Severe COVID-19 Associated With Liver Injury in Patients Without Preexisting Liver Disease

Objective: Coronavirus disease 2019 (COVID-19) is known to disturb liver function tests (LFTs). Not much literature is available regarding the effect of COVID-19 on LFTs in patients without preexisting liver disease. The study aimed to find the effect of COVID-19 in these patients. Methods: This was a single-center, observational study with 142 patients who were admitted with COVID-19 during three months. Seven patients were excluded due to the presence of chronic liver disease. Results: A total of 135 patients were included in the study aged between 18 and 95 years (mean 57.7 ± 15.6); among them, 93 were males (68.9%). Hypertension was present in 74 patients (54.8%), and diabetes was present in 48 patients (35.6%). Fever was the chief complaint in 112 patients (83%), followed by dyspnea in 93 patients (68.9%) and cough in 79 patients (58.5%). Elevated aspartate aminotransferase (AST) was seen in 35 patients (26%), gamma-glutamyl transferase (GGT) in 43 patients (32%), alanine transaminase (ALT) in 18 patients (24%), alkaline phosphatase in 19 patients (14%), bilirubin in six patients (4%), and low albumin in 27 patients (20%). Severe COVID-19, when compared with mild to moderate disease, was associated with elevated AST > two-time upper limit normal (2ULN) (p = 0.002), GGT > 2ULN (0.026), and lower albumin (p = 0.020), higher systemic inflammatory response syndrome (SIRS) (0.045), raised procalcitonin (p = 0.045), higher ferritin (p = 0.005), lower pO2 (p = 0.044), and higher Sequential Organ Failure Assessment score (SOFA) (p = 0.002) pointing to the inflammatory response as cause of liver injury. Factors predicting mortality with COVID-19 were assessed, which showed that direct bilirubin (p = 0.001), low albumin (p = 0.013), tachypnea (0.002), and leukocytosis (<0.001) were independently associated with increased COVID-19-related mortality. Conclusion: Patients suffering from COVID-19 have evidence of liver injury, which appears to be secondary to an inflammatory response that correlates with the severity of COVID-19.


Introduction
The eruption of coronavirus disease-19 (COVID-19) is of great concern globally and has become a substantial challenge for physicians and public health authorities alike [1]. It has affected more than 100 million people worldwide and has been the cause of death of more than two million infected individuals. In Pakistan alone, the number of confirmed cases has surpassed 700,000 [2], crossing a death toll of over 11,000. Although when compared to other countries around the world the number of death is not as high, likely due to the immediate precautions taken by the country's government from an early stage and the possibility of a large number of infected individuals being asymptomatic [3], the number is still significant enough to be seen as a dangerous amount. The virus's rapid spread across the globe has largely been due to its alarming rate of infection, primarily through respiratory droplets and secondarily through physical contact [4]. Its usual clinical manifestations in the general population are a dry cough, fever, and flu-like symptoms, which can develop into acute respiratory distress syndrome and multiple system organ failures [5].
Multiple studies have indicated that there is a progression of deranged liver functions in the infected individuals with COVID-19 previously diagnosed to have liver disease [6]. Fewer studies are showing the deteriorating effects of liver function in those without any preexisting liver-related illness. Therefore, it is essential to further investigate these changes in individuals who do not have any prior history of acute or chronic liver disease to better understand the development of abnormal liver functions during the period of infection.
To investigate this further, we documented the derangement of liver function tests (LFTs) among the patients admitted with COVID-19, followed each case, seeing parameters from inflammatory markers to 1 1 2  1  1   2  2  2  2  2 renal functions, coagulopathy, type of admission ward versus ICU, treatment provided, and outcomes.

Materials And Methods
The study was conducted at Dr.  Continuous variables were expressed as mean and standard deviation, and analysis was performed by Mann-Whitney U test. Categorical data were analyzed using Fisher's exact test. A p-value of <0.05 was considered statistically significant. The statistical analysis was done on the IBM SPSS software version 20.0 (IBM Corp., Armonk, New York).
Liver enzyme derangements were seen in 32% of patients admitted and treated for COVID-19 and correlated with disease severity and outcome as evident in Table 2  Derangement of AST with more than two times ULN (>80) was compared with both mild/moderate and severe COVID as shown in Table 3  Factors predicting mortality with COVID-19 were also assessed as mentioned in Table 4. Stepwise linear regression analysis of continuous variables showed four variables such as direct bilirubin (p = 0.001), low albumin (p = 0.013), increased respiratory rate (0.002), and raised WBC count (<0.001), which were independently associated with increased COVID-19-related mortality.

Discussion
Acute liver injury is defined as a derangement in LFTs due to an acute insult in a previously healthy liver. Many studies have reported patients developing liver injury following COVID-19 infection [7][8][9]. Reasons for this liver injury are still unsure, but multiple studies show it may be due to the expression of angiotensinconverting enzyme II (ACE-II), which is identified as the main receptor for COVID-19 entry into cells. This could suggest that COVID-19 could have a direct effect on the hepatic system along with a sequalae to the ongoing inflammatory response [10,11]. This could also be due to the difference in population and how ACE-II expression analysis differs between ethnicities [12], but further studies are required. Another hypothesis suggests immune-mediated liver injury, i.e., cytokine storm, causing the release of multiple proinflammatory cytokines particularly IL-6 that causes both pulmonary and extrapulmonary damages including the liver [8]. Patients affected with severe disease often have hypoxia and hypotension, and that might also be a causative factor to cause acute liver injury [13]. Lastly, many of the critically ill patients are on a variety of medications and parenteral nutrition, which may also contribute to the ongoing liver insult. All of these are the most commonly highlighted reasons for causing both direct and indirect hepatic damage in COVID-19-affected population.
As hypertension was the most common comorbidity in patients within our study, drug history might be significant in being a cause of worsening deranged liver enzyme levels. The three most common medications patients were using before admission were diuretics (25.9%), angiotensin-converting enzyme (ACE) inhibitors (23%), and beta-blockers (15.6%), followed by statins (14.9%). There is not enough evidence to prove whether the indication of using these drugs before hospitalization is truly significant to deranging liver enzymes [14].
The mean values of LFTs were above the normal range as seen within this study ( Table 2). The liver function enzyme levels of patients with severe cases of COVID-19 were more deranged as compared to the liver enzyme levels of patients with mild or moderate cases. These enzymes, when elevated together, are strong indicators of liver injury. Total bilirubin (TB) and ALP levels were elevated in only a few cases, but overall remained under the normal ranges (mean TB = 0.95, mean ALP = 91.59). These markers in numerous studies have had different results, some in which TB is markedly increased [15,16] and others that coincide with our study [17]. Serum albumin levels were also not severely affected, but a mean decrease was seen in severe cases (Alb = 3.49), which is closer to the lower normal limit as compared to the mild/moderate cases (Alb = 3.82).
AST levels greater than 80 showed more deranged infective markers typically seen in COVID-19 cases as shown in Table 3. AST > 80 showed severely deranged results (mean lactate = 2696.26, mean D-dimer = 2663.31, mean ferritin = 2354.89, mean PCT = 9.24). Although these markers were not as severely deranged in patients with AST < 80, they were deranged nonetheless, showing how this presence of injury to the liver could be secondary to an inflammatory response. This has been seen in multiple studies within our subcontinent [17] and across the globe [18].
SOFA and APACHE II scores were also calculated to assess the prediction of mortality and to cross-reference with the severity of liver enzyme derangement. In severe cases, SOFA and APACHE II scores were markedly raised (mean SOFA = 4.33, mean APACHE II = 12.83), almost three times higher as compared to the scores calculated in mild/moderate cases. These calculations, including the significant increase in infective markers and LFTs, may show an overall systemic effect by the COVID-19 virus and may indicate a positive correlation between an increase in viral, liver injury, and inflammatory response [15].
In our study, COVID-19 mortality was associated with four biomarkers: serum albumin, direct bilirubin, total leucocyte count, and respiratory rate. Serum albumin has been proven to be an acute phase reactant. Under conditions of increased oxidative stress, it can undergo irreversible oxidation leading to increased cellular injury [19]. Patients who were severely affected, admitted to ICU, and those who required ventilatory support reported to have lower serum albumin levels than the mild to moderately affected population (3.37 vs 3.83, p = 0.013). Bilirubin levels are reflective of acute liver injury, and direct bilirubin was increased among the nonsurvivors of our study population (0.29 vs 1.19 p = 0.001). Chai et al. [20] showed that the expression of ACE-2 receptors on cholangiocytes was equivalent to that of the type II alveolar cells; however, the number is 20 times less in the hepatocytes. A study done in China by Liu  showed similar results as well [23].
There are several strengths to this study. First, this study reflects the association between liver derangement and COVID-19 severity, as seen by the deranged LFTs along with COVID-19 inflammatory markers. Second, in this study by removing patients with preexisting and/or newly diagnosed chronic liver disease, we were able to see how COVID-19 affects naïve liver and see the clinical course. Lastly, this study also substantiates the use of AST, GGT, and albumin as COVID-19 severity markers as well as albumin and direct bilirubin as mortality markers along with raised leucocyte count and tachypnoea.
The limitations of this study are as follows: a sample size of 135 cases is smaller as compared to other studies that have been conducted but is still relevant to show a distinct derangement in liver enzyme levels. Furthermore, the cases were all from a single hospital in a certain demographic of Pakistan, which may not show a proper representation of the disease and its effect on liver enzymes on a country-wide basis. Lastly, the laboratory results that were used in this study were results seen on admission, and so the effect of the progress on said laboratory results during a hospital stay has not been utilized.

Conclusions
In conclusion, COVID-19 has multisystemic effects. Liver injury caused by COVID-19 stems majorly from inflammatory response due to cytokines. The degree of liver damage is reflective of the severity and increased mortality, hence showing evidence that liver function enzymes have the potential to be utilized as surrogates to dictate the prognosis of COVID-19 severity.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.