Clinical Features and Lymphocyte Subsets in Recovered Covid-19 Patients With Prolonged Viral Rna Shedding Duration

Abstract

Background:The 2019 novel coronavirus disease (COVID-19) spread in many countries.Data about viral shedding duration, particularly the prolonged ones of the pathogen SARS-Coronavirus-2 (SARS-CoV-2) is scarce. The longest viral RNA sheddingduration reported previously was 37 days. Herein, we report the clinical and immunologic features ofrecovered COVID-19cases with a medium viral RNA shedding duration of 44 days.

Cases presentation: Nine laboratory-confirmed COVID-19 cases from Wuhan with viral RNA shedding duration more than 30 days were included in our study,5 of them were moderate.Althoughinflammatory markers were significantlyhigher, the medium duration in severepatients was similar to that in moderate patients (44.5days vs. 43.6days). Severepatients showed higher NK cells levels, although the T cells and B cells were lower as compared with moderate patients. Contrary to previous reports in influenza, prolonged viralshedding time did not cause poor prognosis in this study.

Conclusions: There could be characteristic immunological dysfunction in COVID-19 patients with prolonged viral shedding durationand interestingly, prolonged viral shedding duration seemed not to be related with poor prognosis.

Background

SARS-Coronavirus-2 (SARS-CoV-2) is the focus of global attention which attributed to an outbreak of a severe respiratory illness since December 2019 in Wuhan, China. This severe respiratory illness originated in Wuhan City in the Hubei province in China and is capable of rapid community transmission (1).

The level and duration of SARS-CoV-2 virus replication are important factors in assessing the risk of trans mission and guiding decisions regarding isolation of patients. A previous study reported that the median duration of viral shedding was 20 days in survivors (2). The longest duration of viral shedding was 37 days in survivors. It has been reported in seasonal or pandemic influenza viral shedding duration that the longer duration of viral RNA shedding was correlated with worse patient prognosis (3–4). It remains unclear whether the SARS-CoV-2 RNA shedding duration affected progression of COVID-19 or not. Here, we report the clinical and immunologic features of a series of COVID-19 cases with prolonged viral RNA shedding duration (the median duration was 44 days).

Cases Presentation

Data Collection

Demographic characteristics (age and gender), clinical features (comorbidities, laboratory findings, diseases severity, treatments and outcomes) were recorded. Patients were followed-up from admission to persistent negative detection of pharyngeal swabs specimens. The SARS-CoV-2 RNA shedding duration was defined as the interval between illness onset and the date of the last pharyngeal swab with a positive finding. Patients were followed-up from admission to 28 days in hospital, hospital discharge, or death, whichever came first.

Results

Total of 9 adult COVID-19 inpatients were included. The median age was 64.3 years old (34–87 yrs). 6 cases were male. The most popular comorbidity was hypertension (5/9). The most common symptoms were fever (6/9) and cough (5/9). All patients were survived at 28-days after admission, with 5 cases discharged. 2 patients were improved in hospital and 2 patients remained receiving invasive machinery ventilation (IMV) and continuous renal replacement therapy (CRRT).

For all patients, the median viral RNA shedding duration was 44 days (30–62 days). The median viral RNA shedding duration in severe or critical patients was similar to that in moderate patients (44.5 days vs. 43.6 days). The severe and critical patients were older, have a lower lymphocyte count (0.58 × 10⁹ /L vs. 1.72 × 10⁹ /L) and ALB levels (29.75 g/L vs. 40.06 g/L), a higher levels of hs-CRP (89.33 mg/L vs. 2.56 mg/L), interleukin 6 (35.62 pg/ml vs. 2.27 pg/ml), d-dimer (1.67 µg/ml vs. 0.36 µg/ml), cTnI (91.03 pg/ml vs. 2.10 pg/ml), and lactate dehydrogenase (402.25 U/L vs. 182.40 U/L).

We also detected the lymphocyte subsets levels in peripheral blood samples. As compared with moderate COVID-19 cases, the severe and critical patients showed a lower levels of CD3⁺CD19⁻ T cells (675.75 N/µl vs. 1370.40 N/µl), CD3⁻CD19⁺ B cells (130.25 N/µl vs. 275.00 N/µl ), CD3⁺CD4⁺ Treg (380.25 N/µl vs. 734.80 N/µl) and CD3⁺CD8⁺ Ts cells (283.50 N/µl vs. 561.40 N/µl ). Interestingly, severe and critical patients have a higher levels of CD3⁻CD16⁺CD56⁺ NK cells (318.25 N/µl vs. 232.20 N/µl ). All patients showed bilateral lung lesion in CT findings (patchy shadowing or ground-glass opacity), 2 patients showed pleural effusion.

Discussion

Our study focused on the clinical features and immunologic manifestations of COVID-19 patients with prolonged viral RNA shedding duration. Duration of infectious virus replication are crucial factors in evaluating the transmission risk (1). The median shedding duration was 44 days, which was far longer than that reported in previous study (2). In this study, the longest viral RNA shedding duration was 62 days. It might be the longest viral shedding duration of COVID-19 to date. The difference might be due to the epidemiological feature of patients enrolled, which were enrolled form the designated hospital for COVID-19 patients of severe or critical illness in Wuhan, the most serious epidemic region in China. Delayed hospital admission was associated with prolonged viral shedding duration, as they received insufficient treatment when outside of hospital. We need more data to determine transmission dynamics and inform our screening practices (5).

Previous reports about influenza A (H7N9) and respiratory syncytial virus revealed that prolonged viral shedding duration associated with risk of complications and poor prognosis (3–4). Contrarily, all patients in our study remain survived although their viral RNA shedding duration exceeded 30 days. It indicated that prolonged viral RNA shedding was not always associated with poor prognosis. This might indicate the specificity of SARS-CoV-2. The stabilizing mutation falling in the nsp2 protein could account for COVID-2019 high ability of contagious, while the destabilizing mutation in nsp3 proteins could suggest a potential mechanism differentiating COVID-2019 from SARS (6–7). The molecular divergence of SARS-CoV-2 evolved into two major types with different characteristics (7). Furthermore, the diversity of mortality in different region of China might be due to the virological dynamics and epidemiological characteristics (8).

Although the inflammatory markers were obviously different (such as hs-CRP and interleukin 6) between severe patients and common patients, the median duration of viral RNA shedding was similar. We did not find this relationship between viral shedding and disease severity. The balance between viral load and immune system was one of the most important factors determined diseases progression or not. SARS-CoV-2 infection can activate innate and adaptive immune responses (9). However, uncontrolled inflammatory innate responses and impaired adaptive immune responses contributed to both locally and systemically harmful tissue damage. Although the severe and critical patients showed obvious lymphocytopenia and dysregulation of lymphocytes subsets, their NK cells levels were higher than common patents. This might be helpful for immune responses against SARS-CoV-2 infection. The differentiation of naive CD4 + T-cells into effector and memory subsets is one of the most fundamental facets of T-cell-mediated immunity (10). Additionally, the antivirus treatment might affect the disease progression.

Conclusion

In conclusion, our study focuses on the clinical and immunologic features in COVID-19 patients with prolonged viral RNA shedding duration. Severe COVID-19 patients have a lower level of CD3⁺CD19⁻T cells and CD3⁻CD19⁺B cells but a higher CD3⁻CD16⁺CD56⁺NK cells levels. Contrary to previous reports in influenza, prolonged viral shedding time did not cause poor prognosis in this study. However, further studies are needed to determine the virological dynamics.

Abbreviations

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Written informed consent for publication of the clinical details and clinical images was obtained from the patient.

Availability of data and materials

Not applicable.

Competing interests:

The authors declare that they have no competing interests.

Funding

This work was supported by Program of Key Talents of Medical Science in Jiangsu Province (QNRC2016745), Suzhou science and technology development plan (SYS202008), Clinical Medicine Center of Suzhou (No.SZZX201502) and Suzhou Key Laboratory for Respiratory Medicine (No. SZS201617).

Author’s contributions

WYZ, KW participated in the collection and check of electronic medical records and patient care resources. YYL analysed the data with the assistance from CGW. DXZ wrote the first draft, JAH and JHJ edited the final draft. All authors had read and approved the final manuscript.

Acknowledgements

Not applicable.

Author details

¹ Department of Pulmonary and Critical Care Medicine, First Affiliated Hospital of Soochow University.

References

1. 10.1016/S0140-6736(20)30744-3
   Chen S, Zhang Z, Yang J, et al. Fangcang shelter hospitals: a novel concept for responding to public health emergencies. Lancet. 2020 Apr 2. pii: S0140-6736(20)30744-3. doi: 10.1016/S0140-6736(20)30744-3.
2. Zhou F, Yu T, Du R, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020;395(10229):1054–62.
3. Lee N, Chan PK, Hui DS, et al. Viral loads and duration of viral shedding in adult patients hospitalized with influenza. J Infect Dis. 2009;200:492–500.
4. Fielding JE, Kelly HA, Mercer GN, et al. Systematic review of influenza A(H1N1)pdm09 virus shedding: duration is affected by severity, but not age. Influenza Other Respir Viruses. 2014;8:142–50.
5. Wilder-Smith A, Chiew CJ, Lee VJ. Can we contain the COVID-19 outbreak with the same measures as for SARS? Lancet Infect Dis. 2020 Mar 5. pii: S1473-3099(20)30129-8. doi: 10.1016/S1473-3099(20)30129-8.
6. Angeletti S, Benvenuto D, Bianchi M, et al. COVID-2019: The role of the nsp2 and nsp3 in its pathogenesis. J Med Virol. 2020 Feb 21. doi:10.1002/jmv.25719.
7. 10.1002/jmv.25719. ew
   Tang X, Wu C, Li X, et al. On the origin and continuing evolution of SARS-CoV-2. National ScienceJ Med Virol. 2020 Feb 21. doi: 10.1002/jmv.25719. ew. https://doi.org/10.1093/nsr/nwaa036.
8. 10.1183/13993003.00562-2020
   Liang WH, Guan WJ, Li CC, et al. Clinical characteristics and outcomes of hospitalised patients with COVID-19 treated in Hubei (epicenter) and outside Hubei (non-epicenter): A Nationwide Analysis of China. Eur Respir J. 2020 Apr 8. pii: 2000562. doi: 10.1183/13993003.00562-2020.
9. 10.1038/s41577-020-0308-3
   Cao X. COVID-19: immunopathology and its implications for therapy. Nat Rev Immunol. 2020 Apr 9. doi: 10.1038/s41577-020-0308-3.
10. Shaw AC, Goldstein DR, Montgomery RR. Age-dependent dysregulation of innate immunity. Nat Rev Immunol. 2013;13(12):875–87.