARS CoV2 RNA was measured using qRT-PCR by Altona Real Star kit. Cellular origin of EV was confirmed using epithelial cells (Epcam+ CK19+ CDh1+), endothelial cells (CD31+CD45-), hepatocytes (ASGPR+) surface markers by Flow cytometry. Results: The COVID19 patients {Mean age 54±23 years;41 males} were having severity between moderate to severe. In patients with cirrhosis, the most common aetiology of liver disease was alcohol (MELD 22±8). In baseline RT-PCR positive patients, SARS-CoV2 RNA inside the EV was present in 53/64 (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 EV from baseline (p=0.008;0.002 respectively). The probability of detecting SARS-CoV2 in EVs in the VTM negative patients was significantly (p=0.001) greater { relative risk ratio 2.25 (95% of CI 1.08 to 4.67;p=0.02, odds ratio 28.1(95% of CI -1.27 to 619.9;p=0.03)}.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 CLDCOVID19 infected patients with significant high levels of EV associated with epithelial 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. It is suggestive of another route of transmission as EV harbour SARS CoV2 RNA. EV associated RNA may determine the ongoing inflammation and clinical course of subjects with undetectable SARS-CoV2 virus and this may also have relevance in management of chronic liver disease patients.