Hepatitis D virus (HDV) is a defective, single-stranded RNA virus that causes an aggressive form of chronic hepatitis. Liver transplantation is one treatment option, among other experimental methods. However, HDV can reactivate after the operation, leading to a recrudescence of chronic hepatitis, albeit typically in a milder form [1]. To counter this reactivation, various prophylactic protocols have been developed. These primarily include the new generation of mutation-resistant nucleos(t)ide analogues (NA; tenofovir alafenamide and entecavir) and may also involve the perioperative and postoperative administration of immunoglobulin against the hepatitis B virus surface antigen hepatitis B virus surface antigen (HBsAg; hepatitis B immunoglobulin [HBIg]) for varying durations. The effectiveness of HBIg in preventing HDV reactivation is not universally agreed upon in the literature, mainly because the available studies did not compare two distinct patient groups (those treated with HBIg and those without), often had small sample sizes, and were descriptive in nature [2–4]. Adil et al. [3] reported no HDV reactivation in 128 liver transplant patients treated for hepatitis B virus (HBV)/HDV-related causes from 2003 to 2013. However, the study did not provide information on the HBIg protocol. In a single-center study, Serin and Tokat [2] found that among 104 patients who received liver transplants for HBV/HDV-related causes between 2014 and 2018, with an average follow-up of 82 months, there was a 14% reactivation rate (n=15) despite the use of HBIg perioperatively and NA on an ongoing basis thereafter.

In this study, we aimed to investigate the role of HBIg in HDV reactivation following liver transplantation. We compared two populations: one that received HBIg during the perioperative period and one that did not. Our ability to carry out this comparison is facilitated by the fact that our center operates a living donor liver transplantation program that incorporates HBIg into its prophylaxis protocol. Additionally, we conduct long-term follow-up of patients who underwent transplantation abroad under both HBIg and non-HBIg protocols.

This study was approved by the Institutional Ethics Committee of The First Central Hospital of Mongolia (approval no. 01/2019). Each patient provided written informed consent.

We invited 89 adult liver transplant patients under our follow-up to participate in the study. These patients had undergone liver transplantation at various centers worldwide and were enrolled in a long-term monitoring program at a high-volume, single-center liver transplant facility. At the time of transplantation, the patients were known to be either HBV monoinfected or HBV+HDV coinfected, with or without hepatocellular carcinoma and/or concurrent hepatitis C virus (HCV) infection. All participants were at least 1 year posttransplantation as of January 1, 2021. Patients were categorized into two groups based on the HBV prophylaxis protocol of their respective transplant centers: the HBIg group and the non-HBIg group. Of the 89 patients, 57 (34 male and 23 female patients, with an average age of 50.3 years) agreed to participate 23 in the HBIg group and 34 in the non-HBIg group. The median and average follow-up periods were 54 and 36 months, respectively, with the longest-surviving patient being 8 years posttransplantation. Serum analyses for HBsAg, anti-HDV, HBV DNA, and HDV RNA were conducted between March and December 2021. HDV RNA was extracted using the BioActGene Virus Purification kit (Bioactiva Diagnostica GmbH), which employs a silica filter spin-column method. Viral nucleic acid quantification was performed using an in-house method, the iTaq universal probes one-step kit (Bio-Rad), along with primers and a pGEM-T Easy Vector (Promega) containing the full-length HDV genome as an RNA standard [5]. Non-HBV/HDV causes were excluded. Individual interviews were conducted with each patient regarding their NA compliance. HBV DNA status prior to the operation was assessed when possible, and the liver function tests for the affected patients (alanine aminotransferase and aspartate aminotransferase, Roche) were performed as part of the routine follow-up.

Every patient was maintained on a tacrolimus immunosuppression regimen that aimed to keep serum trough levels between 2 and 10 ng/mL, depending on the time elapsed since the operation, both before and during the study. In addition, patients were prescribed NA regimens consisting of either tenofovir alafenamide at 25 mg/day, tenofovir desoproxil fumarate at 300 mg/day, or entecavir at 0.5 mg/day. When patients reported noncompliance with their NA regimen, they were strongly encouraged to resume adherence to the regimen as promptly as possible.

The unpaired t-test (Mann-Whitney U-test) was performed to assess group differences in viral reactivations (P<0.05). All statistical analyses were performed using Prism (GraphPad).

The HBIg group consisted of 15 male (65.2%) and 8 female patients (n=23), with an average age of 47.78 years and a follow-up period of 31.56 months. In the non-HBIg group, there were 19 male (55.8%) and 15 female patients (n=34), with an average age of 52.5 years and a follow-up period of 38.4 months. The demographic characteristics of the patients are presented in Table 1.

Table 1. Demographic characteristics of the two groups

Characteristic	HBIg group (n=23)	Non-HBIg group (n=34)	P-value	Total (n=57)
Male sex	15 (65.2)	19 (55.8)	0.48	34 (59.6)
Age (yr)	47.78±10.37	52.50±10.49	0.08	50.59±10.37
Follow-up (mo)	31.56±19.50	38.40±18.36	0.21	36.00±19.65

Values are presented as number (%) or mean±standard deviation.

HBIg, hepatitis B immunoglobulin.

The prevalence of HDV RNA, HBV DNA, HBsAg, and anti-HDV positivity in the HBIg group was 4.3% (1 patient), 17.4% (4 patients), 8.7% (2 patients), and 95.7% (22 patients), respectively. In the non-HBIg group, these rates were 5.9% (2 patients), 8.8% (3 patients), 11.8% (4 patients), and 97.1% (33 patients), respectively. There was no statistically significant difference in the likelihood of HDV reactivation between the HBIg and non-HBIg groups (Table 2). Of the 13 patients previously identified as either HBV monoinfected or HBV/HCV coinfected prior to transplantation, 11 showed positivity for anti-HDV antibodies (84.6%).

Table 2. Summarized results for HBIg and non-HBIg groups

Result	HBIg group (n=23)	Non-HBIg group (n=34)	P-value
HDV RNA positive	1 (4.3)	2 (5.9)	0.99
HBV DNA positive	4 (17.4)	3 (8.8)	0.42
HBsAg positive	2 (8.7)	4 (11.8)	-
Anti-HDV positive	22 (95.7)	33 (97.1)	-

Values are presented as number (%).

HBIg, hepatitis B immunoglobulin; HDV, hepatitis D virus; HBV, hepatitis B virus; HBsAg, hepatitis B virus surface antigen.

For the HBIg group, interview comments, HBV/HDV status prior to liver transplantation, and current liver function tests for those with virus replication can be found in Table 3. Corresponding information for the non-HBIg group is presented in Table 4. The interviews with individuals who experienced reactivation of HBV DNA and/or HDV RNA revealed that all had been noncompliant with their NA regimen at some point. All patients, with one exception, had only one virus actively replicating.

Table 3. Patient interview comments and HBV/HDV status before LT for the HBIg group

Patient no.	Interview comments	HBV/HDV status before LT	Liver function at test (ALT/AST [IU/mL])a)
53	NA regimen noncompliance in 2021	HBV DNA, target not detected HDV RNA, target not detected	141/40
16	NA regimen noncompliance in 2020	No information available	53/48
84	NA regimen noncompliance since LT	HBV DNA, target not detected HDV RNA, target not detected	19.5/28.3
26	NA regimen noncompliance in 2020	HBV DNA, 454,000 IU/mL HDV RNA, target not detected	66/45

HBV, hepatitis B virus; HDV, hepatitis D virus; LT, liver transplant; HBIg, hepatitis B immunoglobulin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; NA, nucleos(t)ide analogue.

^a)Range, 0–40 IU/mL.

Table 4. Patient interview comments and HBV/HDV status before LT for the non-HBIg group

Patient No.	Interview comments	HBV/HDV status before LT	Liver function at test (ALT/AST [IU/mL])a)
88	NA regimen noncompliance during 2013–2016	No information available	45/40
74	NA regimen noncompliance due to pregnancy in 2017	No information available	9.2/16.6
63	NA regimen noncompliance in 2017	No information available	11.4/14.2
37	NA regimen noncompliance due to an episode of toxic hepatitis in 2019	HBV DNA, 6,830 IU/mL HDV RNA, not performed HCV RNA, <15 IU/mL	489/183
2	NA regiment noncompliance in 2021	HBV DNA, 16,500 IU/mL HDV RNA, no information	69/51

HBV, hepatitis B virus; HDV, hepatitis D virus; LT, liver transplant; HBIg, hepatitis B immunoglobulin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; NA, nucleos(t)ide analogue; HCV, hepatitis C virus.

^a)Range, 0–40 IU/mL.

Absence of Spontaneous HDV RNA Reactivation after the Liver Transplantation in NA-Compliant Patients

To our knowledge, this study includes the largest cohort of HDV liver transplant patients observed over the longest follow-up period, with well-matched patient groups receiving HBIg and those not receiving HBIg. Our center’s ability to conduct this study stems from the long-term monitoring of local patients who underwent liver transplantation at various centers worldwide, each employing different HBIg protocols. Major liver transplantation centers in Korea, including our own, incorporate HBIg as a prophylactic measure, whereas some centers in India, Japan, and Singapore do not. This discrepancy creates two comparable groups that are otherwise identical in all respects. In both groups, we observed no HDV replication that could be attributed to spontaneous reactivation of HDV RNA while patients were on the NA regimen. Even in cases where HBV DNA replication was detected prior to transplantation, strict adherence to the NA regimen appeared to effectively suppress viral replication postoperatively. It is only upon discontinuation of NAs that prior HBV replication may influence the clinical outcome.

Adherence to a drug regimen is influenced by the duration of treatment, the number of medications involved, and the patient’s perception of the regimen’s importance. Our study results indicate that after a period of postoperative well-being, there is a decline in compliance among a small subset of patients. Shortening the duration of HBIg therapy or eliminating it entirely, coupled with improved patient education, may improve compliance. For patients with diseases caused by HBV and HDV, adherence to NAs is crucial, and it appears to be more important than HBIg prophylaxis, as demonstrated by this study. The significance of HBIg prophylaxis may be particularly relevant in the uncommon cases of elevated HBV and HDV replication prior to liver transplantation, which can lead to breakthrough infection in the graft despite the presence of NA. Most patients with chronic hepatitis D (CHD) undergoing liver transplantation do not show detectable HBV DNA and typically have a very low HDV RNA replication load after years of chronic illness. Nevertheless, it is advisable to always monitor for viral replication before the operation.

The Need to Test for HBV DNA Replication, Even in the Absence of HBsAg Positivity

We identified four cases of low-level HBV DNA replication in the absence of HBsAg positivity. It is possible for low-level HBV DNA replication to occur even when HBsAg is not detectable in the serum. Consequently, it is important to periodically test for HBV DNA, as this may signify viral genomic integration, which could potentially lead to the development of hepatocellular carcinoma in the future.

Mild, Self-limiting Clinical Course of Chronic Hepatitis D Recrudescence

Ottobrelli et al. [4] reported a mild clinical course of CHD in transplanted patients. We documented a single case of aggressive hepatitis leading to decompensation in a subset of three patients with reactivated HDV RNA. The number of patients willing to participate in the study was significantly reduced due to the coronavirus disease 2019 (COVID-19) pandemic occurring at the time. Many patients declined the invitation or could not consistently travel for follow-up because of strict quarantine measures and the perceived risk of COVID-19 to their immunocompromised condition. Consequently, with the limited number of cases available, the authors are unable to report with statistical certainty the proportion of aggressive CHD, decompensation, or fatal outcomes compared to all patients with HDV RNA reactivation. However, based on our experience, we can assert that the majority of CHD recrudescence cases are mild and tend to resolve on their own after 1–2 years of replication, as evidenced by the liver function tests of patients with reactivation.

The apparent discrepancy between aggressive hepatitis in the general population and a milder course in liver transplant recipients with HDV infection may be attributed to a reduced liver-damaging immune response due to calcineurin inhibitors, such as tacrolimus. Additionally, Iwamoto et al. [6] and Nkongolo et al. [7] have reported an intriguing property of another calcineurin inhibitor, ciclosporin A. In vitro studies indicate that ciclosporin A can inhibit the sodium-taurocholate cotransporting polypeptide (NTCP), which is a key receptor for HBsAg virions [6,7]. Under normal conditions, NTCP is responsible for bile acid transport and can be blocked to varying extents in vitro by several molecules, including bulevirtide (also known as Hepcludex, MYR GmbH) [8], ciclosporin A, metformin or ursodeoxycholic acid [7], among other molecules. Tacrolimus was found to be ineffective in inhibiting NTCP. Ciclosporin A may offer enhanced anti-HDV effects in vivo due to its dual role in modulating the immune response and inhibiting NTCP, compared to tacrolimus. Considering recent research on new treatments for HDV, ciclosporin A could present a promising alternative for nontransplanted patients with chronic HDV infection.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Funding/Support

This study was funded by the Korean Society for Transplantation International Research Grant 2020.

Author Contributions

Conceptualization: AG. Data curation: all authors. Formal analysis: AG. Writing–original draft: AG. Writing–review & editing: all authors. All authors read and approved the final manuscript.

Additional Contributions

The authors wish to thank Dr. Ahn Curie and Dr. Sergelen Orgoi for their help in facilitating this study. They also gratefully acknowledge Ms. Enkhjargal Altangerel for statistical support.