Hepatitis B virus and hepatitis D virus co-infection complicated by autoimmune hepatitis: Two case reports

Abstract

BACKGROUND

Hepatitis D virus-hepatitis B virus (HDV-HBV) co-infection accelerates liver disease progression and increases the risk of hepatocellular carcinoma, but the immunopathogenic mechanism of its combination with autoimmune hepatitis (AIH) has not been clarified. This study reveals for the first time that HDV may induce AIH through abnormalities in immunoregulation in two specific cases. This is the first report of HDV-HBV co-infected patients who did not receive interferon therapy and achieved serological conversion and histological remission by combining antiviral (entecavir) with immunosuppression (prednisone + azathioprine) therapy, providing new evidence of the mechanism of this complex disease.

CASE SUMMARY

A 40-year-old female developed malaise and jaundice with an alanine aminotransferase/aspartate aminotransferase > 20 upper limit of normal (ULN), total bilirubin: 97.20 μmol/L, immunoglobulin G (IgG) 47.1 g/L (> 3 × ULN), HDV RNA 1.6 × 10⁷ copies/mL and liver biopsy showed G3S4. Tenofovir alafenamide combined with prednisone and azathioprine was administered, and three months later the Child-Turcotte-Pugh class C was reduced to class B and IgG decreased to 13.62 g/L. Another 58-year-old male complained of pain in the liver area, anti-nuclear antibody was 1:320, IgG 22.6 g/L (> 1.3 × ULN), and liver biopsy showed G2S3. Entecavir was administered in combination with prednisone and azathioprine, and after 3 months, liver function returned to normal, and IgG reduced to 14.22 g/L.

CONCLUSION

Patients with HDV-HBV co-infection combined with AIH can achieve clinical remission following combination therapy, and the study of immunomodulatory mechanisms should be emphasized.

Key Words: Hepatitis D virus; Hepatitis B virus; Autoimmune hepatitis; Liver biopsy; Pathology; Case report

Core Tip: This study reports, for the first time, two patients with hepatitis D virus (HDV)-hepatitis B virus co-infection combined with autoimmune hepatitis (AIH) who were not treated with interferon and achieved serological conversion and histological remission with antiviral drugs (entecavir/tenofovir alafenamide) in combination with immunosuppression (prednisone + azathioprine). Patients with severe HDV-related liver disease should be routinely screened for autoantibodies to avoid exacerbation by interferon therapy. The combination therapy was effective in controlling HDV-hepatitis B virus co-infection associated AIH, suggesting that aberrant immunoregulation may be an important mechanism of HDV-induced AIH, providing new evidence for elucidating the pathogenesis of this complex disease, and emphasizing the necessity of immune mechanism research.

INTRODUCTION

Hepatitis D virus (HDV) is a satellite virus that requires the envelope proteins of hepatitis B virus (HBV) for particle assembly and propagation. Co-infection with HBV and HDV can lead to severe viral hepatitis, accelerating the progression to liver fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC). HDV possesses a negative-sense, single-stranded RNA genome of approximately 1700 nucleotides, representing the smallest known mammalian virus genome to date[1,2]. A 2020 epidemiological study published in collaboration with the World Health Organization indicated that an estimated 5% of chronic HBV carriers worldwide (15-20 million) are co-infected with HDV[3]. Regions with high prevalence include Central Africa, West Africa, the Mediterranean basin, the Middle East, Eastern Europe, North Asia, certain areas of Southeast Asia, and the Amazon basin in South America. Compared to individuals infected solely with HBV, patients with chronic HDV-HBV co-infection or dual infection exhibit higher incidences of cirrhosis and HCC, as well as increased liver-related mortality rates[4]. Unlike HBV, HDV infection can stimulate the production of interferon (IFN), which effectively suppresses HBV and moderately inhibits HDV[5]. Autoimmune hepatitis (AIH) is an immune-mediated chronic liver disease characterized by elevated levels of autoantibodies, increased immunoglobulin G (IgG) levels, heightened serum transaminase levels, and histological evidence of interface hepatitis with lymphoplasmacytic infiltration[6]. There have been reports of AIH induced in patients with chronic hepatitis D following treatment with pegylated IFN[7]. We report and discuss the clinical and pathological features of two cases where these patients developed AIH without having received INF therapy.

CASE PRESENTATION

Chief complaints

Case 1: A 40-year-old female presented to Department of Hepatology with fatigue and jaundice for one month.

Case 2: A 58-year-old male presented with vague pain in the right side for one month.

History of present illness

Case 1: In September 2011, the patient presented with right-sided distension, pain and malaise. Positive hepatitis B markers were found and a hepatic puncture was performed. The patient refused treatment. In 2012, due to abnormalities in liver function, she underwent a second liver biopsy in an external hospital, which did not rule out primary biliary cholangitis. She was started on ursodeoxycholic acid capsules at 750 mg/day with intermittent follow-up. In 2014, she was started on antiviral therapy with tibivudine. By 2015, due to the diagnosis of AIH, she was advised to switch to antiviral therapy with entecavir along with prednisone. Due to the unknown nature of a pelvic mass, the patient and her family cautiously postponed prednisone treatment. The mass was later clarified to be an ovarian luteal cyst, and no pelvic mass was seen on routine gynecological ultrasound during late follow-up. In 2017, antiviral therapy was switched to tenofovir disoproxil fumarate during pregnancy, and immunosuppressive therapy with azathioprine was added in 2018, and then prednisone 20 mg/day was added in 2019. Following the development of splenomegaly and leukopenia (1.62 × 10⁹/L), her treatment was adjusted to tenofovir disoproxil fumarate plus prednisone 10 mg/day, and azathioprine was discontinued. In 2021, the patient presented to an outpatient hospital and upper abdominal magnetic resonance imaging revealed probable cirrhosis. The treatment regimen was adjusted to tenofovir disoproxil fumarate, methylprednisolone 16 mg/day, merti-mescaline 0.5 g/day, compound glycyrrhizin to protect the liver and reduce enzymes, and tapered down to methylprednisolone 12 mg/day. In July 2023, the patient discontinued the use of ursodeoxycholic acid and methylprednisolone, and continued antiviral therapy with tenofovir disoproxil fumarate only. In August 2023, the patient presented to the clinic with complaints of malaise and jaundice for one month.

Case 2: The patient reported that hepatitis B was identified on physical examination 20 years previously, but no antiviral treatment was given, and since then there has been no regular review. He was admitted to the hospital with the main complaint of pain in the right hypochondrium for one month.

History of past illness

Case 1: The patient had tuberculous pleurisy in 2003 which was cured.

Case 2: The patient has had fatty liver for one year.

Personal and family history

Case 1: The patient was employed as a clerk, with no tobacco or alcohol addiction, and denied any family history of disease or hereditary disease.

Case 2: A smoking history of 20 years, with an average of 15 cigarettes/day, and occasional alcohol consumption in the past, with abstinence for 3 years.

Physical examination

Case 1: Physical examination revealed mild yellowing of the skin, mucous membranes and sclera over the entire body, liver palms (+), and mild edema of both lower limbs.

Case 2: No special features.

Laboratory examinations

Case 1: In 2011, liver function tests showed the following: Alanine aminotransferase 161 U/L, aspartate aminotransferase 118 U/L, alkaline phosphatase 38.6 U/L, and gamma-glutamyl transferase 43.6 U/L. HBV DNA titers were below the limit of detection, quantitative hepatitis B surface antigen was 1387 IU/mL and anti-hepatitis B e antibody was 0.09 IU/mL. Anti-nuclear antibody titer was 1:100 and anti-mitochondrial M2 antibody was positive.

In August 2023, high-sensitivity HBV DNA was 1.12 × 10² IU/mL, alanine aminotransferase 985.00 U/L, aspartate aminotransferase 2115.00 U/L, alkaline phosphatase 198.83 U/L, gamma-glutamyl transferase 112.60 U/L, total bilirubin 97.20 μmol/L, plasminogen time 26.80 seconds, international normalized ratio 2.29, albumin 27.38 g/L, IgG 47.1 g/L, HDV antibody IgG and IgM positive, HDV RNA 1.6 × 10⁷ copies/mL (Tables 1 and 2), and liver biopsy showed G3S4 (Figure 1).

Figure 1 Liver biopsy pathological results for case 1. A: A total of eleven small and medium-sized confluent areas were seen in the perforated liver tissue, with disorganized lobular architecture (hematoxylin and eosin); B: The main lesions were enlargement of most of the confluent areas, moderate inflammatory cell infiltration, predominantly single nucleated cells, and plasma cells were seen, often forming foci of lymphocyte aggregation, and mild-moderate interfacial inflammation (hematoxylin and eosin); C: The small bile ducts were discernible, and the biliary epithelium was irregularly arranged, and a mild-moderate fine biliary reaction was seen in the periphery of the confluent areas; fibroblastic tissue hyperplasia was seen in the confluent area, and the fibrous intervals of the bridge were formed, which destroyed the hepatic lobules by separating the hepatic parenchyma. Hepatocellular lipoatrophy and ballooning were seen in the lobules (hematoxylin and eosin); D: With focally necrotic and apoptotic vesicles, and mild inflammatory cell infiltration in the sinusoids, with some hepatocytes with ground-glass lesions (hematoxylin and eosin).

Table 1 Laboratory tests, cirrhosis status and treatment for case 1.

Test item	August 2023	November 2023
Current antiviral medication	Tenofovir propofol fumarate	Tenofovir propofol fumarate
Cirrhosis stage	Decompensated	Decompensated
ALT (U/L)	985.00	17.20
AST (U/L)	2115.0	23.50
GGT (U/L)	161.61	28.93
ALP(U/L)	198.83	57.50
Alb (g/L)	27.38	41.10
TBIL (μmol/L)	97.20	45.90
DBIL (μmol/L)	60.22	23.60
IBIL(μmol/L)	36.98	22.30
LDH (U/L)	653.11	267.88
CHE (U/L)	2240	1310.00
TBA (μmol/L)	126.40	176.20
TP (g/L)	80.56	61.80
Glb (g/L)	53.18	20.70
PT (seconds)	26.80	22.20
PTA (%)	27.70	40.50
INR	2.29	1.73
WBC (109/L)	1.55	1.57
HGB (g/L)	105.00	73.00
PLT (109/L)	103.00	60.00
IgG (g/L)	47.10	13.62
AFP (ng/mL)	16.80	3.84
HBsAg (IU/mL)	12286.00	14214.00
HBeAg (IU/mL)	0.12	0.13
HBV DNA (IU/mL)	1.12 × 102	< 20
Anti-HDV IgM	Positive	Positive
Anti-HDV IgG	Positive	Positive
Liver stiffness (kPa)	16.5	17.6
Fat attenuation (db/m)	268	246
Non-alcoholic fatty liver disease	Present	Present

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: Gamma-glutamyl transferase; ALP: Alkaline phosphatase; Alb: Albumin; TBIL: Total bilirubin; DBIL: Direct bilirubin; IBIL: Indirect bilirubin; LDH: Lactate dehydrogenase; CHE: Cholinesterase; TBA: Total bile acid; TP: Total protein; Glb: Globulin; PT: Prothrombin time; PTA: Prothrombin time activity; INR: International normalized ratio; WBC: White blood cell; HGB: Hemoglobin; PLT: Platelet; IgG: Immunoglobulin G; AFP: Alpha-fetoprotein; HBsAg: Hepatitis B surface antigen; HBeAg: Hepatitis B e antigen; HBV: Hepatitis B virus; HDV: Hepatitis D virus; IgM: Immunoglobulin M.

Table 2 Laboratory and pathological results related to autoimmune hepatitis.

Test/feature		Case 1	Case 2
Anti-nuclear antibody		1:320	1:320
IgG (g/L) elevation		47.10	22.60
Liver kidney microsomal type 1 antibody		-	+
Anti-soluble liver antigen antibody		-	-
Pathology	Portal inflammation	Moderate	Moderate
	Interface hepatitis	Mild to moderate	Moderate
	Plasma cell infiltration	Present	Present
	Rosette formation	Absent	Absent
	Piecemeal necrosis	Absent	Absent
Inflammation grade (G)		G3	G2
Fibrosis stage (S)		S4	S3

IgG: Immunoglobulin G.

Case 2: Alanine aminotransferase 77.20 U/L, aspartate aminotransferase 60.80 U/L, alkaline phosphatase 142.50 U/L, gamma-glutamyl transferase 93.58 U/L, HDV antibody IgG and IgM positive, HDV RNA negative, high-sensitivity HBV DNA < 20 IU/mL, quantitative hepatitis B surface antigen 7755.00 IU/mL, quantitative hepatitis B e antigen 0.09 IU/mL, anti-liver and kidney microsomal type 1 antibody positive, antinuclear antibodies IgG 1:320, IgG 22.60 g/L (Tables 2 and 3), and liver biopsy showed G2S3 (Figure 2).

Figure 2 Liver biopsy pathological results for case 2. A: The liver biopsy tissue revealed seven portal areas of medium to small size with disorganized lobular architecture (hematoxylin and eosin); B: The primary lesions include expansion of some portal areas, moderate inflammatory cell infiltration dominated by mononuclear cells (hematoxylin and eosin); C: Plasma cells are easily visible in small clusters (CD38), and moderate interface hepatitis observed in some portal areas; D: The bile ductules are discernible with disorganized epithelial lining and infiltration of inflammatory cells into the interductal epithelium, accompanied by a mild bile duct reaction around the portal areas. There is also proliferation of interstitial fibrous tissue in the portal areas, leading to the formation of local fibrous septa that segment the hepatic parenchyma and disrupt the lobular structure (Masson).

Table 3 Laboratory tests, cirrhosis status and treatment for case 2.

Test item	June 2023	January 2024
Current antiviral medication	Entecavir	Entecavir
Cirrhosis stage	Compensated	Compensated
ALT (U/L)	77.20	47.60
AST (U/L)	60.80	32.90
GGT (U/L)	93.58	86.89
ALP (U/L)	142.50	100.40
Alb (g/L)	40.90	36.40
TBIL (μmol/L)	13.20	7.20
DBIL (μmol/L)	2.90	1.80
IBIL (μmol/L)	10.30	5.40
LDH (U/L)	218.10	214.00
CHE (U/L)	7380	6470.00
TBA (μmol/L)	5.60	21.10
TP (g/L)	78.80	67.90
Glb (g/L)	37.90	31.50
PT (seconds)	13.20	13.50
PTA (%)	83.30	95.0
WBC (109/L)	4.70	6.53
HGB (g/L)	144.00	134.00
PLT (109/L)	143.00	129.00
IgG (g/L)	22.60	14.22
AFP (ng/mL)	10.07	7.40
HBsAg (IU/mL)	7755	9605.00
HBeAg (IU/mL)	0.09	0.12
HBV DNA (IU/mL)	< 20	< 20
Anti-HDV IgM	Positive	Positive
Anti-HDV IgG	Positive	Positive
Liver stiffness (kPa)	21.6	22.7
Fat attenuation (db/m)	294	283
Non-alcoholic fatty liver disease	Present	Present

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: Gamma-glutamyl transferase; ALP: Alkaline phosphatase; Alb: Albumin; TBIL: Total bilirubin; DBIL: Direct bilirubin; IBIL: Indirect bilirubin; LDH: Lactate dehydrogenase; CHE: Cholinesterase; TBA: Total bile acid; TP: Total protein; Glb: Globulin; PT: Prothrombin time; PTA: Prothrombin time activity; WBC: White blood cell; HGB: Hemoglobin; PLT: Platelet; IgG: Immunoglobulin G; AFP: Alpha-fetoprotein; HBsAg: Hepatitis B surface antigen; HBeAg: Hepatitis B e antigen; HBV: Hepatitis B virus; HDV: Hepatitis D virus; IgM: Immunoglobulin M.

Imaging examinations

Case 1: Nuclear magnetic resonance imaging showed cirrhosis with diffuse intrahepatic regenerative nodules, splenomegaly, portal hypertension, splenic varicose vein, and open umbilical vein.

Case 2: Enhanced computed tomography of the upper abdomen showed cirrhosis, splenomegaly, portal hypertension and periumbilical vein opening.

FINAL DIAGNOSIS

Case 1

The patient was diagnosed as HBV, HDV, and AIH.

Case 2

The patient was diagnosed as HBV, HDV, and AIH.

TREATMENT

Case 1

Treatment consisted of hepatoprotective and anti-yellowing, anti-infective, artificial liver support therapy, and antiviral therapy with prednisone 40 mg/day and tenofovir disoproxil fumarate 25 mg/day.

Case 2

The patient was treated with antiviral therapy with entecavir 0.5 mg/day and immunosuppressive therapy with prednisone acetate 30 mg/day and azathioprine 50 mg/day.

OUTCOME AND FOLLOW-UP

Case 1

Currently, prednisone has been reduced to 20 mg/day, and the Child-Turcotte-Pugh score has improved to grade B. The patient’s condition was stable.

Case 2

The patient’s liver function and immunoglobulin level were normalized and his condition was stable.

DISCUSSION

Chronic HDV infection accelerates the disease progression in patients with chronic hepatitis B. A meta-analysis showed that for every 3% increase in HDV prevalence, the detection rate of HDV RNA increased by 10%, with HDV accounting for 18% (95% confidence interval: 10%-26%) of cirrhosis cases and 20% (95% confidence interval: 8%-33%) of HCC cases[8]. Drug use and male homosexual behavior are high-risk factors for HDV infection[9]. Patients co-infected with HDV typically exhibit higher levels of transaminases compared to those with HBV mono-infection. Compared to patients with HBV and hepatitis C virus (HCV) co-infection, those with HBV and HDV co-infection are likely to experience more severe liver disease, accelerated fibrosis progression, and up to a threefold increased risk of developing cirrhosis and HCC[10]. The two cases reported here progressed to the cirrhosis stage, corroborating these research findings to some extent.

There are also studies on the co-occurrence of AIH in viral hepatitis. One study analyzed the clinical characteristics of patients with HBV-HCV co-infection and AIH, suggesting that in cases of HBV or HCV with unexplained increases in transaminase and IgG levels (especially in patients previously treated with IFN-α), where liver histology is consistent with AIH, and non-organ-specific autoantibodies, particularly anti-nuclear antibodies and anti-smooth muscle antibodies, or other more AIH-specific antibodies are detected, along with low or absent viremia, concurrent AIH should be highly suspected[11]. Similarly, another study found that patients with chronic hepatitis D are more likely to exhibit high non-organ-specific autoantibodies titers compared to those with chronic hepatitis B, suggesting that autoantibodies are often detected as a co-infection characteristic in chronic hepatitis D patients[4].

The pathogenesis of HDV infection has not been fully elucidated, but its close association with abnormalities in immune regulation is widely recognized. As the first line of defense in the innate immune system, the host uses pathogen-associated molecular pattern recognition to achieve self-vs-non-self-differentiation and pathogen identification through a surveillance network mediated by pattern recognition receptors[12]. Recent studies have shown that HDV RNA may be demonstrated in the cytoplasm by pattern recognition receptor family member MDA5, which activates mitochondrial antiviral signaling proteins and initiates a downstream signaling cascade, including activation of transcription factors such as IFN regulatory factor 3/7 and nuclear factor-kappa B, which ultimately induces the synthesis of type III IFN (IFN-β/λ), whereas the induction of IFN-α is unaffected[2,13].

Notably, there appears to be a paradoxical immune exploitation strategy for HDV. The IFN-β/λ-mediated enhancement of HBV antigen presentation may instead reinforce the inhibitory effect of T cells on HBV, thus creating a more favorable replicative environment for HDV. How HDV maintains its own replication in the IFN-activated state remains an unanswered question. Existing hypotheses mainly include: (1) HDV RNA replicates in the nucleus, forming a physical barrier to circumvent intrinsic cytoplasmic immune sensing; (2) The hepatitis B surface antigen-coated ribonucleoprotein complex blocks the pattern recognition receptor recognition pathway; and (3) HDV viral proteins may directly intervene in the IFN signaling pathway[14,15]. IFN plays a dual role in HDV infection and AIH pathogenesis[16]. Clinical data show that 4%-19% of patients treated with IFN-α develop secondary autoimmune diseases[17], while AIH patients have significantly elevated levels of IFN-β/λ in peripheral blood[18]. Although IFN-α/β induction has been shown to be associated with the development of AIH[19,20], AIH was present in the current two cases who did not receive antiviral therapy, suggesting that there may be multiple immune-mediated mechanisms for HDV-associated liver injury (Figure 3).

Figure 3 Hepatitis D virus, hepatitis B virus and autoimmune hepatitis interaction mechanism diagram. HBV: Hepatitis B virus; HDV: Hepatitis D virus; IFN: Interferon; MHC: Major histocompatibility complex; TNF: Tumor necrosis factor; LC: Liver cancer; IgG: Immunoglobulin G; NTCP: Sodium taurocholate co-transporting polypeptide; MDA5: Melanoma differentiation-associated protein 5; cccDNA: Covalently closed circular DNA.

Viral hepatitis can induce autoimmunity through molecular mimicry[21] and the immune system uses the major histocompatibility complex to detect foreign pathogens. This locus encodes the human leukocyte antigen (HLA) genes and various immune response genes that defend against pathogens. There are two main types of HLA antigens, HLA class I and class II. The role of HLA class I is to recognize infected cells, while HLA class II molecules deliver peptides to receptors on CD4+ helper T cells. HDV is fully adapted for recognition by CD8+ T cells, while CD8+ T cells are restricted by conventional HLA class I alleles. Furthermore, in persistent HBV-HDV co-infection, the rapid progression of cirrhosis is attributed to immune-mediated mechanisms, initiated by innate pathways of the IFN system or triggered by dysfunctional T cells[22,23]. The molecular mimicry theory provides an important explanation for the autoimmune mechanism. HLA class I molecules are responsible for presenting endogenous antigens to CD8+ T cells and their expression is significantly upregulated in HDV-infected hepatocytes[24]. This enhanced antigen-presenting capacity, superimposed on the adaptation of HDV to the conventional HLA class I antigen-presenting pathway, may induce a cytotoxic immune response. The release of IFN-γ and tumor necrosis factor-α by activated CD8+ T cells may not only exacerbate autoimmune attacks against infected hepatocytes[20,25], but also extend inflammatory damage through bystander effects.

CONCLUSION

Clinical case analyses revealed the following key issues. Both patients included in this report progressed to the stage of cirrhosis despite effective control of viral replication with immunosuppressive therapy. The persistent liver function abnormalities in the HDV co-infected case (case 1) suggest that virus-induced immune activation may outweigh direct antiviral benefits, whereas progression of the patient with no HDV RNA (case 2) suggests the presence of non-viral immunopathological damage. This phenomenon supports that HDV infection accelerates hepatic fibrosis through two pathways. On the one hand, virus-specific immune responses partially inhibit HBV replication, and on the other hand, the persistently activated immune system maintains the chronic inflammatory state of the liver.