Serological diagnosis of hepatitis B and delta virus (HBV/HDV) coinfection

Diagnosis of acute hepatitis B virus/hepatitis delta virus (HBV/HDV) coinfection is currently based on detection of anti-HD, however this antibody may be undetectable during the acute phase of hepatitis. To evaluate the entity of misdiagnosis of HBV/HDV coinfection in acute HBsAg-anti-HBc IgM positive hepatitis we examined sera from 245 consecutive patients obtained at admission and day 30, 60, 120, 210 and 400 of their follow-up. Anti-HD was detected in the serum of 26 out of 245 patients (10.6%). In 15% of cases it was present at admission, while in 92% it was found after 30 days. The combined detection of HDV-RNA, HDAg and IgM anti-HD in acute phase sera allowed a correct etiologic diagnosis in 69% of the cases. These findings suggest that the prevalence of HBV/HDV coinfection is underestimated when anti-HD is the only marker to be detected during the acute phase of disease. A correct etiologic diagnosis can only be made by testing acute phase sera for all the available markers of HDV. However, the best cost-effective procedure is to test any patient with HBV markers at presentation for anti-HD, 30-40 days after the onset of symptoms.     

Chronic hepatitis delta virus (HDV) infection

The Hepatitis Delta Virus (HDV) is a small RNA virus which replicates only in patients who are concurrently infected with hepatitis B virus (HBV). Delta hepatitis is endemic particularly in countries in the Mediterranean basin. In other parts of the world, HDV infection occurs among intravenous drug addicts and persons who receive multiple blood transfusion. HDV superinfection in a chronic HBV carrier often leads to severe chronic hepatitis and cirrhosis, whereas acute HDV and HBV co-infection is frequently associated with fulminant hepatitis. Diagnosis is usually based on the detection of HDV antigen in liver tissue and antibody to HDV (anti-HD) in serum. Nowadays, HDV antigen can also be detected in serum using immunoblot assay. The presence of HDV-RNA can now be confirmed by molecular biology techniques including polymerase chain reaction (PCR). Interestingly, up to 50% of patients with chronic HDV infection have in the serum autoantibodies against microsomal antigens of the human liver and kidney (LKM) and antibodies against the basal cell layer of the rat forestomach. Therapeutic approaches including interferon therapy and liver transplantation are still under discussion. This review discusses in detail some of the main features of chronic HDV infection.     

Quantification of serum markers of hepatitis B (HBV) and Delta virus (HDV) infections in patients with chronic HDV infection

The interplay between hepatitis B (HBV) and delta (HDV) viruses is complex and not always characterized during chronic HDV infection. We assessed the clinical usefulness of new quantitative assays for HBV and HDV serum markers in a retrospective cross‐sectional study. Sera obtained from 122 HDV genotype 1 and HBV genotype D coinfected, anti‐HIV‐negative patients (71 males; median age 49.8 [21.7‐66.9] years), recruited consecutively in two geographical areas (Italy 69 patients, Romania 53 patients) with different HBV and HDV epidemiology, were tested for HBsAg, HBV‐DNA, HBcrAg, total anti‐HBc, HDV‐RNA, IgM and total anti‐HDV using quantitative assays. Cirrhosis, which showed comparable prevalence in the two cohorts, was diagnosed in 97 of 122 (79.5%) patients. At multivariate analysis, cirrhosis was associated with lower total anti‐HBc/IgM anti‐HDV ratio (OR 0.990, 95% CI 0.981‐0.999, P = .038), whereas disease activity was associated with higher total anti‐HDV (OR 10.105, 95% CI 1.671‐61.107, P = .012) and HDV‐RNA levels (OR 2.366, 95% CI 1.456‐3.844, P = .001). HDV‐RNA serum levels showed a positive correlation with HBV‐DNA (ρ = 0.276, P = .005), HBsAg (ρ = 0.404, P < .001) and HBcrAg (ρ = 0.332, P < .001). The combined quantitative profiling of HBV and HDV serum markers identifies specific patterns associated with activity and stage of chronic hepatitis D (CHD). HDV pathogenicity depends on the underlying active HBV infection in spite of the inhibition of its replication. HDV‐RNA, IgM anti‐HDV, total anti‐HDV, total anti‐HBc, HBsAg and HBcrAg serum levels qualify for prospective studies to predict progressive CHD and identify candidates to antiviral therapy.     

Serologic markers of hepatitis B virus (HBV) and hepatitis D virus infection in carriers of hepatitis B surface antigen who are frequently exposed to HBV

Serum hepatitis B e antigen (HBeAg) and HBV DNA are indicators of active replication of HBV, whereas IgM antibody to hepatitis B core antigen (IgM anti-HBc) may indicate an active immune response to chronic HBV infection. Fifty-eight carriers of hepatitis B surface antigen (HBsAg) who had frequent parenteral exposures were studied for the presence of HBeAg, HBV DNA, IgM anti-HBc and hepatitis delta virus (HDV) serologic markers. Active replication of HBV was detected in 36.2% (25% of drug addicts, 16.7% of thalassemia patients, and 46.9% of hemodialysis patients) and seropositivity for IgM anti-HBc in 55.2% of the HBsAg carriers. Among the 39 HBsAg carriers who were negative for HBeAg, IgM anti-HBc was detected significantly more frequently than HBV DNA (46.1% vs. 5.1%, p less than 0.001). Serologic evidence of HDV infection was detected in 35% of drug addicts, 50% of thalassemia patients and in 9.4% of hemodialysis patients. These data revealed that continued replication of HBV was more frequent in hemodialysis patients than in drug addicts and thalassemia patients who are HBsAg carriers and the opposite was true for the prevalence of HDV infection.     

Chronic hepatitis D virus (HDV) infection in hepatitis B virus carrier chimpanzees experimentally superinfected with HDV

Chimpanzees that were chronic hepatitis B virus carriers and that were superinfected with hepatitis D virus (HDV) apparently developed acute, self-limited type D hepatitis. Reevaluation with a sensitive hybridization-based assay using RNA probes specific for the HDV genome, however, demonstrated that greater than 50% of these animals still had detectable signs of ongoing HDV replication an average of 2.4 y (range, 1-6.4 y) after inoculation. The only positive marker for the presence of HDV was serum HDV RNA; HDV antigen was undetectable in both serum and the liver by an immunoblot assay and immunofluorescence, respectively. Because the detected amount of viral genome was very low, the previous failure to identify the chronic HDV carrier state in the chimpanzee can be attributed to the lower sensitivity of previously described assays.     

Reappearance of hepatitis D virus (HDV) replication in chronic hepatitis B virus carrier chimpanzees rechallenged with HDV

Four chronic hepatitis B virus (HBV) carrier chimpanzees, which had apparently cleared hepatitis D virus (HDV) after a first experimental challenge with HDV, were reinoculated with a homologous strain of HDV. All animals had reappearance of low levels of serum HDV RNA and transient, mild alanine aminotransferase (ALT) elevations, which in two cases correlated with HDV RNA positivity. Plasmas obtained from two chimpanzees after rechallenge were inoculated into two other chronic HBV carrier animals that had recovered from a previous HDV infection. A similar reappearance of HDV RNA in serum (without ALT elevation) was noticed. These same plasmas, however, when inoculated into a chronic HBV carrier chimpanzee never exposed to HDV caused a severe acute hepatitis D. Rechallenge with HDV in chimpanzees apparently recovered from a first HDV infection resulted in the reappearance of HDV replication, sometimes associated with hepatitis. This can be interpreted as reinfection with HDV, but other explanations are possible. Although the serum level of HDV RNA observed after rechallenge with HDV is low, its transmission to individuals susceptible to HDV infection can result in severe acute hepatitis D.     

Chronic HDV (hepatitis delta virus) hepatitis. Intrahepatic expression of delta antigen, histologic activity and outcome of liver disease

The expression of intrahepatic delta antigen (HDAg) was studied in relation to the morphologic features of HDV hepatitis and the outcome of liver disease. The study was performed in 101 patients followed up for an average of 12 years; one or more liver biopsies were available from each patient, giving a total of 167 specimens. The histologic features were assessed using numerical scores. A significant positive relation was observed between the number of HDAg-positive cells and the extent of portal inflammation (Spearman's rank coefficient 0.75). The highest degree of inflammation and intrahepatic expression of HDAg was found before the elimination of the virus, while the outcome of HDV disease was unrelated to the severity of the initial morphologic lesion. These results suggest that the individual immune response may play an important role in the pathogenesis of HDV hepatitis.     

Interaction between HDV and HBV infection in HBsAg-chronic carriers

Summary We studied the interaction between HBV and HDV infection in 149 consecutive subjects with HBsAg positive chronic hepatitis and in 22 chronic HBsAg healthy carriers. Liver HBcAg was detected in 52 (30.4%) of the 171 subjects. Of these 52, 35 were HBV-DNA and HBeAg positive, 11 HBV-DNA positive only; two HBeAg positive only and four were negative for both HBeAg and HBV-DNA. None of the 119 HBcAg-negative subjects had detectable HBV-DNA in serum. HD-Ag in hepatocytes was detected in 31 of the 171 subjects (18%); it was detectable in none of the 22 HBsAg healthy carriers, in four of the 56 patients with chronic persistent hepatitis (7.2%), in six of the 24 patients with chronic lobular hepatitis (25%), in 16 of the 40 patients with chronic active hepatitis (40%) and in five of the 29 with cirrhosis (17%). A presence of anti-HD in serum in the absence of liver HD-Ag was found in 54 of the 171 subjects (32%). This condition was observed not only in patients with a progressive disease (37.7% of chronic active hepatitis or cirrhosis and 33% of chronic lobular hepatitis), but also in healthy carriers (36%) and in chronic persistent hepatitis patients (21.4%). Liver HBcAg was detected in 6.4% of the 31 HD-Ag-positive patients, in 12.9% of the 54 HD-Ag-negative/anti-HD positive, but in 50% of the 86 with no marker of HDV infection. HDV appears to inhibit HBV genome and such inhibition may persist even when anti-HD is the only HDV marker detectable.

---

Interaktionen zwischen HDV und HBV bei chronischen HBsAg-Trägern

Zusammenfassung Bei 149 nacheinander eingewiesenen chronischen HBsAg-positiven Patienten mit chronischer Hepatitis und 22 gesunden HBsAg Trägern wurde die Interaktion zwischen der HBV- und HDV-Infektion untersucht. Bei 52 der insgesamt 171 Personen (30,4%) fand sich HBsAg in den Leberzellen. Bei 35 dieser 52 Fälle wurde HBV-DNA und HBeAg nachgewiesen, in 11 Fällen HBV-DNA allein. Bei den 119 HBcAg-negativen Personen waren HBV-DNA in keinem Fall im Serum nachweisbar. Bei 31 der 171 Personen wurden HD-Ag in Hepatozyten entdeckt (18%). Die 22 gesunden HBsAg-Träger waren alle HD-Ag negativ. Von den 56 Patienten mit chronisch persistierender Hepatitis waren vier positiv (7,2%); von den 24 Patienten mit chronischer lobulärer Hepatitis sechs (25%), von den 40 Patienten mit chronisch aktiver Hepatitis 16 (40%) und von den 29 Patienten mit Zirrhose fünf (17%). 54 der 171 Untersuchten (32%) wiesen im Serum anti-HD auf, ohne daß sich HD- Ag in der Leber nachweisen ließ. Dabei handelte es sich nicht nur um Patienten mit progressivem Krankheitsverlauf (37,7% der Patienten mit chronisch aktiver Hepatitis und 33% derer mit chronischer lobulärer Hepatitis) sondern auch um gesunde Träger (36%) und Patienten mit chronisch persistierender Hepatitis (21,4%). Bei 6,4% der 31 HD-Ag positiven Patienten ließ sich HBcAg in der Leber nachweisen; positiv waren auch 12,9% der 54 HD-Ag-Negativ/anti-HD-Positiven und 50% der Personen ohne Marker für eine HDV-Infektion. Offensichtlich hemmt HDV das HBV-Genom; diese Hemmwirkung kann bestehen bleiben, auch wenn anti-HD der einzige nachweisbare Marker für eine durchgemachte HDV-Infektion ist.

---

---

This study was supported by MURST — Cirrhosis Project, Rome, Italy.     

Expression of hepatitis B virus (HBV) markers in chronic liver disease positive for antibody to hepatitis C virus (HCV)

We investigated expression of HBV markers in chronic liver disease positive for antibody to HCV (anti-HCV). Sera from 107 patients with chronic non-A, non-B liver disease, 65 HBs antigen carriers with chronic liver disease and 14 asymptomatic HBV carriers were tested for the presence of anti-HCV. Anti-HCV was detected in 83 (78%) patients with chronic non-A, non-B liver disease, irrespective of the past history of blood transfusion, and anti-HCV prevalence was similar in each category of chronic liver disease. Fifty-three (64%) out of these 83 sera positive for anti-HCV has also antibodies to HBV. Anti-HBc antibody was detected frequently in liver cirrhotics with hepatocellular carcinoma than in chronic persistent hepatitis, chronic active hepatitis and cirrhotics without hepatocellular carcinoma. In addition, titers of anti-HBc antibody were significantly higher in cirrhotics with hepatocellular carcinoma than in the other groups. On the other hand, anti-HCV was detected in 7 out of 65 patients with HBV-related liver disease. Four out of these 7 were patients with HBV-related hepatocellular carcinoma. Anti-HCV was detected in none of asymptomatic HBV carriers. These findings suggest that infection with both HBV and HCV is likely to cause more serious liver disease than infection with a single agent.     

Mutation at codon 130 in hepatitis B virus (HBV) core region increases markedly during acute exacerbation of hepatitis in chronic HBV carriers

Mutations within T-cell or B-cell epitopes are suggested to have some influence on the clinical course of chronic hepatitis B virus (HBV) infection. To investigate the relationship between liver cell injury and heterogeneity of the HBV core gene, we focused on the sequence of codon 130, which is located on both T- and B-cell epitopes, and serially analyzed the proportion of mutant virus (core130Thr) to wild-type virus (core130Pro) during the exacerbation of chronic hepatitis B. Sera obtained serially from five HBV carriers who had exacerbation of hepatitis, and three asymptomatic HBV carriers (ASCs) with persistently normal serum aminotransferase (ALT) values were studied, using the restriction fragment length polymorphism (RFLP) method. Core130Pro predominated in the sera in the remission state, but core130Thr increased markedly in parallel with ALT elevation and decreased again after the ALT peak, followed by the predominance of core130Pro, in all the five patients. In one patient, the ratio of core130Thr/core130Pro (Thr/Pro) was more than 70% at the ALT peak. On the other hand, in sera from the three ASCs core130Pro always predominated, and no divergence was identified in the ratio of Thr/Pro. Our data suggest that codon 130 is one of the most important immunogenic regions in the HBV core gene and that elevation of Thr/Pro could be the result of immune selection. Received: January 21, 2000 / Accepted: October 6, 2000     

Prevalence of hepatitis delta virus (HDV) infection in chronic hepatitis B patients in eastern Turkey: still a serious problem to consider

Summary. Hepatitis delta virus (HDV) is a serious cause of liver‐related morbidity and mortality. Coexistent infection with HDV tends to aggravate the course of hepatitis B virus (HBV)‐associated liver disease. The aim of this study was to determine the prevalence of HDV infection among patients chronically infected with HBV in the Elazig region, which is in eastern Turkey. A group of 282 patients infected with chronic HBV were investigated for the study. Anti‐HDV seropositivity was evaluated in all patients. The anti‐HDV‐positive patients were further tested for HDV RNA. Severity of liver disease was assessed by liver biopsy. Regression analysis was used to determine the relationship between independent variables and HDV positivity. Of 282 chronic HBV patients, 192 were men (68.1%) and 90 were women (31.9%). The mean age was 43.8 ± 12.7 (between 18 and 73 years). Anti‐HDV was positive in 45.5% of the patients (128/282). Among the 128 anti‐HDV‐positive patients, 116 were checked for HDV RNA and 56.9% were found positive (66/116). Chronic HDV infection rate was therefore present in at least 23.4% of the whole study group (66/282). There were 83 patients with cirrhosis (29.4%) in the study group. Anti‐HDV seroprevalence and HDV RNA presence were higher in those with cirrhosis (61.4% and 42.2%, respectively). No significant relationship was found between anti‐HDV seropositivity and demographic factors such as age, sex and operation or transfusion history except family history. HDV‐RNA‐positive patients had significantly higher ALT and lower albumin levels when compared to HDV‐RNA‐negative patients. HDV‐RNA‐positive patients also had a significantly higher fibrosis stage. In conclusion, these findings demonstrated that HDV infection is endemic and still a serious problem in the Elazig region of eastern Turkey. HDV infection is significantly related to the family exposure and increases the risk of severe liver fibrosis in this region.     

Liver disease activity and hepatitis B virus replication in chronic delta antigen-positive hepatitis B virus carriers

Delta antigen is currently thought to reflect superinfection of the liver with a defective RNA virus (delta agent), requiring helper function from hepatitis B virus for its replication. To assess the influence of delta agent on hepatitis B virus replication in patients persistently infected with both viruses and showing chronic liver disease, we measured serum and liver hepatitis B virus DNA in HBsAg-positive chronic liver disease patients who were either positive or negative for delta antigen in the liver. Hepatitis B virus DNA was assayed in the serum of 21 patients with delta antigen-positive/HBsAg-positive chronic liver disease and in 21 patients with delta antigen-negative/HBsAg-positive chronic liver disease matched for HBeAg/anti-HBe status and underlying liver histology. HBcAg and delta antigen in liver was determined by immunofluorescence or immunoperoxidase staining. In delta antigen-positive/HBsAg-positive chronic liver disease, serum hepatitis B virus DNA was detected transiently in 4 of 21 cases (19%) and was present in these patients at low levels (trace to 2+). In contrast, 9 of 21 (43%) delta antigen-negative/HBsAg-positive chronic liver disease patients were serum hepatitis B virus DNA positive, and five of these had high serum hepatitis B virus DNA levels (3+ to 4+). Serum HBsAg and anti-HBc titers were significantly lower in delta antigen-positive cases and correlated with reduced amount of HBcAg in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of antibodies to proteins encoded by the X-region of hepatitis B virus (HBV) in sera of patients with chronic HBV infection: correlation with other HBV markers

The genome of hepatitis B virus (HBV) contains a fourth open reading frame (ORF), designated X-region. It was the aim of our study to test sera of patients with chronic HBV infection for the presence of antibodies reactive with a 17 kd gene product of the X-ORF. For this purpose, a 35S-X-ORF-encoded protein, synthesized in vitro, was applied as antigen for the detection of antibodies to HBx proteins in sera of 86 individuals. Antibodies reacting with a gene product of the X-ORF were present in 10 out of 24 HBsAg-positive patients with hepatocellular carcinoma (PLC) or liver cirrhosis and in one out of 8 HBsAg-carriers. In addition, the antibodies could also be detected in 6 out of 35 sera from patients with PLC or cirrhosis negative for HBsAg but positive for anti-HBc and anti-HBs. Antibodies to a gene product of the X-ORF can be detected in sera of patients with chronic HBV-related liver disease, independently of HBsAg and the HBeAg/anti-HBe system.     

HBV carriers and HDV superinfection--studies on the cases of HBsAg clearance

We studied the annual clearance rates of hepatitis B surface antigen (HBsAg) and the annual seroconversion rates of HBsAg (HBs seroconversion rates), and the correlation between HBsAg clearance and hepatitis delta virus (HDV) superinfection in hepatitis B virus (HBV) carriers in Japan. Out of 1,029 HBV carriers followed for more than 36 months, 56 cases were cleared of HBsAg from the sera, and 24 of these cases developed hepatitis B surface antibody. The annual clearance rate of HBsAg was 0.94% and the annual HBs seroconversion rate was 0.27%. These rates increased with aging, especially above 30 years of age. Antibody to HDV was detected in three cases with increased serum alanine aminotransferase activity preceding HBsAg clearance. These data indicate that HDV superinfection may play a role in induction of the HBsAg clearance in HBV carriers in Japan.     

乙型肝炎相关肝移植受体供肝植入前后乙型肝炎病毒标志物的变化

目的 了解乙型肝炎相关受体在拉米夫定或拉米夫定 乙型肝炎免疫球蛋白(HBIG)预防下供肝植入前后移植物乙型肝炎病毒(HBV)标志物的变化，探讨移植物HBV再感染的可能机制，为预防复发寻找切入点。方法用酶联放射免疫法、HBVDNA荧光定量法、免疫组织化学LSAB法定期检测78例受体手术前后血清及90例供肝活检组织，重点观察供肝植入前后HBV标志物在肝组织及血清中的动态变化。结果移植时无论受体为HBV活跃复制状态或非活跃复制状态，供肝植入后2h内活检肝组织既无HBVDNA又无HBsAg、HBcAg阳性证据。结论在现有预防措施下，无论受体术前HBV复制状态如何，健康供肝无术中HBV颗粒直接感染的证据，其机制及在远期HBV再感染中的意义尚待进一步研究。