Prevalence of human herpesviruses in biliary fluid and their association with biliary complications after liver transplantation

Liver transplantation (LT) is to date the only curative option for patients with end-stage liver disease. Liver transplantation requires lifelong immunosuppression to prevent allograft rejection and subsequent graft failure. Immunosuppression predisposes solid organ recipients to various opportunistic infections. The role of many of these opportunistic pathogens in the development of complications after LT is unknown. Human herpesviruses (HHV) 1–8 are enveloped, double-stranded DNA and human host specific viruses that proliferate in lymphocytes and neuronal or epidermal cells. They can persist lifelong in the host and reactivate under circumstances of immunosuppression. Active disease can be detected by polymerase chain reaction (PCR) of HHV 1–8 DNA [1]. The incidence of Cytomegalovirus (CMV), HHV-6 and HHV-7 in blood after LT has been reported as high as 70, 33 and 42%, respectively [2]. CMV is the most common infectious pathogen after LT [3] and can infect various organs, including the allograft itself. CMV in bile has been associated with early graft loss and biliary complications after LT [4]. HHV-6 and HHV-7, together with CMV (HHV-5), comprise the group of beta-herpesviruses. Human herpesvirus 6 has been linked to hepatitis post LT, but most HHV-6 infections after LT are asymptomatic [5, 6]. The presence of HHV-6 and HHV-7 viremia in blood after LT has inconsistently been linked to reduced graft survival. However, detecting HHV-6 DNA in liver biopsies has been associated with graft hepatitis and reduced graft survival [7‐9]. Epstein-Barr virus (EBV) and CMV detection in liver biopsies had no effect on overall survival after LT [7]. To prevent the reactivation of CMV after LT, patients receive valganciclovir chemoprophylaxis for three to 6 months after LT [10]. For HHV-6 and -7, there are no recommendations for prevention. In vitro, HHV-6 and HHV-7 are less susceptible to ganciclovir than CMV [11]. The objective of this study was to examine the association between herpesviruses in different body fluids and tissues after LT and to elucidate their role in graft complications after LT.

Biliary fluids can routinely be assessed after ERC but are rarely subject to scientific investigation. This is the first single-center case-control study to investigate herpesvirus 1–8 prevalence in human bile samples and its association with biliary complications after LT. We found a high prevalence of CMV, HHV-6 and HHV-7 in biliary fluids in LT patients both with and without biliary complications. The rate of positivity of HHV-6 correlated with poor re-LT-free survival after ERC. The persistence of beta-herpesviruses in epithelia after LT has been described frequently [16]. Cytomegalovirus in bile has already been implicated in biliary lesion formation after LT, but its significance remains controversial [4, 17, 18]. Interestingly, in our study, the rates of detection of CMV, HHV-6 and HHV-7 in bile have been considerably higher than in serum or liver biopsies (Table 2). In bile we found the highest prevalence for HHV-6 (34.2%) and HHV-7 (20.5%) followed by CMV (18.3%) and EBV (16.4%) (Table 2). Presence of HHV-6 and HHV-7 often coincided possibly due to viral coactivation, as 9/15 patients positive for HHV-7 in bile also tested positive for HHV-6 [19]. We found that patients with NAS and ACR were much more likely to test positive for HHV-6 and HHV-7 in bile than patients of the control group (Table 4). Interestingly, neither HHV-6 nor HHV-7 in liver biopsy or serum were associated with biliary complications after LT. In Kaplan-Meier analysis HHV-6 was significantly associated with death and retransplantation after ERC. This significance was lost however in multivariate cox proportional hazard analysis after controlling for known risk factors such as type of stricture in the multivariate analysis (Fig. 2 and Table 6), however HHV-6 in bile remained the strongest risk factor for decreased re-LT free survival among the included. If HHV-6 in bile is causally related to biliary strictures and thus graft survival or a mere bystander of damage of the biliary epithelium remains to be determined. We are not the first group to report the relation of HHV-6 and decreased graft survival. In a cohort of pediatric LT recipients chromosomally integrated HHV-6 DNA was associated with worse graft survival [20]. HHV-6 has also been associated with worse LT graft survival in an adult population after diagnosis of graft hepatitis with higher intrahepatic HHV-6 amplification levels [7]. The mechanism for this association remains unclear. However HHV-6 has been described to exert immunomodulatory effects such as increased secretion of interferon gamma, tumor-necrosis factor alpha or CCL5 [21‐24]. HHV-6 has also been implicated in numerous autoimmune diseases such as multiple sclerosis, connective tissue disorders or autoimmune thyroiditis in part through molecular mimicry [25‐29]. HHV-6 could thus exert immunomodulatory effects in the liver allograft leading to a host versus graft reaction leading to further damage of the biliary ducts.

The preexisting biliary damage of the donor organ at time of transplantation has been shown to predict subsequent risk for biliary complications [30]. We therefore assessed labMELD and cold ischemia time as established risk factors for mortality and associated with early allograft loss, which were evenly distributed in our study.

Increased recipient morbidity at time of transplantation likewise seems to play a role for biliary HHV-6 infections. We recorded labMELD score at transplantation as a surrogate marker for recipient morbidity at transplantation [31]. Patients positive for HHV-6 in bile had a higher median labMELD (16.4 vs. 22.5, p = 0.02, Table 4) but not a higher eMELD score at LT. Patients with a higher labMELD score at the time of transplantation presented more severe hepatic and renal impairment. This precondition might predispose patients to opportunistic infections and, in our study, might in part explain the increased prevalence of herpesvirus infections. The association of HHV-6 with graft complications and reduced survival poses the important question of possible prevention of HHV-6 reactivation/infection. Interestingly, valganciclovir prophylaxis did not reduce the incidence of HHV-6 prevalence nor the mean viral count in bile (38.2% vs. 27%, p = 0.25, Table 5). This might be due to a lower responsiveness of HHV-6 and HHV-7 than CMV to valganciclovir or ganciclovir treatment [32]. In a study by Humar et al. of CMV, HHV-6 and HHV-7 coinfected patients, valganciclovir treatment did reduce the CMV but not the HHV-6 and HHV-7 viral load [32]. Consistent in our study CMV prophylaxis was sufficient in our patients to prevent CMV infection. This might explain why in our study CMV in bile was not related to NAS, contrasting with prior data published by our group [4]. In the current study, bile retrieval was undertaken early after LT while many patients (47.8%) were under CMV prophylaxis. When excluding patients on prophylactic therapy, there was a numerical though statistically insignificant higher prevalence of non-anastomotic strictures in patients who tested positive for CMV in bile (50% vs. 33%, ns).

Two important findings conclude our study. The first is the striking discrepancy of herpesvirus detection of the different compartments (bile, serum, tissue). Currently, blood serum is used for routine HHV-6 detection; indeed biliary samples might be more sensitive and specific for HHV-6 detection in the liver allograft. This finding is not limited to herpesviruses but might extend to all DNA-viruses and bacteria. We thus hypothesize that testing biliary fluid might mirror local infective processes of the hepatobiliary compartment with a higher sensitivity than serum or biopsy specimen. The second important finding is the correlation between locoregional HHV-6 (and possibly CMV in the later course) detection and severe liver allograft complications. We further hypothesize that routine CMV prophylaxis with valganciclovir is not sufficient to prevent HHV-6 (and HHV-7) infection, which raises important questions for clinical practice [33, 34].

Our study however poses several important limitations. HHV-6 positivity in bile did not significantly correlate with positivity in the liver biopsy. Even samples with very high virus counts in bile that where taken on subsequent days to the liver biopsy showed no virus amplification in the biopsy tissue. It could be possible, that the liver biopsy was non representative of the donor organ. The focal distribution of HHV-6 in infected liver tissue is not known. The design of the study was a retrospective case-control study wherein different cohorts were randomly selected from a tissue and fluid biobank. Concordant biopsy, serum and bile samples were not always available, and when they were available, they were not always taken on the same day.

The methods for DNA detection were not established for biliary fluids, and stability data on viral DNA in biliary fluid is lacking. Furthermore, viral loads for HHV-6 were low. Only 4 out of 25 patients had viral loads > 1000 copies/ml, the threshold for consideration as an active HHV-6 infection in serum samples [35]. While this finding could reflect a high percentage of latent infection and explain why viral DNA in those cases could not be detected in serum samples from the same recipients, this could also be due to DNA degradation in our bile samples. Our study warrants further exploration in a prospective experimental setting. If confirmed, treatment of occult HHV-6 and HHV-7 infection in cases of sustained and progressive biliary damage after LT might lead to a reduced risk of graft loss and need for re-LT. Further understanding of the role of non-CMV beta-herpesviruses in LT might also affect recommendations for the routine use of valganciclovir in the initial months after LT.

The present study shows that CMV, EBV and HHV-6 and -7 are highly prevalent in biliary fluid. Overall beta-herpesvirus detection was more frequent in bile than in liver biopsy or serum. HHV-6 is associated with biliary complications after LT. Further studies are needed to assess the potential connection of HHV-6 detection in bile and biliary complications after LT and the general value of routine viral diagnostics in bile compared to serum and liver biopsy.