Prolonged existence of SARS-CoV-2 RNAs in the extracellular vesicles of respiratory specimens from patients with negative reverse transcription-polymerase chain reaction

Background and aim:Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is primarily in the respiratory tract,particularly in patients with underlying comorbidities.This study aimed to investigate the presence of the virus inside the extracellular vesicles(EVs)in patients with and without chronic liver disease(CLD).Methods:Eighty patients with positive SARS-CoV-2,including twenty-four patients with CLD and fiftysix patients without CLD,and five healthy controls with negative SARS-CoV-2 were enrolled.Nasal swab specimens were tested for the detection of SARS-CoV-2 using reverse transcription-polymerase chain reaction(RT-PCR).Patients with coronavirus disease 2019(COVID-19)were followed up on days 7 and 14.Nasal swab,collected in viral transport media(VTM),and plasma samples were investigated at each time point.EVs were isolated from the nasal swabs(collected in VTM)and plasma using differential ultracentrifugation and estimated at each time point.The transmission or replication by the EVs was assessed in Vero E6 cells.Results:In patients with baseline RT-PCR positive,SARS-CoV-2 RNAs inside the EVs were found in 68/80(85%)patients with higher viral load in the nasal swabs than in the EVs(cycle threshold(Ct)value,23.4±5.7 vs.30.3±5.0,P<0.001).On follow-up at day 7,of the 32 patients negative for COVID-19,15(46.9%)had virus persistence in the EVs(Ct value,30.7±2.7),and on day 14,of the 56 patients with negative SARS-CoV-2,16 patients(28.6%)had positive SARS-CoV-2 RNAs in the EVs(Ct value,31.4±3.0).The mean viral load decreased on days 7 and 14 compared to baseline in the nasal swabs(P<0.001)but not in the EVs.Additionally,SARS-CoV-2 RNAs were undetectable in the plasma,but 12.5% of patients were positive in the plasma EVs.Significantly prolonged and high viral load was found in the EVs on day 14 in COVID-19 patients combined with CLD compared with COVID-19 patients(P?0.0004).We found significant higher levels of EV-associated with endothelial cells and hepatocytes in the COVID-19 t CLD group than COVID-19 group(P?0.032 and P?0.002,respectively),suggesting more endothelial cells and hepatocytes cellular injury in liver disease patients with COVID-19.Interestingly,we also found EVs could transmit SARS-CoV-2 RNAs into Vero E6 cells at 24 h post-infection.Conclusions:The identification of SARS-CoV-2 RNAs in the EVs in patients with negative RT-PCR indicates the persistence of infection and likely recurrence of the infection.It is suggestive of another route of transmission as EVs harbor SARS-CoV-2 RNAs.EV-associated RNAs may determine the ongoing inflammation and clinical course of subjects with undetectable SARS-CoV-2 virus and this may have relevance to better management of patients with CLD.