key: cord-0905317-7ot9ds34
authors: Lei, Chunliang; Lin, Weiyin; Deng, Xilong; Hu, Fengyu; Chen, Fengjuan; Cai, Weiping; Li, Yueping; Wen, Chunyan; Guan, Yujuan; Wang, Jian; Chen, Xiaoting; Cao, Yi; Li, Feng; Tang, Xiaoping; Li, Linghua
title: Factors associated with clinical outcomes in patients with Coronavirus Disease 2019 in Guangzhou, China
date: 2020-10-14
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104661
sha: 88ec4804c62755ded0c19e6d65ee8093df78e19f
doc_id: 905317
cord_uid: 7ot9ds34

BACKGROUND: Coronavirus Disease 2019 (COVID-19) is threatening billions of people. We described the clinical characteristics and explore virological and immunological factors associated with clinical outcomes. METHODS: 297 COVID-19 patients hospitalized in Guangzhou Eighth People's Hospital between January 20 and February 20, 2020 were included. Epidemiological, clinical and laboratory data were collected and analyzed. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA in respiratory tract, blood samples and digestive tract was detected and lymphocyte subsets were tested periodically. RESULT: Among the 297 patients (median age of 48 years), 154 (51.9 %) were female, 245 (82.5 %) mild/moderate cases, and 52 (17.5 %) severe/critical cases. 270 patients were detected for SARS-CoV-2 RNA in anal swabs and/or blood samples, and the overall positive rate was 23.0 % (62/270), higher in severe/critical cases than in mild/moderate cases (52.0 % vs. 16.4 %, P < 0.001). The CD4/CD8 ratio on admission was significantly higher in severe/critical cases than in mild/moderate cases (1.84 vs. 1.50, P = 0.022). During a median follow-up period of 17 days, 36 (12.1 %) patients were admitted to intensive care unit (ICU), 16 (5.4 %) patients developed respiratory failure and underwent mechanical ventilation, four (1.3 %) patients needed extracorporeal membrane oxygenation (ECMO), only one (0.34 %) patients died of multiple organ failure. Detectable SARS-CoV-2 RNA in anal swabs and/or blood samples, as well as higher CD4/CD8 ratio were independent risk factors of respiratory failure and ICU admission. CONCLUSIONS: Most of COVID-19 patients in Guangzhou are mild/moderate, and presence of extrapulmonary virus and higher CD4/CD8 ratio are associated with higher risk of worse outcomes.

Since December, 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an outbreak of respiratory illness termed Coronavirus Disease 2019 (COVID- 19) globally. 1, 2 The disease has become a global pandemic, threatening billions of people and leading to over 730,000 death worldwide. 3 At the late March, the epidemic had been under control in China with the strong and sustained efforts of the whole country. Despite many previous articles report mainly concentrating on the epidemiological findings and clinical characteristics of patients with COVID-19 in Hubei province and outside of Hubei, 4-6 the clinical experience in Guangzhou, a non-epidemic area but with high risk of imported cases from abroad, is still very valuable for controlling this emerging disease.

Previous study shows that most cases of COVID-19 are mild with good prognosis, however, the proportion of organ failure, especially respiratory failure and mortality rate of severe patients is considerable. 7 One of the main challenges for the clinicians is how to quickly identify COVID-19 patients at high risk for worse outcomes. However, effective early warning indicators are still limited so far.

In this study, we comprehensively described the clinical characteristics, and J o u r n a l P r e -p r o o f explore virological and immunological factors associated with outcomes of hospitalized patients confirmed with COVID-19, exploring valuable experiences for controlling COVID-19 in non-endemic regions.

This was a retrospective, observational study conducted at Guangzhou Eighth People's Hospital. We analyzed hospitalized COVID-19 patients between January 20 and February 20, 2020. The end of follow up was June 1st, 2020, or the day when patients recovered and discharged from hospital, or transferred to the designated hospital for critically ill patients, or died.

People's Hospital (Approval No. 202001134). Written informed consent was obtained from all patients.

The medical records, nursing records and laboratory reports of all patients with COVID-19 were retrospectively collected and reviewed by two physicians, and all radiological images were reviewed by two radiologists. Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of studied patients. PBMCs were stained with the following antibodies: CD3-Pacific-Blue, CD4-APC/CY7 and CD8-Percp/CY5.5 to obtain percentage of CD3+ T cells, CD4+ T cells and CD8+ T cells, and CD4/CD8 ratio by flow cytometry.

The specimens of throat swabs, anal swabs and blood samples were J o u r n a l P r e -p r o o f obtained every 3 to 6 days. SARS-CoV-2 nucleic acid was detected by realtime fluorescence reverse transcriptional polymerase chain reaction (RT-PCR) on the platform of Da'an Gene Corporation, Sun Yat-sen University, Guangzhou, China, which has been described. 8 

Based on "Diagnosis and treatment of pneumonitis caused by new coronavirus (trial version 7)" issued by the National Health Commission of China on March 3, 2020, clinical classifications of patients with COVID-19 were defined as follows. 11 Mild status was defined as having mild clinical symptoms but no signs of pneumonia on imaging. Moderate status was defined as having fever and respiratory symptoms, and pneumonia on imaging. Severe status must meet any of the following conditions: 1) experiencing respiratory distress, RR ≥30 times/minute; 2) in the resting state, the oxygen saturation ≤93%; 3) arterial blood oxygen partial pressure (PaO2)/oxygen concentration (FiO2) 

Data were expressed as counts and percentages for categorical variables and as mean and standard deviation or median and interquartile range (IQR) for continuous variables. Qualitative and quantitative differences between subgroups were analyzed using χ 2 test or Fisher's exact tests for categorical parameters and the Student's t test or Mann-Whitney U-test for continuous parameters, as appropriate. Cox regression model was performed to analyze the association of baseline parameters with clinical outcomes. All statistical tests were 2-sided. Statistical significance was taken as P <0.05. All analyses were performed with SPSS software, version 26.0 (IBM, Armonk, NY).

From January 20, 2020 to February 20, 2020, 297 consecutive patients who had been confirmed with COVID-19 and admitted to Guangzhou Eighth People's Hospital were included in this study. The median age was 48 years, and 154 (51.9%) were female. 245 (82.5%) patients were diagnosed with mild/moderate cases, 52 (17.5%) severe/critical cases. The baseline characteristics are shown in Table 1 .

A total of 270 patients were detected for SARS-CoV-2 RNA in anal swabs J o u r n a l P r e -p r o o f 8 / 25 and/or blood samples, and the overall positive rate was 23.0% (62/270), higher in severe/critical cases than in mild/moderate cases (52.0% vs. 16 181 patients were detected for viral RNA both in anal swabs and blood samples, and 6 (3.3%) patients were double positive (all were male, age ranged from 30 to 82 years). Notably, three of the six double positive patients were critical cases who developed respiratory failure and required mechanical ventilation in intensive care unit (ICU), two patients were severe cases who need high-flow nasal cannula and close monitoring, only one patient was moderate case.

286 of the 297 patients (96.3%) were analyzed for the percentage of CD3 + T cells, CD4 + T cells and CD8 + T cells, and CD4/CD8 ratio by flow cytometry in peripheral blood mononuclear cell (PBMC). Compared to the mild/moderate cases, the severe/critical cases had lower percentage of CD3 + T cells (52.6% vs. 61.4%, P<0.001) and CD8 + T cells (33.2% vs. 36.5%, P=0.034), but higher J o u r n a l P r e -p r o o f percentage of CD4 + T cells (62.8% vs. 55.8%, P=0.016) (Figure 2A) 

Among the 297 patients, nearly two thirds received oxygen inhalation, antibiotic therapy, traditional Chinese medicine and antiviral therapy (including lopinavir/ritonavir, arbidol and chloroquine phosphate). In addition, 66 (22.2%) patients were treated with oseltamivir, 65 (21.9) with corticosteroids, and 35 (11.9%) with immunoglobulin, respectively (Supplemental Table 1 ).

During a median follow-up period of 17 days (IQR, 13-24), 36 (12.1%) patients were admitted to ICU for high-flow nasal cannula or higher-level oxygen support measures to correct hypoxemia ( Table 2) . 16 As of June 1st, 2020, 284 (95.6%) patients had recovered and discharged from Guangzhou Eighth People's hospital, one (0.3%) 82-year-old patient died of multiple organ failure even though receiving ECMO treatment, twelve (4.0%) patients were transferred to the designated hospital for critically ill patients in Guangzhou due to the deterioration of their illness within 8 days (IQR, [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] after admission ( Table 2 ). All of the twelve transferred patients had recovered and discharged from hospital. No medical staff in our hospital had nosocomial infection of COVID-19 since January.

Cox regression model was performed to analyze the association of baseline parameters including age, gender, comorbidities, clinical symptoms, laboratory index, imaging findings and extrapulmonary virological detection with the probability of respiratory failure ( SARS-CoV-2 RNA can be detected not only in respiratory tract, but also in blood, digestive tract and feces. [12] [13] [14] Patients with more severe disease tended to have a higher detection rate of extrapulmonary SARS-CoV-2 RNA. 15 Recently published research showed that SARS-COV-2 RNA in serum was associated with multiple organ damages and higher mortality rate. 16 Our previous cross-sectional study indicated that detectable SARS-CoV-2 RNA in blood was an indicator for the further clinical severity, another study showed that detectable SARS-CoV-2 RNA in the digestive tract was a potential warning indicator of severe disease. 8, 9 In this larger sample size longitudinal study, we found patients with detectable extrapulmonary SARS-CoV-2 RNA had an Lymphocytopenia is often found in patients with COVID-19, especially in severe ones. 7, 17 Chen et al found lower CD4 + T cells count was associated with ICU admission. 18 Liu et al found the more serious the disease and the worse the prognosis, the lower were the T cell, CD4 + T cell, and CD8 + T cell counts on admission. 19 A recent published study found CD4/CD8 ratio was significantly higher in critically ill than in non-critically ill patients. 20 We found severe/critical patients had lower levels of lymphocytes, lower percentage of CD3 + T cells and CD8 + T cells but higher percentage of CD4 + T cells than mild/moderate ones.

Notably, higher CD4/CD8 ratio was independently associate with higher risk of respiratory failure and ICU admission. This result indicated that lymphocyte subsets could be a useful parameter for early prediction of prognosis of COVID-19. We also found that there was no significant change in the percentage of In this study, around 75% of mild/moderate patients and nearly 100% of the severe/critical patients received antiviral treatment including Lopinavir/ritonavir J o u r n a l P r e -p r o o f (LPV/r), abidol and chloroquine. However, these antiviral regimens perform little benefit for improving the clinical outcomes including virus clearance of hospitalized mild/moderate COVID-19 beyond supportive treatment [12] [13] [14] [15] . [21] [22] [23] The median days from admission to positive-to-negative conversion of viral RNA exceeded 10 days (11 days [IQR, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] ) in severe/critical cases disregard LPV/r or abidol or chloroquine, which indicates the antiviral failure of these medicines. Even so, our study in another way implies that comprehensive therapy scheme could successfully prevent and treat COVID-19 even without effective antiviral regimens currently.

Our study has some limitations. First, the comparison of different antiviral regimens was not randomized nor blinded, the baseline health status between antiviral and non-antiviral patients were not comparable. Second, we did not collect information on absolute values of T cells, B cells, NK cells, monocytes and dendritic cells. In addition, we were unable to provide information on the changes in the absolute number of immune cells in these patients. Further studies are needed to explore the relationship between these immunological indexes and outcomes of COVID-19. 

All authors declare that they have no conflict or competing interests. J o u r n a l P r e -p r o o f

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C-reactive protein (IQR) -mg/L 10

J o u r n a l P r e -p r o o f 16 / 25 We thank all patients who participated in this study and all staff of Guangzhou