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

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-conrmed COVID-19 cases from Wuhan with viral RNA shedding duration more than 30 days were included in our study,5 of them were moderate.Althoughin�ammatory markers were signi�cantlyhigher, 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 in�uenza, 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 in uenza 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).

Methods and Patients
This retrospective study enrolled 9 laboratory-con rmed COVID-19 adult inpatients admitted to Wuhan Tongji Hospital from February 09 to March 09, 2020.
Diagnosis and clinical classi cation of COVID-19 were according to the Chinese management guideline for COVID-19 (version 7.0) and the World Health Organization interim guidance.Severe and critically ill COVID-19 patients were identi ed by reviewing and analyzing admission logs and histories of all available electronic medical records and patient care resources by two physicians.This study was approved by the institutional review boards at Wuhan Tongji Hospital and the First A liated Hospital of Soochow University.As an emerging infectious disease, written informed consent was exempted.

Data Collection
Demographic characteristics (age and gender), clinical features (comorbidities, laboratory ndings, 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 de ned as the interval between illness onset and the date of the last pharyngeal swab with a positive nding.Patients were followed-up from admission to 28 days in hospital, hospital discharge, or death, whichever came rst.

Statistical analysis
Continuous data with normal distribution were presented as mean ± standard deviation.Frequency data were expressed as proportions.Data were analyzed using SPSS 25.0.

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).

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 insu cient treatment when outside of hospital.We need more data to determine transmission dynamics and inform our screening practices (5).
Previous reports about in uenza 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 speci city 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 in ammatory 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 nd 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 in ammatory 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.

Figures Figure 1 A:
Figures

Table 1
The clinical and immunologic characteristics of all patients.