{'fraction_non_alphanumeric': 0.041177051446627824, 'fraction_numerical': 0.02127974721042757, 'mean_word_length': 5.115640096618358, 'pattern_counts': {'":': 0, '<': 5, '<?xml version=': 0, '>': 3, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Effects of metformin, insulin on COVID-19 patients with pre-existed type 2 diabetes: A multicentral retrospective study

Aims Type 2 diabetes is considered to be one of the essential risks of adverse outcomes in coronavirus disease 2019 (COVID-19).1 Metformin and insulin were suggested to affect the outcomes. However, divergent views are still expressed. We aim to gain further insight into metformin and insulin in both pre-admission and in-hospital usage in COVID-19 patients with pre-existed type 2 diabetes. Main methods This is a multicentral retrospective study of the hospital confirmed COVID-19 patients between January 19 to April 09, 2020, who admitted to 3 main hospitals in Xiangyang city, China. The effect of type 2 diabetes, metformin, and insulin on COVID-19 were analyzed, respectively. Clinical characteristics, blood laboratory indices, clinical observational indices, and outcomes of these cases were collected. Key findings A total of 407 confirmed COVID-19 patients (including 50 pre-existed type 2 diabetes) were eligible in our study. COVID-19 patients with type 2 diabetes had more adverse outcomes than non-diabetes (OR2: mortality: 1.46 [95% CI3 1.11, 1.93]; P < 0.001). Pre-admission metformin usage showed a declined intensive care unit admission rate in a dose-dependent fashion (OR 0.04 [95% CI 0.00, 0.99]; adjust P = 0.049). While in-hospital insulin usage attempted to increase the invasive ventilation (8 [34.8%] vs. 1 [3.7%], adjust P = 0.043), independent of age and blood glucose. Significance Our study indicated that pre-admitted metformin usage may have beneficial effects on COVID-19 with pre-existed type 2 diabetes, insulin should be used sparingly in the hospital stay.

the World Health Organization since the start of the pandemic [1]. The numbers of confirmed infections and death cases are consistently increasing due to the lack of specific medicine for SARS-CoV-2 infection. Although the current large-scale vaccine production has brought dawn to mankind, the globalization of vaccination [2] and virus mutation [3] are still challenging to eliminate SARS-CoV-2.
Among them, patients with type 2 diabetes mellitus (T2DM) facing on the higher risks of adverse outcomes on COVID-19 than non-diabetes [4]- [6]. Besides, diabetes has been suggested as the risk factor of structural and pulmonary ventilation changes in the lungs in severe COVID-19 patients [7]. The antidiabetic medicine treatment regimen may have impacted the progression [8,9]. Metformin and insulin are the most commonly used antidiabetic medicine. Theoretical evidence has determined that metformin might have a beneficial on antivirus through reducing the proinflammatory, profibrotic states [10,11] and improve the immune response [12]. Insulin treatment may have the potential risk of poor prognosis with an increase in renal angiotensin converting enzyme 2 (ACE2) expression in mice with diabetes [13]. Several clinical studies have assessed the impact of metformin and insulin, whereas, their opinions were somehow discrepant [14]- [17]. The divergence of views may be attributed to the disparity objects, which was either inhistory or in-hospital drug usage rather than both.
Our aims of this study were to carry out a retrospective analysis of the effect of metformin/insulin in COVID-19 patients with T2DM in both pre-admission and in-hospital usage. We considered the discharge or death as the primary endpoint; poor prognosiseither intensive care unit (ICU) admission or invasive ventilationas the secondary study endpoint, to validate the role of T2DM in COVID-19 and to gain an insight into metformin and insulin treatment in COVID-19.

Subjects
Other than Wuhan, Xiangyang city is another central traffic-hub-city in Hubei province, China, which led to the severe influence of SARS-CoV-2. The hospital confirmed patients with COVID-19 who were admitted from January 19 to April 09, 2020, were collected. To reduce the potential sources of bias, the confirmed patients were collected from Xiangyang No.1 People's Hospital, Xiangyang Central Hospital, and Xiangyang Xiangzhou People's Hospital, which are the three main hospitals for receiving and treating COVID-19 patients, and admitted in approximately half of the confirmed COVID-19 patients in Xiangyang city.

Study design
In-hospital confirmed COVID-19 patients were divided into T2DM and non-diabetes, T2DM defined by fasting venous or capillary glucose ≥ 7.0 mmol/L (≥126 mg/dL); two-hour postprandial venous plasma glucose or random plasma glucose ≥ 11.1 mmol/L (≥200 mg/dL) according to the World Health Organization diagnostic criteria [18].
Patients with pre-existed T2DM who historically had metformin till admission were included in the pre-admission metformin group. Patients who received metformin alone or with other antidiabetic medications after admission were considered into the hospital metformin group. The definitions of pre-admission and in-hospital insulin group were consistent with metformin. Due to the lack of the data of HbA1c, we use the median of random blood glucose levels, which were tested within 24 h of admission, to assess the preadmission blood glucose control status.
The baseline was defined by the time when patients were admitted to the hospital. For the endpoint, we considered the discharge or death as the primary endpoint, and poor prognosis as the secondary study endpoint, including ICU admission and invasive ventilation.
The study protocol was approved by the local ethics committee (Ethics Committee of Xiangyang No. 1 People's Hospital Affiliated to Hubei University of Medicine, 2020KY002), and waived informed consent from study participants because of the study's retrospective design.
All clinical investigations were conducted in accordance with the guidelines of the Declaration of Helsinki.

Inclusion criteria
Referring to the diagnostic criteria by Novel Coronavirus Pneumonia Diagnosis and Treatment Plan (6th) by the National Health Commission of China [19], confirmed COVID-19 patients were included in our study.
Confirmed cases were: a positive result of real-time reverse transcription-polymerase chain reaction detection of novel coronavirus nucleic acid in suspected cases, who having epidemiological history (having a history of contact with the Wuhan area or contact with the confirmed cases in the last 14 days), or clinical presentations (having fever and/or respiratory tract symptoms or having the novel coronavirus pneumonia features through chest computed tomography scan (CT)).
Confirmed patients were defined into four forms: mild form, moderate form, severe form, and critical form, in accordance with the Novel Coronavirus Pneumonia Diagnosis and Treatment Plan (6th) [19].

Exclusion criteria
Patients were excluded who were re-detected positive, whose prognosis could not be tracked, patients with less data, or the hospital stay less than 3 days.

Criteria for hospital discharge
Patients considered for discharge need to achieve three criteria: 1) three or more than three days of normal body temperature with ameliorated respiratory symptoms, 2) substantially improved chest radiology of acute exudative lesions, 3) taken at least 24 h apart of two consecutive negative nucleic acid tests using respiratory tract samples.

Statistical analysis
The results are reported as the median (interquartile range [IQR]) for performed in comparing categorical data, with Bonferroni correction in multiple comparisons. Mann-Whitney U test was performed in comparing non-normal continuous data. In all COVID-19 populations, univariable and multivariable logistic regression were performed to determine the odds ratios (OR) and 95% confidence intervals (CIs) for factors associated with clinical outcomes. To avoid bias, data less than half of the total cases in the cohort will not be performed in multiple regression and subgroup analysis. To perform the comorbidities status, considering each of the comorbidity was indicated a significant association with outcomes in univariable logistic regression, we included the numbers of comorbidities (T2DM, Hypertension, cardiovascular disease, chronic kidney disease) into the multivariable analysis, rather than each individual. Depending on the multivariable logistic regression in all patients, age and blood glucose were closely correlated with the outcomes, therefore, were chosen as confounders to adjust during the analysis of the T2DM cohort. The liner regression and logistic regression were performed in adjusting in continuous variables and categorical variables, respectively. P < 0.05 was considered statistically significant. Statistical analysis was carried out with SPSS (IBM Corp, Armonk, NY, USA), version 23.0.

Demographics and characteristics of the study population
From January 19 to April 09, 2020, 441 cases were collected, among them, 34 were excluded for the following reasons: 18 patients who were re-detected positive, 4 patients whose prognosis could not be tracked, 3 patients with few data, and 9 patients in hospital stay no more than 3 days. Finally, 407 confirmed patients were eligible in our study (Fig. 1) [7.0%], P < 0.001) was seen in T2DM cohort. Proportion of gender had no difference between two groups (P > 0.05) ( Table 2).
For the clinical observational indices, even greater rate of corticosteroid was utilized in T2DM cohort  (Table 2).
Based on the aforementioned significant difference parameters between groups, the numbers of comorbidities and further seven factors had chosen into multivariable analysis, which contains: age, blood glucose, lymphocytes%, r-GGT, BUN, CRP, and the use of corticosteroid (yes/no). In multivariable analysis, age and blood glucose were still significantly associated with mortality (age: OR 1. 23 (Fig. 2).     (Table 3). After adjusting the age and blood glucose levels, no difference in fever days, hospital LOS, mortality, and invasive ventilation utilization between groups, but with a dramatically lower rate of ICU admission (1 [5.6%] vs. 14 [43.8%], adjust P = 0.005) in metformin patients compared with the non-metformin group. Interestingly, the dose of metformin (per day) in pre-admission showed a negative association with the rate of ICU admission after controlling (OR 0.04 [95% CI 0.00, 0.99]; adjust P = 0.049), as shown in Table S1. Whereas, no pronounced difference in clinical observational indices and outcomes between insulin and non-insulin usage before admission (Fig. 3).

Hospital metformin or insulin usage
During hospitalization, 22 of 50 cases with T2DM had metformin. Patients had in-hospital metformin with a median of 1.0 g per day [IQR 0.92-1.27]. After adjusting the age and blood glucose, there is no difference in all characteristics at admission (adjust P > 0.05) ( Table 4).
However, after the non-divergent baseline, lower CK-MB, LDH, and ddimer, but higher urea levels were observed in hospital metformin users at the endpoint (Table S2) (Table 4). However, under the comparable COVID-19 treatment protocols, higher LDH, more deteriorated inflammation including higher CRP; ESR, and CT scores, but lower r-GGT and urea levels were seen in hospital insulin users compared with noninsulin users at the endpoint (Table S2).
Regarding the clinical observations and outcomes, after adjusting the age and blood glucose, no difference was seen in fever days, hospital LOS and outcomes between in-hospital non-metformin and metformin users (adjust P > 0.05). In comparison, longer fever days  (Fig. 4). However, the dose of inhospital insulin usage had positively association with the rate of invasive ventilation (OR 1.05 [95% CI 0.01, 1.10]; P = 0.027), but no association was found after controlling the age and blood glucose (OR 1.04 [95% CI 0.99, 1.09]; adjust P = 0.088), as shown in Table S1.

Discussion
This retrospective study is aiming to explore the effects of both pre- admission and in-hospital use of metformin and insulin in COVID-19 patients with pre-existed T2DM. We validated the risk of T2DM in COVID-19 patients, which had increased mortality and poor prognosis (ICU admission and invasive ventilation) than non-diabetes. Age and blood glucose were indicated as the risk factors of poor outcomes after multivariable regression. Notable, after controlling the age and blood glucose, pre-admission metformin instructive for declining the ICU admission rate in a dose-dependent fashion in COVID-19 patients with pre-existed T2DM; whereas, in-hospital insulin usage inversely increased the rate of invasive ventilation.
Regarding the mortality in COVID-19 patients with diabetes, several lines of evidence have suggested that diabetes is one of the important risks of mortality in COVID-19 patients [4,21,22]. The underlying mechanism supposed to be the microvascular disease, endothelial dysfunction, severe pneumonia, and inflammatory storm underlies the adverse consequences of COVID-19 [5,23]. Older age and increased CRP Table 3 Characteristics, baseline laboratory indices, clinical observational indices, and outcomes in pre-admission metformin or insulin users.  PCT, procalcitonin; CT, computed tomography; COVID-19, coronavirus disease 2019. Data were n (%) or median (IQR). P-values were shown as χ2 test or Fisher's exact test or Mann-Whitney-U test. P < 0.05 was considered significant (bolded). a The concentration of metformin presenting in "gram" per day. b The concentration of insulin presenting in "unit" per day.
were considered contribute to the increased mortality in COVID-19 patients with diabetes [16]. Uniformly, in our study, older age and more deteriorated inflammation were seen in T2DM. Moreover, exacerbated hepatic-renal functions were observed in T2DM, which may also contribute to the dramatically increased mortality and poor prognosis in comparison with non-diabetes. It was suggested that un-well glycemic control was associated with poor outcomes compared with those of wellcontrolled patients [24,25]. In accordance, we demonstrated that age and admission blood glucose were the independent risk factors of mortality and poor prognosis in COVID-19 patients. In order to eliminate the influence of these two factors on the outcomes, we had controlled the age and blood glucose in the analysis of metformin and insulin subgroups.
Metformin is considered the first-line T2DM treatment agents nowadays [26]. Initially, it was found from the plant Galega officinalis and used for treating flu and malaria [26]. In recent years, metformin has attracted attention again for its antiviral effect; several studies have suggested its anti-inflammation roles in several pathology situations [11,12]. Recently, Sharma et al. suggested that the possible machination of metformin on COVID-19 might ascribe to the declining of proinflammatory and profibrotic states, release acute lung injury via activation of adenosine monophosphate-activated protein kinase [10].
Notwithstanding, in our study, analogous to the study of Chen et al. [16], pre-admission metformin usage had no impact on mortality. Whereas, the attenuated inflammatory response (decreased neutrophils (%)) with benefits on immune-modulatory effects (increased lymphocytes, lymphocytes (%)), decreased LDH were observed in pre-admission metformin in our study. Besides, pre-admission metformin was negatively associated with ICU admission in a dose dependent fashion, independent of age and blood glucose. These results implicate that preadmission metformin usage may have the protecting effects of COVID-19 patients with T2DM without affecting the mortality.
In comparison, in-hospital metformin had no impact on mortality or poor prognosis in our study. Whereas, the decreased LDH, CK-MB at the endpoint were observed, may involve in the cardiac protecting function, which has been raised in other literature [27]. Besides, a decreased d-dimer in hospital metformin cases was found, which was considered instructive for ameliorated outcomes of COVID-19 patients [28]. However, it is worth noting that the urea levels were increased in in-hospital metformin users, which could be a risk of acidosis, especially in patients with a history of nephrotic disease. Regarding this, X. Cheng et al. have turned out that in-hospital metformin usage could have the risk of increasing the incidence of acidosis [15]. Hence, the in-hospital metformin usage may have cardiac protecting and ameliorated coagulating function, but the monitoring of renal function is needed for metformin patients, and severe nephrotic patients should use metformin sparingly.
As for the COVID-19 patients with diabetes who utilized the insulin. A study from New York City implicated that an increased risk of mortality in confirmed COVID-19 cases with diabetes who had prior admission insulin treatment [29]. Similar results were found in 120 COVID-19 patients with diabetes in Wuhan China [16]. However, instead of pre-admission insulin usage, we found in-hospital insulin usage was closely associated with a greater rate of invasive ventilation. And the greater rate of invasive ventilation might among the consequences of deteriorated inflammation (greater CT score, higher CRP, ESR levels) and higher LDH at the endpoint in insulin users compared with those of non-insulin users. A diabetic mice model study indicated that the ACE2 expression increased under insulin treatment [13], which might result in an exacerbated outcome. Interestingly, the r-GGT and urea levels were lower in-hospital insulin group, which suggests that insulin might have less risk of haptic-renal impairment, but the mechanisms are still unclear.
The limitations of this study should be noted. First, due to the limitation of diabetes samples, our results may have been obtained by chance; the results must be confirmed in a more extensive study. Second, the complexity of anti-diabetic agents, purely single antidiabetic medicine groups or purely pre-admission/in-hospital antidiabetic medicine groups were hard to achieve, and the results might be confounded by other antidiabetic medicine. Last, data of HbA1c were not enough to analyze and the confounders we included were limited owing to the sample sizes, may also influence the outcomes. Large and long-term randomized controlled trials with follow-up evaluation are needed to

Conclusion
In this study, the hospital confirmed COVID-19 patients with T2DM had adverse outcomes, which might among the consequences of deteriorated inflammation, impaired immunity, and exacerbated hepaticrenal function than non-diabetes. Among COVID-19 patients with T2DM, pre-admission metformin usage was associated with declining the rate of ICU admission, whereas, in-hospital insulin usage had the risk of increasing the invasive ventilation after controlling age and blood glucose. In aggregate, metformin might achieve greater benefit for the COVID-19 patients with T2DM than insulin. Further studies are needed to confirm the present results. BMI, body mass index; ALT, alanine aminotransferase; AST, aspartate aminotransferase; r-GGT, r-gamma-glutamyl-transferase; LDH, lactate dehydrogenase; BUN, blood urea nitrogen; CK, creatine kinase; CK-MB, creatine kinase-myocardial band; MYO, myoglobin; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; PCT, procalcitonin; CT, computed tomography; COVID-19, coronavirus disease 2019. Data were n (%) or median (IQR). P-values were shown as χ2 test or Fisher's exact test or Mann-Whitney-U test. P < 0.05 was considered significant (bolded). a The concentration of metformin presenting in "gram" per day. b The concentration of insulin presenting in "unit" per day.

CRediT authorship contribution statement
Xueqi Cheng, Xingjian Zhou designed the study. Xueqi Cheng, Siyi Xin, analyzed and interpreted the data and completed the final manuscript. Xingjian Zhou revising it critically for important intellectual content. Yaqi Chen, Leyu Li, Wanjun Chen, Wenjia Li, Baoan Zhou, Chenxia Li, Gong Yu, Fei Li, Peng Duan, collected and sorted the data and drafting the article. All authors read and approved the final manuscript. Xinjian Zhou has primary responsibility for the final content.

Declaration of competing interest
No financial or commercial conflict of interest exists.