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

  1. P. Debishree Subudhi
  2. Sheetalnath Rooge
  3. Swati Thangariyal
  4. Sivang Goswami
  5. Reshu Agarwal
  6. Ekta Gupta
  7. Sukriti Baweja

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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.

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  1. Version published to 10.1101/2021.12.16.21267912v2 on medRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.12.16.21267912: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: Institutional Ethics committee approved the study (F.37/ (1)/9/ILBS/DOA/2020/20217/513) and patients were enrolled after taking informed written consent.
    Consent: Institutional Ethics committee approved the study (F.37/ (1)/9/ILBS/DOA/2020/20217/513) and patients were enrolled after taking informed written consent.
    Field Sample Permit: Detailed protocol of sample collection, processing and isolation were followed according to the special guidelines by ISTH for extracellular vesicles to avoid any artifacts as described previously by our group (10).
    Euthanasia Agents: After 10 minutes, excess sample was washed twice with 100µl of decarbonated water and dried at room temperature for 2 mins.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Co-infection of Vero Cells by Infected EVs from COVID19 patients in Cell Culture: Vero cells were cultured in DMEM medium (GIBCO/BRL, Grand Island, NY, USA), supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Multiparameter flow cytometry was performed according to a standard protocol, and the data was acquired using a FACS Verse flow cytometer and analyzed using FlowJo software (Treestar Inc., Ashland, OR, USA)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Co-infection of Vero Cells by Infected EVs from COVID19 patients in Cell Culture: Vero cells were cultured in DMEM medium (GIBCO/BRL, Grand Island, NY, USA), supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum.
    GIBCO/BRL
    suggested: None
    Statistical Analysis: All the data were analysed using GraphPad Prism 7 (GraphPad Software, Inc. La Jolla, CA, USA) and SPSS.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    The present detection tools have its own limitation and the risk factors predict SARS-CoV-2 reactivation in patients is not been known. In a recent report it has been confirmed that in a significantly proportion of COVID-19 patients, SARS-CoV-2 reactivation developed after discharging from hospital (9%)(13). Also, reported the clinical characteristics of these patients with SARS-CoV-2 reactivation which were similar to those of non-reactivated patients with COVID-19 infection (14). In our findings, presence of SARS CoV2 in associated form with extracellular vesicles reveals for the first time the hidden form of RNA. Notably, based on few reports there is currently an evidence to suggest that a proportion of recovered COVID-19 patients again got COVID-19 positivity (15). We also found that even in RT-PCR negative patients, SARS-COV2 RNA was detectable even after 14 days inside the EVs. The results of the SARS-CoV-2 RNA tests, in such cases are fluctuant. May be because, no research has yet accurately established the contagious period of COVID-19. Our study will be first of its kind, to identify the new route of transmission or infectivity. Besides patients and asymptomatic carriers, those in convalescence may also be infectious. SARS-CoV-2 RNA from respiratory tract specimens may be persistent or recurrently positive during the course of this disease (16,17). Furthermore, Angiotensin-converting enzyme-2 (ACE-2), identified as the cell entry receptor of SARS-CoV-2, was highly e...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a protocol registration statement.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.12.16.21267912v1 on medRxiv