The decrease of CD4 T cell is associated with mortality in critical inpatients with COVID-19

Background The 2019 novel coronavirus (SARS-CoV-2) has caused an outbreak in the world. The critically ill patients had a high mortality. However, the possible risk factors of critical patients with coronavirus disease 2019 (COVID-19) are not fully known. We aim to investigate the risk factors in critical patients with COVID-19 and to address their role in predicting disease progression. Methods In this single-centered, retrospective, observational study, we enrolled 91 critically ill adult patients with COVID-19 in Renmin Hospital of Wuhan University between Jan 20, 2020 and Feb 28, 2020. Data were collected using a standard method including clinical records and laboratory ndings. Results 39 patients (42.9%) were dead and 52 patients (57.1%) were cured and discharged before Mar 22, 2020. CD4 T cell count, CD8 T cell count and glomerular ltration rate were signicantly lower in non-survivors than in survivors. However, the non-survivors presented a higher proportion of D-dimer, Cardiac troponin and immunoglobulin G than in survivors. Intravenous immunoglobulin was more common in survivors than in non-survivors. On multivariate analysis, D-dimer ( (cid:0) 1 µg/mL, OR = 9.53, 95% CI, 2.53– 35.88), CD4 + T count ( (cid:0) 200/µl, OR = 9.68, 95%CI, 2.76-40.00 ) and cardiac troponin ( (cid:0) 0.04 ng/mL, OR = 5.73, 95% CI, 1.86–17.66) were independent risk factors for mortality. Conclusion The decrease of CD4 T cell is associated with higher risk mortality in critical inpatients with COVID-19. Intravenous immunoglobulin was more common in survivors than in non-survivors.


Introduction
At present, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outbreak and spread to many countries in the world. The current best estimate is that about 81% of people with coronavirus disease 2019 (COVID-19) have mild disease and never require hospitalization [1]. In s singlecenter case series of 138 hospitalized patients with COVID-19 in Wuhan, results showed that the mortality of patients was 4.3% [2]. However, in another study, Yang reported that the mortality was 62% among critically ill patients with COVID-19 and 81% among those requiring mechanical ventilation in Wuhan [3].
Zhou and colleagues provide further insight into the mortality risk of critical inpatients with COVID-19.
They found that older age, high sequential organ failure assessment (SOFA) score, and d-dimer greater than 1 µg/L are associated with the mortality risk of critical inpatients. In this study, not all laboratory tests were done, including lactate dehydrogenase, in ammatory and immune factors. Studies have suggested that a substantial decrease in the levels of lymphocytes indicates that coronavirus consumes many immune cells and inhibits the body's cellular immune function [4,5]. Damage to T lymphocytes might be an important factor leading to exacerbations of patients [6]. In this study, we aim to investigate the risk factors in critical patients with COVID-19 and to address their role in predicting disease progression.

Study design
This single-centre, retrospective, observational study was done at Renmin Hospital of Wuhan University (Wuhan, China). 91 critical inpatients with COVID-19 from January 20, 2020 to Feb 28, 2020 were enrolled. This study was approved by the Ethics Committee of Renmin Hospital of Wuhan University. All critical inpatients were diagnosed with SARS-CoV-2 pneumonia, according to WHO interim guidance [7].
All cases matched clinical classi cation of critical type.

Data collection
The demographic characteristics, clinical records, initial hospitalized laboratory ndings, computed tomography and outcome data were extracted from electronic medical records using the standardised data collection form. Two experienced clinicians entered data and reviewed the data. We collected data on age, sex, chronic medical histories (hypertension, chronic cardiac disease, chronic pulmonary disease, diabetes), the rst symptoms, electrocardiogram and vital signs (heart rate, respiratory rate and Oxygen saturation), laboratory values on admission (blood routine examination, coagulation function, biochemical criterion, humoral immunity, cellular immunity, myocardial injury index and other indexes), treatment (antiviral drug: Arbidol/ Ribavirin/ interferon α), intravenous immunoglobulin, Chinese patent drug (XueBiJing/ TanReqing/ YanHuNing), Methylprednisolone), as well as living status.

Outcomes
The outcome was death or discharged. Discharge standard were de ned according to the guidance of New coronavirus pneumonia prevention and control program in China (6th edition) [8]. Critical patient selection criteria (at least one of the following criteria): Pulse oxygen saturation ≤ 93% in resting state or shortness of breath (respiratory rate ≥ 30 bpm, or CT scan of the lungs suggesting that a lesion progression greater than 50% within 48 hours.

Statistical analysis
Continuous variables are expressed as means ± standard deviation. The statistical differences were made using the unpaired and two-tailed t-test. Categorical data were compared using Pearson Chi-Square test or a Fisher exact test when expected values were less than 5 cases. Binary logistic regression analyses were used to assess the possible death predictors. Prespeci ed relevant clinical data was tested rst in a univariate and then in a multivariate regression model by LR with a forward stepwise variable selection. P value < 0.05 was considered to indicate a statistically signi cant difference. The statistical analysis was performed using the SPSS 22.0 software.

Results
Baseline and treatment characteristics were dead and 52 patients (57.1%) were cured and discharged before Mar 22, 2020. The mean age was 63.9 ± 14.1 years (range, 29-85 years), and 52 (57.1%) were male. Baseline characteristics of the survivors and death are shown in Table 1. Overall, there was no signi cant difference in the proportion of hypertension, diabetes, chronic obstructive pulmonary disease (COPD), rst symptoms and respiratory rate between survival group and death group. In addition to common symptoms such as fever, 11% of patients had diarrhea and 4.4% had nausea and vomiting, and one patient in death group has diarrhea as single symptom. The arrhythmic events, such as sinus tachycardia and atrial or ventricular arrhythmia showed no difference between the two groups. Furthermore, there was no signi cant difference abnormal ST segment and T wave between the two groups. There was no signi cant difference in the treatment of antiviral drug, Chinese patent drug (XueBiJing/ TanReqing/ YanHuNing) and Methylprednisolone between survival group and death group. However, intravenous immunoglobulin was more common in survivors than in death (p = 0.009). Compared with the survival group, patients in death group were older (70.4 ± 12.9 years vs 59.1 ± 13.2 years, p < 0.001), and more likely to have low Oxygen saturation (≤ 93%, 87.2% vs 69.2%), p = 0.044). Moreover, coronary heart disease (23.1% vs 5.8%) was present more often in non-survival group (p = 0.026).

Laboratory ndings
The laboratory ndings are shown in Table 2  In the death group, almost all patients (97.4%) have a higher D-dimer level that more than 0.55 µg/mL, and 79.7% patients have a more higher D-dimer level that more than 1 µg/mL, and the percentage signi cantly elevated than in the survival group (p 0.001). The non-survivors also presented a higher proportion of increased prothrombin time (p = 0.003), Lactate dehydrogenase (p = 0.022). B-type natriuretic peptide precursor (p = 0.07), high-sensitivity CRP (p = 0.021), and procalcitonin level (p = 0.006) than those survivors. Furthermore, the proportion of abnormal myocardial injury index of death was higher than that of survivors.

Discussion
We report on 91 critically ill inpatients with con rmed SARS-CoV-2 infection. This present retrospective study identi ed several risk factors for death. In particular, CD4 T cell count less than 200/µl, d-dimer levels greater than 1 µg/mL, and cardiac troponin greater than 0.04 ng/mL on admission were associated with higher odds of critical in-hospital death. Our study also demonstrated that older age, high-sensitivity C-reactive protein, and lactate dehydrogenase were more commonly seen in severe COVID-19 illness.
This study, to our knowledge, is the rst report to investigate the relationship between immune factors and death. Studies have shown that the clinical features of SARS-CoV-2 infection bear some resemblance to SARS-CoV and middle east respiratory syndrome coronavirus (MERS-CoV) infections [9,10]. The 2019-nCoV infection appears to be initially associated with an increased Th2 response, which might re ect a physiological reaction to curb overt in ammatory responses [11]. Reduced CD4 T cell and CD8 T cell number has been shown in SARS patients [12]. Levels of CD4 T cell and CD8 T cell were lower in severe COVID-19 patients than mild patients [13]. However, the association between CD4 T cell, CD8 T cell and mortality in patients with COVID-19 is limited. In the present study, the results showed that CD4 T cell and CD8 T cell decreased in critical inpatients with COVID-19. Furthermore, we found that low CD4 T cell level is an independent risk factor for mortality in critical inpatients with COVID-19. Previous study has found that the lower CD4T lymphocyte count is an important predictor for the prognosis and adverse clinical outcomes of cryptococcosis [14]. In HIV/HCV-coinfected individuals the death risk is also related to the level of CD4 + cells [15]. The loss of CD4 Th1 cells directly reduced numbers and function of antiviral CD8 T cell in persistent viral infection [16]. Interestingly, in the present study, we found that there was no signi cant difference in the treatment of antiviral drug and Methylprednisolone between the two group, while intravenous immunoglobulin was more common in survivors than in death. Whether immunoglobulin treatment can reduce mortality in critical inpatients with COVID-19 should be further to investigated.
Previous studies have shown that older age, d-dimer greater than 1 µg/mL and greater cardiac troponin are the potential risk factors of inpatients with COVID-19 [17,18]. The greater levels of D-dimer and cardiac troponin occurring in non survivors in the current study are similar to the ndings of other studies. However, we found that older age is not the independent risk factor for mortality in critical inpatients with COVID-19. The different results between our results and other studies may be related to different criteria for collecting cases. In the present study, all the patients were the critical inpatients with COVID-19 according to the guidance of New coronavirus pneumonia prevention and control program in China (6th edition) [8]. Taken together, these results showed that older age and may be related to the severity of the disease.

Limitations
There are several limitations to this study. First, some patients were transferred from the other hospitals.
The different treatment in the early stage of patients may affect the progress of patients. Second, due to the retrospective study design and the limited patients, data from larger populations and multiple centers is warranted to further con rm the outcomes of cellular immunity in COVID-19. Third, in the present study, the surviving patients were not followed up after discharge, so our results only show the risk of mortality during hospitalization.

Conclusions
The decrease of CD4 T cell is associated with higher risk mortality in critical inpatients with COVID-19. Intravenous immunoglobulin was more common in survivors than in non-survivors.