Association between non‐alcoholic fatty liver disease with the susceptibility and outcome of COVID‐19: A retrospective study

Abstract This study aims to evaluate the effect of non‐alcoholic fatty liver disease (NAFLD) on the susceptibility and consequences of coronavirus disease 2019 (COVID‐19). We retrospectively collected data from 218 adult COVID‐19 patients who showed no evidence of excessive alcohol consumption and underwent abdominal ultrasound examinations. Of these patients, 39.4% patients had been diagnosed with NAFLD, which indicates a much higher prevalence of NAFLD than that reported in the general population. Significantly elevated white blood cell count (p = 0.008), alanine aminotransferase (p = 0.000), aspartate aminotransferase (p = 0.006) and C reactive protein (p = 0.012) were found in the patients with NAFLD. These patients also had significantly higher proportions of hypertension (p = 0.006) and diabetes (p = 0.049) than the non‐NAFLD cases. No significant differences existed in the severity, mortality, viral shedding time and length of hospital stay between patients with or without NAFLD in the sample population. However, subgroup analyses found that in patients with normal body mass index (BMI), NAFLD sufferers were more likely to experience a severe event (30.0% vs 11.5%, p = 0.021). Kaplan‐Meier curve (log‐rank p = 0.017) and Cox regression (HR = 3.26, 95% CI: 1.17–9.04, p = 0.023) analyses confirmed that before and after adjusting for gender, age and comorbidities, NAFLD patients with normal BMI had a higher incidence of suffering severe events. People with NAFLD may have a higher proportion of COVID‐19. NAFLD may be correlated with the severity of COVID‐19 patients in the normal BMI group.

COVID-19. 9 Liver impairment has been reported in up to 50% of patients with COVID-19, and more than 60% of severe cases. 10 Histological examination in the liver of a COVID-19 patient showed moderate micro-vesicular steatosis and mild lobular and portal activity. 11 However, the causes of liver injury in COVID-19 cases and the effects of liver-related comorbidities on the susceptibility and outcomes of COVID-19 remain unclear.

| Study Design and Participants
The study protocol was subject to approval by the institutional ethics board of the Second Xiangya Hospital of Central South University (No. 2020001). We retrospectively collected data from a cohort of subjects who had laboratory-confirmed  were patients in the Public Health Treatment Center of Changsha, China, before 14 March 2020. Fatty liver was diagnosed based on the ultrasound parameters (such as parenchymal brightness, deep beam attenuation and liver-to-kidney contrast) by abdominal ultrasonography with a 3.5-MHz transducer (S9, SonoScape, China).
Ultrasonography was performed by the experienced radiologists for medical indications in the Public Health Treatment Center of Changsha, China. We used the following criteria to diagnose NAFLD: (1) The presence of fatty liver; (2) The absence of excessive alcohol consumption (average alcohol intake ≥30 g/day for men and ≥20 g/ day for women).

| Data Collection
Two authors carefully collected and reviewed the individual medical records of the patients. Detailed information on demographic data, body mass index (BMI), underlying comorbidities, medical history, symptoms, laboratory parameters, chest computed tomographic (CT) scans and outcomes were recorded. For BMI data, the medical records of 18 patients did not include height and weight.

| Statistical Analysis
Because the continuous variables in this study were non-normal distributed, we used the Fisher's exact test (χ2 test) and Mann-Whitney F I G U R E 1 Flow chart of the study population test to compare the differences between the categorical variables and the continuous variables, separately. The Kaplan-Meier (KM) method and the log-rank test were applied to assess the association between NAFLD and the outcomes. Cox regression was conducted to further evaluate the effect of NAFLD after adjusting for other risk factors. All analyses were performed using IBM SPSS Statistics version 26.0 software.
The two patients who died did not suffer from NAFLD.
Next, to investigate whether the effect of NAFLD on the outcomes of COVID-19 patients was dependent on gender, age, BMI or comorbidities, we divided our patients into subgroups according to these factors. We found that in patients with normal BMI (18.5 ≤ BMI <24 kg/m 2 ), NAFLD patients were more likely to develop severe events than those without NAFLD (30.0% vs 11.5%, p = 0.021).
However, no significant differences were found in patients with low (< 18.5 kg/m 2 ) or high (≥ 24 kg/m 2 ) BMI values ( Table 2) (Table 3). Thus, a KM analysis was performed and confirmed that NAFLD patients were more likely to experience a severe event (log-rank p = 0.017; Figure 2). After adjusting for gender, age, hypertension, cardiovascular disease, diabetes and chronic liver disease, NAFLD is significantly correlated with the severity of COVID-19 (HR =3.26, 95% CI: 1.17-9.04, p = 0.023; Table 4) in the normal BMI group.
Moreover, the results showed that in non-elderly patients (< 60 years), those with NAFLD had a higher incidence of severe events (21.2% vs 10.5%, p = 0.036; Table 2). Subsequently, a KM analysis confirmed that non-elderly NAFLD patients had a significantly elevated risk of suffering a severe event compared with those without NAFLD (log-rank p = 0.035; Figure S1). However, after adjusting for gender, hypertension, cardiovascular disease, diabetes and chronic liver disease, NAFLD had no significant influence on the likelihood of the severe events in non-elderly patients (HR =1.79, 95% CI: 0.72-4.44, p = 0.208; Table S2).
No significant effects of NAFLD were found on the virus shedding time or length of hospital stay in the patient cohort. Thus, we performed subgroup analyses according to gender, age, BMI and the common comorbidities. Results showed that in elderly patients, the virus shedding time was significantly lower in NAFLD patients than in non-NAFLD individuals (16 days [4-38] vs 23 days , p = 0.011; Table 2). In terms of laboratory findings among elderly patients, alanine aminotransferase was conspicuously higher in patients with NAFLD, compared to those without (25.0 U/L [12.8-58.1] vs 15.6 U/L [4.9-69.4], p = 0.002; Table S1).

| DISCUSS ION
This study investigated the susceptibility of patients with NAFLD to SARS-CoV-2 and the association between NAFLD and outcomes of COVID-19. We found that individuals with NAFLD may be more susceptible to SARS-CoV-2 than those without NAFLD. In patients with normal BMI, those with NAFLD who were infected by COVID-19 may have a higher proportion of developing severe diseases than other patients.
Despite no evidence of increased liver uptake of SARS-CoV-2 in NAFLD patients, 25 in our study, the COVID-19 cases with NAFLD had higher levels of alanine aminotransferase and aspartate aminotransferase than the other patients. Moreover, patients with abnormal liver tests are at a higher risk of developing to the severe events. 26 It has been reported that patients with NAFLD have a higher risk of disease progression, 21 while NAFLD patients with an increased fibrosis-4 index and NAFLD fibrosis score are at a higher risk of suffering a severe event due to  However, the influence of NAFLD on the COVID-19 in subgroups of age, gender, BMI and comorbidities remains unclear.
The prevalence and severity of NAFLD vary between different age groups. 15,28 A large sample study in China found that, compared to non-elderly adults (18-59 years), the elderly (≥ 60 years) showed a lower overall prevalence of NAFLD, but a higher proportion of NAFLD accompanied with diabetes, hypertension and hyperlipidaemia. 29 Elderly NAFLD patients are more likely to develop nonalcoholic steatohepatitis and fibrosis than non-elderly patients. 30 Moreover, NAFLD is considered to be associated with increased mortality in elderly people. 31 Interestingly, in the non-elderly group The susceptibility to COVID-19 of NAFLD sufferers increases linearly with BMI. It increases 5-to 10-fold in obese patients and by a factor of 10-to 14-fold in the morbidly obese. 32 However, a significant proportion of NAFLD patients have relatively normal BMI, and it has been reported that obese NAFLD has different clinical characteristics from non-obese NAFLD. 33 Therefore, in this study, we performed a subgroup analysis on BMI and found that in patients with a normal BMI (18.5-24 kg/m 2 ), NAFLD was associated with an increased risk of severe disease. However, we did not find similar results in patients with higher and lower BMI, which may be related to their different metabolic states. This is an interesting result, but well-designed studies with larger sample sizes are required to investigate this assumption.
This study contains several limitations. Firstly, we analysed the effect of NAFLD on the susceptibility and outcome of COVID-19 in one of the nearest provincial capitals to Wuhan, but we did not have the data on the prevalence of NAFLD in Changsha, China. Also, in most of the subgroups that were created according to the common comorbidities, no significant differences were found. However, the results did show certain trends. Univariate analysis indicated that non-elderly NAFLD patients had a higher incidence of severe events.
Nevertheless, after adjusting for gender, hypertension and BMI, the difference became statistically insignificant. This may be because of the small sample size and the high incidence of comorbidities.
Another limitation is that in the subgroup analysis on BMI, we only divided our patients into three groups. Overweight and obese patients were pooled together in one group (BMI ≥24 kg/m 2 ), due to the small number of obese patients (BMI ≥28 kg/m2). Finally, as this study was retrospective, data regarding abdominal adiposity was not available. Therefore, well-designed studies with larger sample sizes are still needed to more clearly demonstrate the association between NAFLD and the outcomes of COVID-19.
In summary, people with NAFLD may have a higher proportion of SARS-CoV-2 infection, with a higher rate of liver injury, as well as a higher incidence of severe COVID-19 in normal-weight patients. People who have NAFLD may need stronger protection and more aggressive treatment to decrease the risk of infection, reduce the chance of liver injury and improve the outcome.
However, due to the small sample size, especially for elderly patients, further studies with a larger sample size are required to confirm these findings.

ACK N OWLED G EM ENT
Not available.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Abbreviation: NAFLD, non-alcoholic fatty liver disease.