CMV reactivation was associated with mortality in liver failure and high glucocorticoid doses intravenously seemed to be the most important risk factor for CMV reactivation in liver failure. Thus, monitoring CMV DNA dynamically may be considered when using glucocorticoid.

The Cytomegalovirus (CMV) is a common β-herpesvirus that establishes lifelong latency following primary infection. After initial infection, CMV persists within the body. The CMV seroprevalence rate in healthy adults reportedly ranges from 40% to 100%, whereas the number in the Chinese population is nearly 100% (Cannon et al., 2010; Marsico and Kimberlin, 2017). Typically, immunity against the virus controls its replication, although intermittent viral shedding can continue to occur in a seropositive immunocompetent person (Gandhi and Khanna, 2004). However, in immunocompromised populations, CMV has become one of the most important opportunistic pathogens, such as in patients undergoing organ transplantation or hematopoietic stem cell transplantation (HSCT), who have the highest risk of developing CMV-associated complications (Singh et al., 2020). CMV diseases exist in a wide spectrum, ranging from asymptomatic viremia to severe systemic and gastrointestinal tract disease, which has been regarded as a major cause of morbidity and mortality in immunocompromised populations (von Muller et al., 2007; Berman and Belmont, 2017).

Systemic inflammation is a key feature of liver failure, and poor disease outcome is closely associated with exacerbated systemic inflammatory responses (Clària et al., 2016). Similar to sepsis, a cytokine storm can occur in liver failure, especially in acute-on-chronic liver failure (ACLF). Owing to associated immune disorders, coinfections with other typical and opportunistic pathogens may be common and related to liver failure prognosis. Previous studies indicated that CMV might be reactivated in patients with cirrhosis, thereby inducing fulminant hepatic failure (Arai et al., 2009; Hsu et al., 2014; Rosi et al., 2015). While numerous studies have reported CMV in patients undergoing organ transplantation or HSCT, few studies are available regarding CMV infection in patients with liver failure (Cho et al., 2019). A retrospective study conducted in China analyzed the risk factors for both CMV and Epstein-Barr virus infection and their influence on the liver function of patients with ACLF-based hepatitis B virus, indicating that 5% (5/98) of the patients presented with CMV DNA (Hu et al., 2018). However, the study was conducted on a small scale, and the influence of prognosis on CMV infection was not confirmed. Currently, the full spectrum of CMV disease, risk factors for reactivation, and long-term clinical implications are not well understood in patients with liver failure. An improved understanding of CMV reactivation in patients with liver failure is important to elucidate the role of virologic surveillance, prophylaxis, and antiviral therapy.

The aim of this study was to describe a 10-year retrospection of patients with liver failure and CMV reactivation and to explore potential risk factors for CMV reactivation and impact on outcome.

While CMV reactivation is a known major cause of morbidity and mortality in transplant recipients, its role as a pathogen in patients with transplant-free liver failure is not well understood. In this study, we showed that CMV reactivation occurred at a high rate (16.7%) in patients with liver failure. Clinical manifestations and liver functions were comparable between the CMV reactivation and non-reactivation groups, resulting in neglected virologic surveillance. However, CMV reactivation may triple mortality in patients with liver failure, thereby demonstrating its important effect on prognosis. Similar to findings of previous studies in other populations, such as patients with rheumatic diseases, we found that the use of high glucocorticoid doses, especially those administered intravenously, might lead to CMV reactivation (Gardiner et al., 2019; Li et al., 2019). To the best of our knowledge, this is the first report to investigate the risk factors of CMV reactivation and their effect on prognosis in patients with transplant-free liver failure. Our study supports the role of CMV virologic surveillance in liver failure.

CMV reactivation has been recognized as a cause of poor prognosis in other patient populations, such as those with hematological malignancies, HSCT, HIV infection, and rheumatic diseases (Strippoli et al., 2006; Fujimoto et al., 2013; Hardie et al., 2013). Furthermore, CMV reactivation was an independent predictor of pneumocystis pneumonia in solid organ transplant recipients, in association with a poor prognosis (Hosseini-Moghaddam et al., 2018). However, the risk factors and outcomes of CMV reactivation in liver failure patients remain to be extensively investigated. Previous study indicated that CMV coinfection was a possible risk factor for the progression of liver fibrosis (Ibrahim et al., 2017). A cytokine storm may be induced, leading to the development of sepsis in patients with liver failure, who are susceptible for opportunistic pathogens such as CMV (Sarin and Choudhury, 2018). In the present study, CMV reactivation occurred in liver failure patients at a high rate of more than 15%, which was higher than that reported in a previous study in China (Hu et al., 2018). The higher rate may be due to the fact that the previous study focused on liver failure caused by Hepatitis B virus, while our study included liver failure with multiple underlying causes. We indicated that liver failure caused by hepatotropic virus infection, such as Hepatitis B, seemed to be less prone to CMV reactivation, compared to other causes. Actually, compared to the patients with liver failure caused by drug-induced or autoimmune, who may receive glucocorticoids, the patients with liver failure caused by hepatitis virus showed lower proportion with CMV reactivation. Noteworthy, both clinical manifestations and liver function were not evidently different between the CMV reactivation and non-reactivation groups, which resulted in neglected CMV detection. In our retrospective study, CMV DNA tests results were unavailable in one third of all patients. It was unclear whether the low numbers of CMV-positive patients were due to the rarity of this combination of events, or because of under-diagnosis owing to the lack of testing in this population.

Immunosuppression is a widely recognized risk factor for CMV reactivation (Tong et al., 2013). Consistent with the published literature, the use of high doses of glucocorticoids was found to contribute to CMV reactivation in liver failure patients (Choo et al., 2019). Furthering previous studies, we explored the effects of different glucocorticoid doses and forms on CMV reactivation. We found that the dosage of glucocorticoids in the CMV-positive group was significantly higher than that in the CMV-negative group. It seemed that the use of intravenous glucocorticoids was more likely to reactivate CMV, while oral glucocorticoid intake with a low dose maintenance had no effect on CMV reactivation (Gardiner et al., 2019). In the present study, CMV reactivation was less frequent in patients with hepatotropic virus infection, which might be associated with lower use of glucocorticoids in liver failure caused by viral hepatitis. Furthermore, a novel finding of the current study is the association of higher white blood cell counts with CMV reactivation in liver failure patients, but such an association may be secondary to a higher dose of glucocorticoids or coinfection with bacteria in CMV reactivation patients. As shown in Table 1 , the co-infection rate in the CMV-positive group was significantly higher than that in the CMV-negative group (57.6% vs 42.4%, P=0.033), which confirmed that immunosuppression would lead to co-infections with opportunistic pathogens. CMV reactivation through NF-κB mediated activation has been found to be linked to co-infection by pathogens through the inflammation, DNA damage and oxidative stress notably (Hummel and Abecassis, 2002; Liu et al., 2016). By inhibiting the NF-κB signaling pathway, co-infection following CMV reactivation will be alleviated (Chen et al., 2018).

The composition of lymphocyte subsets can reflect the immune status of an individual to some extent. In the present study, we explored the changes in lymphocyte subsets in the study population and found that the percentage of CD8⁺ T lymphocytes in the CMV-positive group was significantly higher than that in the CMV-negative group, and that the absolute counts were relatively higher. The results were plausible, because CD8⁺ T cells play a major role in the control of CMV viremia (Attaf et al., 2020). A previous study showed that patients with low B cell counts before HSCT might be at a risk of developing infections (Gernert et al., 2020). Sepsis non-survivors exhibited reduced B cell numbers at the onset of sepsis, compared to sepsis survivors (Krautz et al., 2018). In concordance, comparison of CMV reactivation patients with non-reactivation patients showed significant differences in B cell percentages and B cell numbers in our study. The percentages of B lymphocytes and B cell numbers in the CMV-positive group were significantly lower than those in the CMV-negative group. This result suggested that low B cell counts were associated with a higher possibility of CMV reactivation, which necessitates careful monitoring of patients with low B cell numbers.

There were several limitations in this study. First, this was a single-center, retrospective study with statistical power limited by the sample size. Despite extending our search over a 10-year study period, we could only identify 33 patients with liver failure and proven CMV reactivation for comparison. A further prospective multicenter study with a larger sample size is required for validation. Second, although we enrolled patients who were CMV IgG antibody-positive and demonstrated detectable CMV DNA as CMV reactivation cases, we could not ascertain reactivation in all cases, apart from confirmation of primary disease, because we could have missed the time window when IgG status remained negative for primary disease. However, given the known high seroprevalence rate of CMV, particularly amongst older adults, we assumed that most patients were likely to be CMV seropositive from childhood.¹ Third, we only performed qualitative detection of CMV DNA; hence, we could not analyze the relationship between CMV DNA load and severity of prognosis. PCR methodology will be improved, and we will address this issue in our future studies. Finally, quantification of glucocorticoid use was difficult, as steroid regimens could be complex and often change with varied responses of patients to therapy. However, after examination of glucocorticoid usage in multiple ways, it was evident that CMV reactivation cases had received significantly higher amounts compared to those received by controls.

In conclusion, we found that CMV reactivation was associated with mortality in liver failure. High glucocorticoid doses intravenously seemed to be the most important risk factor for CMV reactivation in liver failure. Thus, monitoring CMV DNA dynamically may be considered when using glucocorticoids in patients with liver failure. Once CMV DNA is identified, early intervention by therapeutic antivirals may improve liver failure prognosis.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by Institutional Review Board of Huashan Hospital affiliated with Fudan University. Since no patient’s privacy data was showed, informed consent was not required.

Conception or design of the work (QY, LS, and JZ). Execution of CMV DNA viral load testing (YC and AL). Statistical analysis and interpretation of data (QY, ZZ, XY, and BZ). Drafting the manuscript (QY, ZZ, and XY). Accountability for all aspects of the work and final approval of the version to be submitted (LS, JZ, and WZ). All authors contributed to the article and approved the submitted version.

This work was supported by research grants from the Natural Science Foundation of Shanghai, China (grant number 19ZR1407800) and the Key Laboratory Project of Shanghai Science and Technology Commission [grant numbers 20dz2210400].

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.