Higher Survival Rate Associated with Thymosin Alpha 1 use in Critical COVID-19: A Retrospective Multicenter Cohort Study

Background Currently no satisfactory pharmaceutical intervention is available for COVID-19. This retrospective study aimed to determine the therapeutic effect of thymosin alpha1 in critical COVID-19. Results We enrolled 109 critically ill severe acute respiratory syndrome-related coronavirus-2 RNA positive patients from 15 hospitals. The mortality rate in critical patients treated with thymosin alpha1 was 11%, compared to 56% in critical patients not treated with thymosin alpha1. With confounding factors adjusted in multivariate logistic regression, thymosin alpha1 treatment was identied as a protective factor for critical COVID-19. Conclusion Our observation advocates the treatment of critical COVID-19 with thymosin alpha1. p are from Mann-Whitney U test or Student's t test, appropriate. NLR = neutrophil-to-lymphocyte ratio. APTT = activated partial thromboplastin time. ESR = Erythrocyte sedimentation rate. CRP = C-reactive protein. BUN = blood urea nitrogen. AST = aspartate transaminase. ALT = alanine aminotransferase. Data are median (IQR). p values comparing thymosin treatment and non-thymosin treatment are from Mann-Whitney U test or Student's t test, as appropriate. NLR = neutrophil-to-lymphocyte ratio. APTT = activated partial thromboplastin time. ESR = Erythrocyte sedimentation rate. CRP = C-reactive protein. BUN = blood urea nitrogen. AST = aspartate transaminase. ALT = alanine aminotransferase. SOFA = Sequential Organ Failure Assessment.


Introduction
The clinical presentations of the Coronavirus Disease 2019 (COVID-19) have two con icting sides. On one side, the excessive response of the immune system, represented by high levels of proin ammatory cytokines and chemokines, is considered the major mechanism for critical illness and mortality of COVID-19 [1,2]. For this reason, suppression of patients' immune system with glucocorticoid has been common in the management of COVID-19 [3][4][5]. On the other side, severe lymphocytopenia was frequently observed in COVID-19, especially in aged and severe cases [1,6]. Recently, Liu et al. reported that thymosin alpha 1 (Ta1) may bene t COVID-19 patients by restoring their immune capacity [7]. However, this study did not address the potential in uences of the confounding factors.
To determine the therapeutic effect of Ta1 in critical COVID-19, we conducted a retrospective cohort study and found that use of Ta1 caused bene cial outcomes including a much lower mortality rate.

Methods
We reviewed the medical records of 410 suspected COVID-19 patients. Excluding 56 records for negative RNA test, 24 for duplicated records, 219 for non-critical condition, and 2 for the lack of relevant data, we enrolled 109 critically ill [8] severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) RNA positive patients hospitalized at 15 hospitals in Jingzhou and Dongguan, China, from January 16 to March 30, 2020 (Fig. 1).
Treatment of COVID-19 followed the Diagnosis and Treatment Guideline for COVID-19, National Health Commission of the People's Republic of China. Most patients were treated with oxygen supplementation, Page 4/19 respiratory support, antibiotics, usually Moxi oxacin, and antivirus drugs, usually Lopinavir and Ritonavir.
Mechanical ventilation was conducted when indicated by exacerbating or persistent hypoxemia and dyspnea after non-invasive oxygen supplementation. Based on physician's preference, 18 of the critical patients exhibiting persistent positive results for SARS-CoV-2 RNA or lymphocyte count < 1.5 × 10 9 /L were treated with Ta1 (Ta1 group) at a dosage of 1.6 mg per 12 or 24hr subcutaneously for the length of critical illness. The other 91 critical patients were not treated with Ta1 (non-Ta1 group).
Multivariate logistic regressions were performed to identify clinical features and treatments associated with Ta1 treatment and mortality, respectively, and were adjusted with potential confounders identi ed from univariate regressions.

Results
Eighteen of the 109 critically ill COVID-19 patients (17%) were treated with Ta1. Compared to the 91 critical patients not treated with Ta1, the Ta 1 treated patients were more often from Dongguan People's Hospital (Guangdong Province) (Table 1), exhibited a lower serum procalcitonin level on admission (Table 2), and more often treated with immunoglobulin and albumin (Table 1).  Two patients (11%) in the Ta1 died compared to 51 patients (56%) in the non-Ta1 group. Similarly, 3 patients (17%) in the Ta1 group required mechanical ventilation, compared to 43 patients (47%) in the non-Ta1 group (Table 1). After adjusting for potential confounding factors including age, gender, location of the hospitals, comorbidities, treatment with immunoglobulin and albumin, Ta1 treatment was identi ed as a protective factor for mortality, with an adjusted odds ratio of 0.02 (95%CI: 0.00-0.35) (Table 3). However, no correlation was observed between Ta1 treatment and the requirement for mechanical ventilation after adjustment for confounding factors (Table 4).  Most of the critical patient in both study groups exhibited similarly severe lymphocytopenia on admission. Before discharge, the lymphocyte counts of many patients treated with Ta1 were restored to the normal range, and were signi cantly higher than those in the non-Ta1 group (  The laboratory results before discharge were similar to those on admission in that both study groups exhibited elevated in ammatory markers such as C-reactive protein (CRP) and Erythrocyte sedimentation rate ( Table 2, 5). Importantly, the CRP level was signi cantly lower in the Ta1 group compared to the non-Ta1 group before discharge. Similarly, other in ammatory markers such as the neutrophil count and the neutrophil-to-lymphocyte ratio fell into the normal range in all patients treated with Ta1 (Table 5). Before discharge, markers of tissue damage for liver, kidney, and muscle in the Ta1 group exhibited a trend of decreased level compared to the non-Ta1 group, in line with the lower Sequential Organ Failure Assessment (SOFA) score in Ta1 group (Table 5).

Discussion
With our patients, Ta1 is the best pharmaceutical intervention for critical COVID-19 achieving a much reduced mortality rate (11%), compared to the mortality rate in the non-Ta1 group (56%). Our data also indicated that the reduced mortality rate by Ta1 is linked with the increased counts of lymphocytes and the prevention of infections and injuries of multiple organs. Therefore, Ta1 may have protected the patients by boosting the antiviral capacity of the immune system.
Our observations are in concert with a recent report that Ta1 increased lymphocyte counts and thymus output, and reversed T cell exhaustion in severe COVID-19 [7]. Similar outcomes were observed with in uenza virus infected mice. When treated with Ta1, these mice exhibited increased NK-cell activity, CD4 and CD8 cell counts and activity in the lung, reduced viral titers and increased survival [9]. Besides viral infection, Ta1 has been successful in the treatment of severe sepsis, a condition known for high mortality rate. Patients with severe sepsis often present multi-organ failure in septic shock characterized by overstimulation of the immune system in response to infection, and are commonly treated with immune suppressive drugs such as glucocorticoids. Counter-intuitively, several studies have demonstrated bene ts of Ta1 in the treatment of severe sepsis, including higher survival rates [10].
The timing of the Ta1 treatment may be critical, because evidence has been presented for over-active immune system in causing the critical illness and mortality in COVID-19 [1,2]. Our patients received Ta1 at times when severe symptoms presented with persistent viral infection or lymphocytopenia, likely in a status of immune exhaustion. Monitoring serum cytokine levels may provide additional help in making the decision on whether to use and when to use Ta1.
The therapeutic mechanism of Ta1 remains largely unknown. Through up-regulation of TLR2 and TLR9 expression, the pleiotropic effects of Ta1 include not only enhanced functions of NK cells, CD4 + and CD8 + T cells [9,11], but also FoxP3 + Treg cells that promotes immune tolerance [12][13][14]. Before further evidence from mechanistic study is available, we cannot rule out the possibility that the bene ts of Ta1 in critical COVID-19 was mediated by the Ta1 stimulation of the proliferation of FoxP3 + Treg cells, which consequently led to a more balanced immune activity.

Limitations
Our study has several limitations. Firstly, this is a retrospective study based on chart review, wherein the validity of the diagnosis and the evaluation factors might have led to information bias. Secondly, COVID-19 is a sudden epidemic, the supply of medical resources might affect the treatment which may led to selection bias. Thirdly, there was a relatively small sample size in Ta1

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