Laboratory Profile of COVID-19 Patients with Hepatitis C-Related Liver Cirrhosis

Patients with cirrhosis are known to have multiple comorbidities and impaired organ system functioning due to alterations caused by chronic liver failure. In the past two years, since the COVID-19 pandemic started, several studies have described the affinity of SARS-CoV-2 with the liver and biliary cells. Considering hepatitis C as a significant independent factor for cirrhosis in Romania, this research was built on the premises that this certain group of patients is susceptible to alterations of their serum parameters that are yet to be described, which might be useful in the management of COVID-19 in these individuals. A retrospective cohort study was developed at a tertiary hospital for infectious disease in Romania, which included a total of 242 patients with hepatitis C cirrhosis across two years, out of which 46 patients were infected with SARS-CoV-2. Stratification by patient weight and COVID-19 status identified several important laboratory serum tests as predictors for acute-on-chronic liver failure and risk for intensive care unit admission. Thus, white blood cell count, lymphocyte count, ferritin, hypoglycemia, prothrombin time, and HCV viral load were independent risk factors for ACLF in patients with COVID-19. High PT, creatinine, BUN, and HCV viral load were the strongest predictors for ICU admission. Inflammatory markers and parameters of gas exchange were also observed as risk factors for ACLF and ICU admission, including procalcitonin, CRP, IL-6, and D-dimers. Our study questions and confirms the health impact of COVID-19 on patients with cirrhosis and whether their laboratory profile significantly changes due to SARS-CoV-2 infection.


Introduction
The clinical manifestations of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) range from asymptomatic infection to life-threatening illness [1]. It is still uncertain why some patients infected with SARS-CoV-2 evolve to develop severe, lifethreatening forms of coronavirus disease 2019 (COVID- 19), whilst others have a mild or even asymptomatic clinical history. Numerous risk factors for severe COVID-19 illness have been discovered, including sociodemographic characteristics and concurrent disorders [2]. Hepatic involvement is common in SARS-CoV-2 infection, presenting clinically as increased liver function tests or sudden hepatic failure [3]. In patients with pre-existing chronic liver diseases or cirrhosis, SARS-CoV-2 infection has had inconsistent effects. Previously published small-scale studies from tertiary referral centers suggested that patients with cirrhosis who were infected with SARS-CoV-2 died at a rate of up to 40% [4]. However, other studies have shown that patients with cirrhosis who tested positive for SARS-CoV-2 infection died at a rate equivalent to those hospitalized with cirrhosis-related complications who did not test positive for SARS-CoV-2 infection [5].
In the 2018 census, more than 37 thousand new cases of hepatitis C were recorded in countries of the European Union [6], bringing the overall prevalence to 19 million cases [7] and over 70 million cases globally [8]. Since the pandemic began in 2019, there has been concern that pre-existing chronic liver disease (CLD) may predispose individuals to poor outcomes following SARS-CoV-2 infection, particularly given the overlapping risk factors for severe COVID-19 and CLD, such as advancing age, obesity, and diabetes [9,10]. The presence of a pre-existing liver condition associated with SARS-CoV-2 infection, such as hepatitis C virus (HCV), may be critical for patient prognosis, since chronic hepatitis C continues to be a health burden in a number of European nations. While the precise effect of COVID-19 on the liver is unclear, abnormalities in liver biochemistry are common in COVID-19 cases, occurring in 15-65% of SARS-CoV-2-infected individuals [11].
In our present research, we focused on determining the possible changes in serum parameters and liver function markers, which show abnormalities in routine laboratory testing, and the relationship between cirrhosis and the severity of COVID-19. We describe the possible relationship between the clinical course, laboratory findings, and the outcome of 46 patients admitted in our department with documented cirrhosis and SARS-CoV-2 infection.

Materials and Methods
We conducted a retrospective cohort study to determine the risk of severe COVID-19 infection in patients with post-hepatitis C cirrhosis who had been infected with SARS-CoV-2. We used clinical and laboratory data from patients hospitalized at Timisoara's Infectious Diseases and Pulmonology Hospital, "Victor Babes". The data gathering period was from 1 April 2020 to 1 December 2021. We discovered 46 instances of SARS-CoV-2 infection among 242 patients with cirrhosis who were being followed at our clinic.
Patients with hepatitis C cirrhosis exhibit unique clinical signs and symptoms such as ascites, abdominal wall vascular collaterals, hypertrophic osteoarthropathy, clubbing, and asterixis on physical examination [12]. Weakness, weariness, anorexia, and weight loss are all constitutional signs [13]. The hospitals also conducted ultrasonography and liver biopsies, and microscopically interpreted sample slides revealed evidence of regenerating nodules of hepatocytes, surrounded by fibrous connective tissue that bridges between portal tracts, confirming the diagnosis of cirrhosis. The inclusion criteria were set for patients diagnosed exclusively with non-alcoholic cirrhosis and non-overlapping diagnosis codes of chronic hepatitis B or other primary or secondary causes of cirrhosis. The diagnosis in all patients was priorly established through liver biopsy, so all patients with a secondary cause of cirrhosis other than chronic hepatitis C infection were excluded from the study after proper histopathologic immunohistochemical analysis of the liver samples [14,15].
Data of patients with post-hepatitis C cirrhosis were compared before and after SARS-CoV-2 infection using the IBM SPSS v.26 statistical software. The χ 2 test and Fisher exact test were used for comparing proportions of categorical variables depending on the expected frequency assumption. The unpaired Student's t-test was used to compare average values of normally distributed data for the COVID and non-COVID study groups. We utilized the Mann-Whitney U-test to compare median values of continuous variables and the Shapiro-Wilk test to measure the departure of data from normality. A multivariate analysis was performed to determine independent risk factors for developing ACLF and ICU admission.

Results
Patients with hepatitis C cirrhosis were divided in two groups based on their COVID-19 status at the time of study. A total of 46 patients were included in the COVID group and 196 in the non-COVID group. Table 1 describes the general characteristics of the patients involved in the study. It was noted that patients in the COVID groups had significantly lower average BMI values (p-value = 0.035), while the proportion of underweight patients was higher in the same COVID group (p-value = 0.041). More than half of our patients with cirrhosis had a history of portal hypertension, among other complications specific to their chronic liver disease, whereas hepatic encephalopathy was observed in a statistically significant higher proportion in patients infected with SARS-CoV-2 (54.3% vs. 36.2%, p-value = 0.023). The study participants were also grouped by the Child-Pugh score, having no significant difference in proportions of Child-Pugh A, B, or C. However, we noticed significant higher number of cirrhosis patients that developed acute-on-chronic liver failure while being infected with SARS-CoV-2 (19.6% vs. 5.1% p-value < 0.001). Similar findings were observed in the rate of ICU admissions in patients with COVID-19 (26.0% vs. 8.2%, p-value < 0.001), and in the mortality rate (15.2% vs. 6.1%, p-value = 0.039).
The inflammatory profile was analyzed only in patients positive for SARS-CoV-2 infection. Therefore, data during the acute phase of COVID-19 were obtained from 46 patients, while the second evaluation was performed in 39 patients, since seven of them did not survive. Alterations of the studied parameters can be observed in Table 3. The majority of inflammatory markers were on average higher than the upper limits of the normal range both at the initial evaluation when patients were confirmed with COVID-19, as well as at 4 weeks after being tested negative for SARS-CoV-2. An important finding is the higher mean value of procalcitonin in patients at the second evaluation (0.5ug/L vs. 1.2ug/L, p-value < 0.001), signifying the risk of bacterial infection after the acute viral infection. The other inflammatory markers were elevated in patients during the acute phase of COVID-19, including CRP levels (56mg/L vs. 12mg/L, p-value < 0.001), IL-6 (49pg/mL vs. 17pg/mL, p-value < 0.001), fibrinogen (5.1g/L vs. 3.7g/L, p-value < 0.001), and D-dimers (331ng/mL vs. 262ng/mL, p-value < 0.001). Arterial blood gases also showed significant alterations from the normal range. Patients with COVID-19 had statistically significant higher levels of arterial CO 2 (49mmHg vs. 43mmHg, p-value < 0.001) and lower O 2 saturation (89% vs. 92%, p-value = 0.028).
The clinical profile was also evaluated at 4 weeks after SARS-CoV-2 infection (Table 4), without any important findings, except pleural effusions that were found in a significantly increased number of patients (15.2% vs. 33.3% at 4 weeks, p-value = 0.049).  Figures 1 and 2 describe the association of laboratory parameters with ACLF and ICU admission in underweight and normal weighted patients with cirrhosis from the two study groups, and the association of inflammatory markers and arterial blood gases with ACLF and ICU admission in underweight and normal weighted patients with cirrhosis from the two study groups, respectively. The odds ratio and 95% confidence intervals are developed based on BMI values that initially showed significant differences between study groups (COVID and non-COVID). The multivariate analysis described significant interactions between WBC and ferritin in all subgroups as predictors for the risk of developing ACLF. Other significant associations with ACLF were observed only in patients with COVID-19 with elevated lymphocytes and ALT levels, respectively, and low fasting glucose levels. Elevated PT, creatinine, BUN, and HCV viral load were associated with an increased risk for ICU admission in cirrhosis patients, regardless of their SARS-CoV-2 status, although the risk was higher once COVID-19 was diagnosed, and stratification by BMI indicated that underweight patients had a higher chance of being admitted to the ICU. Lastly, underweight patients with cirrhosis and COVID-19 were in the group with the highest risk factor for developing ACLF and ICU admission, where procalcitonin levels along with CRP, IL-6, and fibrinogen were all significant independent risk factors. Additionally, d-dimers, pCO 2 , and SaO 2 were all independent risk factors for ICU admission, but not for ACLF. Table 3. Dynamics in inflammatory markers and blood gases of patients with cirrhosis at 4 weeks after SARS-CoV-2 infection.

Discussion
By the nature of this study, it is one of the first to describe a thorough evaluation of laboratory data in cirrhosis patients after a SARS-CoV-2 infection and to present a stratified risk analysis for developing acute-on-chronic liver failure and being admitted to an intensive care unit, while recording the nutritional status of the patient and SARS-CoV-2 infection using serum parameters. During the investigation of laboratory profiles in our cohort, we observed many instances of blood parameters' departure from normality, where both COVID and non-COVID patients with cirrhosis showed abnormal values such as preexistent anemia, hypoglycemia, elevated liver enzymes, poor liver function tests, nutritional deficiency, kidney failure, and hypolipidemia. Similar findings were separately described in different studies until present day, such as anemia in cirrhosis [16], hypoglycemia in cirrhosis [17], or hypolipidemia suffered by patients with cirrhosis [18]. However, all these studies did not describe their findings in conjunction with the presence of SARS-CoV-2.
As observed in the current study, patients with cirrhosis exhibit significant alterations in lipid profile compared to healthy individuals. However, SARS-CoV-2 infection in these patients did not seem to trigger important differences in serum lipid studies. Our research findings reveal that blood lipid levels, including total cholesterol, triglycerides, HDL-C, LDL-C, VLDL-C, and total lipids, are considerably lower in patients with HBV-cirrhosis, indicating hypolipidemia. Because HDL-C is generated in the liver, significant damage to hepatocytes, such as those induced by alcohol use, chronic viral hepatitis, or liver cirrhosis, may result in impaired liver function and a drop in total cholesterol and HDL-C levels, as was previously established [19]. Reduced cholesterol levels in individuals with cirrhosis showed the degree of liver cell damage, which is connected with the impairment of the liver's capacity to synthesize [20].
Although about 30% of patients with cirrhosis have diabetes mellitus (DM) [21], it is not mediated through insulin resistance triggered by obesity. Instead, patients with cirrhosis have a tendency towards malnutrition and cachexia but can develop DM from different metabolic pathophysiologic alterations [22]. Nevertheless, the tendency towards emaciation through sarcopenia [23,24] and the cachexia of the cirrhotic patient was the reason for stratifying data in our research by the BMI of patients involved.
From the earliest reports to the present day, hemostasis abnormalities with a tendency toward hypercoagulability have been observed in COVID-19 patients [25]; in particular, increased D-dimer and fibrinogen levels have been associated with a poor prognosis in hospitalized patients, primarily due to pulmonary embolism or disseminated intravascular coagulation [26]. However, blood samples were often taken after hospitalization, and nothing is known regarding the importance of clotting parameters in identifying individuals with a more severe form of COVID-19, defined by ARDS, and their association to thromboembolic events at their initial presentation. Considering this, we evaluated all the coagulation parameters made available to our lab and found higher than normal fibrinogen, d-dimers, and PT, which were all proven individual risk factors for ICU admission, while fibrinogen alone was also a risk factor for developing ACLF. The evaluation of acute-onchronic liver failure was another aim of this study, since it was observed that significantly more patients with cirrhosis develop this pathology [27].
Lastly, the arterial blood gases were evaluated, both due to the lung and liver affinity of the SARS-CoV-2 [28], and due to the mild hypoxemia encountered by approximately one-third of patients with chronic liver disease [29]. This anomaly can be explained by the findings that arterial venous anastomoses and communications between the portal and arterial circulations, as well as between the bronchial and pulmonary veins, are more likely to be functional in patients with cirrhosis. These anastomoses and communications account for hypoxemia and perfusion defects seen on lung scans in patients with cirrhosis. Shunts reported in individuals with severe liver illness that result in blood gas changes may be a consequence of portal hypertension development, intrapulmonary arteriovenous shunting, or ventilation-perfusion inequality [30].

Conclusions
Our study asks and confirms whether a laboratory profile of patients with cirrhosis significantly changes due to SARS-CoV-2 infection. Based on the presented results, we prove that low BMI is a good stratifier for risk analysis in developing acute-on-chronic liver failure and being admitted to the intensive care unit. Several inflammatory markers, such as procalcitonin, CRP, IL-6, fibrinogen, and d-dimers, show strong interactions with SARS-CoV-2 infection, being significant risk factors for ICU admission. Nevertheless, the laboratory profile of patients with cirrhosis is altered by the intrinsic mechanisms of cirrhosis, and it is still being modified further by the presence of the SARS-CoV-2 virus, indicating that WBC, lymphocyte count, creatinine, ferritin, BUN, and ALT levels rise significantly in patients with COVID-19.  Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the "Victor Babes" Clinical Hospital for Infectious Diseases and Pulmonology in Timisoara, approved on 15 December 2021, with approval number 12570.
Informed Consent Statement: Written informed consent has been obtained from the patients to publish this paper.