Hepatitis B variants among Egyptian patients undergoing hemodialysis.

Hepatitis B virus (HBV) infection is a serious nosocomial infection that affects patients undergoing hemodialysis (HD). However, certain HBV variants are not detected by routine serological tests in Egyptian dialysis units because of mutations that change important viral antigens (Ags). Of note, these mutations can result in the appearance of different HBV variants with different clinical manifestations. Thus, the present study aimed to assess different clinical forms of HBV infections and viral genotypes among patients undergoing HD in the Ismailia governorate of Egypt. To this end, serum samples were collected from 150 patients undergoing HD and screened for HBV-DNA using a nested PCR technique. Positive samples were then screened for HBV serological markers (hepatitis B core antibody [HBcAb], hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B e antigen and hepatitis B e antibody) using ELISA and the HBV viral load quantitated by qPCR. HBV genotypes were detected by direct sequencing of the partial surface (S) gene. The most common clinical form of HBV infection in our study cohort was overt HBV infection (10%); followed by seropositive occult hepatitis B infection (7.3%), most of whom had an isolated HBcAb. The least common form was the precore mutant (1.3%). All HBV isolates were genotype D. This study reveals the importance of HBcAb and PCR in screening for HBV, especially for detection of occult hepatitis B infection.

Basal core promoter and precore mutations among hepatitis B virus circulating in Brazil and its association with severe forms of hepatic diseases

BACKGROUND In Brazil, few studies have investigated the prevalence of infection with the precore (PC) and basal core promoter (BCP) mutants of the hepatitis B virus (HBV). OBJECTIVES This study aimed to analyse the frequency of PC and BCP mutations among patients infected with HBV and to evaluate the association between the variants and advanced hepatic disease. METHODS A total of 161 patients infected with HBV were studied. To identify PC and BCP mutations, a 501-bp fragment of HBV DNA was amplified and sequenced. FINDINGS PC and BCP regions from HBV strains were successfully amplified and sequenced in 129 and 118 cases, respectively. PC and BCP mutations were detected in 61.0% and 80.6% of the cases, respectively. The A1762T/G1764A variant was identified in 36.7% of the patients with grade 1 and 2 liver fibrosis (29/79) and in 81.8% of the patients with grade 3 and 4 liver fibrosis (9/11) (p < 0.01); in 76.9% of the patients with cirrhosis (10/13) and in 38.1% of the patients without cirrhosis (40/105) (p = 0.01); and in 77.8% of the patients with hepatocellular carcinoma (HCC) (7/9) and in 39.4% of the patients without HCC (43/109) (p = 0.03). MAIN CONCLUSIONS A high prevalence of HBV PC and BCP mutants was found. The A1762T/G1764A variant was independently associated with advanced forms of liver fibrosis, hepatic cirrhosis, and HCC.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication.

Prodromal fever indicates a high risk of liver failure in acute hepatitis B.

OBJECTIVES: The role of prodromal fever in the clinical course of acute hepatitis B virus (HBV) infection is still largely unclear. This study was conducted to investigate the factors associated with prodromal fever and its role in the development of acute liver failure (ALF) in patients with acute hepatitis B (AHB). METHODS: Inpatients with AHB diagnosed between January 2006 and December 2010 were evaluated and followed. Clinical manifestations, results of laboratory tests, and outcomes were compared between patients with and without prodromal fever. The diagnosis of AHB was based on the discrete onset of symptoms, jaundice, abnormal liver function tests, the detection of high-titer IgM antibody to hepatitis B core antigen (anti-HBc), and a compatible clinical history. RESULTS: A total of 618 AHB inpatients were identified during the study period, of whom 102 (16.5%) had prodromal fever and 41 (6.6%) developed ALF. Prodromal fever indicated more severe liver injury and was independently associated with hepatitis B e antigen (HBeAg) negativity. The occurrence of ALF was more common in febrile patients than in non-febrile patients (18.6% vs. 4.3%, p<0.001). Multivariate logistic regression showed prodromal fever and temperature >38.0°C to be independently associated with the risk of ALF, with an odds ratio (95% confidence interval) of 3.5 (1.4-8.6) and 7.1 (2.6-19.7), respectively. CONCLUSIONS: AHB patients with prodromal fever, which is associated with a lack of HBeAg due to HBV mutation, are at high risk of ALF. Febrile patients with AHB should be managed with particular care.

Severe de novo Hepatitis B Recovered from Late-Onset Liver Insufficiency with Prolonged Ascites and Hypoalbuminemia due to Hepatitis B Virus Genotype Bj with Precore Mutation.

De novo hepatitis B is associated with a high risk of hepatic failure often resulting in fatal fulminant hepatitis even when nucleotide analogues are administered. A 77-year-old female developed de novo hepatitis B after R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) treatment for diffuse large B-cell lymphoma. Hepatitis B virus (HBV) isolated from the patient was of genotype Bj, with a precore mutation (G1896A) exhibiting an extremely high viral load at the onset of hepatitis. She showed markedly high levels of transaminase with mild jaundice on admission and rapid decrease of prothrombin activity after admission. Although acute liver failure was averted by the administration of entecavir and corticosteroid pulse therapy, liver volume decreased to 860 ml, and marked hypoalbuminemia accompanying massive ascites occurred 2 months after the onset of hepatitis and persisted for 3 months with high levels of HBV DNA and mild abnormal alanine aminotransferase levels. Frequent infusions of albumin solution, nutrition support, and alleviation therapy showed limited effect. However, overall improvement along with HBV DNA reduction was observed after increasing the dose of entecavir and completion of prednisolone that was administered with a minimum dose for adrenal insufficiency. An immediate and sufficient suppression of virus replication with potent antiviral therapy is critical, particularly in patients infected with HBV precore mutation (G1896A) and/or Bj genotype, which may have a high viral replication and direct hepatocellular damage.

Precore/basal core promoter mutants quantification throughout phases of hepatitis B virus infection by Simpleprobe.

AIM: To investigate precore/basal core promoter (PC/BCP) mutants throughout hepatitis B virus (HBV) infection and to determine their relationship to hepatitis B early antigen (HBeAg) titers. METHODS: We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012. None of the patients received antiviral therapy. HBV DNA from serum, was quantified by real-time PCR. The HBV genotype was determined by direct sequencing of the S gene. We used the Simpleprobe ultrasensitive quantitative method to detect PC/BCP mutants in each patient. We compared the strain number, percentage, and the changes in PC/BCP mutants in different phases, and analyzed the relationship between PC/BCP mutants and HBeAg by multiple linear regression and logistic regression. RESULTS: Patients with HBV infection (n = 191) were assigned to groups by phase: Immune tolerance (IT) = 55, Immune clearance (IC) = 67, Low-replicative (LR) = 49, and HBeAg-negative hepatitis (ENH) = 20. Of the patients (male, 112; female, 79) enrolled, 122 were HBeAg-positive and 69 were HBeAg-negative. The median age was 33 years (range: 18-78 years). PC and BCP mutation detection rates were 84.82% (162/191) and 96.86% (185/191), respectively. In five HBeAg-negative cases, we detected double mutation G1896A/G1899A. The logarithm value of PC mutant quantities (log10 PC) significantly differed in IT, IC, and LR phases, as well as in the ENH phase (F = 49.350, P < 0.001). The logarithm value of BCP mutant quantities (log10 BCP) also differed during the four phases (F = 25.530, P < 0.001). Log10 PC and log10 BCP values were high in the IT and IC phases, decreased in the LR phase, and increased in the ENH phase, although the absolute value at this point remained lower than that in the IT and IC phases. PC mutant quantity per total viral load (PC%) and BCP mutant quantity per total viral load (BCP%) differed between phases (F = 20.040, P < 0.001; F = 10.830, P < 0.001), with PC% and BCP% gradually increasing in successive phases. HBeAg titers negatively correlated with PC% (Spearman's rho = -0.354, P < 0.001) and BCP% (Spearman's rho = -0.395, P < 0.001). The negative correlation between PC% and HBeAg status was significant (B = -5.281, P = 0.001), but there was no such correlation between BCP% and HBeAg status (B = -0.523, P = 0.552). CONCLUSION: PC/BCP mutants become predominant in a dynamic and continuous process. Log10 PC, log10 BCP, PC% and BCP% might be combined to evaluate disease progression. PC% determines HBeAg status.

Spontaneous reactivation of hepatitis B virus (HBV) infection in patients with resolved or occult HBV infection.

Reactivation of a former hepatitis B virus (HBV) infection can be triggered by immunosuppressive therapy, diseases associated with an immunocompromised state, organ transplantation or the withdrawal of antiviral drugs. Despite the absence of such risk factors, a spontaneous reactivation of HBV replication occurred in two elderly patients with resolved or occult HBV infection. A 73-year-old male underwent coronary artery bypass grafting in October 2008, and was negative for HBsAg but positive for anti-HBs. In July 2009, his serum became positive for HBsAg, HBeAg and HBV DNA (6.4 log copies/ml; genotype C), but negative for anti-HBc IgM, with abrupt elevation of the liver enzymes. The entire genomic sequence of HBV recovered from this patient revealed no mutations in the core promoter and precore regions that interfere with HBeAg production. A 76-year-old male with a history of endoscopic mucosal resection for esophageal cancer in 2002 and an initial diagnosis of diabetes mellitus in 2009, at which time he was negative for HBsAg. He was found to be positive for HBsAg in September 2012 during a laboratory examination performed prior to the resection of recurrent esophageal cancer, despite a low HBV load (2.1 log copies/ml). Three months later, without the administration of any anticancer drugs, the HBV DNA (genotype B) level increased to 5.1 log copies/ml. A precore G1896A variant with high quasispecies diversity was recovered from the patient. Aging, surgical stress and complication of disease(s) associated with compromised immunity, such as cancer, arteriosclerosis and diabetes mellitus may trigger spontaneous HBV reactivation.

Molecular analysis of the interspousal transmission of hepatitis B virus in two Japanese patients who acquired fulminant hepatitis B after 50 and 49 years of marriage.

A 71-year-old (C1I) and 69-year-old (C2I) Japanese female contracted fulminant hepatitis B after 50 and 49 years of marriage, respectively. Both index cases exhibited high levels of anti-HBc IgM antibodies (24.2 and 31.5 S/CO, respectively), suggestive of acute hepatitis B virus (HBV) infection, although they had no discernible risk factors for HBV infection, except for chronically HBV-infected spouses with detectable HBV DNA (3.3 log copies/ml [C1S: 72-year-old] and 7.2 log copies/ml [C2S: 71-year-old]). The HBV genotype/subgenotype was identical in each couple (B/B1 or C/C2). The HBV isolates from the index cases and spouses shared a nucleotide sequence identity of 99.5% and 99.7%, respectively, over the entire genome, and these four isolates had the highest nucleotide sequence identity of only 97% to HBV isolates deposited in DNA databases. Phylogenetic trees confirmed a close relationship of the HBV isolates between C1I and C1S and between C2I and C2S, supported by a high bootstrap value of 100% within each couple, indicating the transfer of HBV infection between spouses. These four isolates shared a precore mutation of G1896A known to be associated with fulminant hepatitis B. Although the history of sexual contact within a reasonable incubation period was obscure for one stable, monogamous couple (C1I and C1S), the other couple had a monogamous sexual relationship within six months prior to disease onset. This study indicates that two elderly Japanese patients with fulminant hepatitis B acquired HBV infection via interspousal (most likely sexual) transmission during long-lasting marriages.

[Basic core promoter and precore mutations of hepatitis B virus]. / Les mutants précore et du promoteur basal du core du virus de l'hépatite B.

Since hepatitis B virus (HBV) replication involves an error-prone reverse transcription step, the rate of nucleotide changes during replication is higher than that found in other DNA viruses. As a result, HBV has a "quasispecies" distribution in infected individuals. Such selection processes to allow the emergence of variant viruses, such as precore (PC) and basal core promoter (BCP) mutants. The dominant PC variant is a point mutation of G to A at nucleotide 1896 (G1896A) that produces a premature stop codon, which terminates translation of the precore protein, resulting in the lack of HBe-antigen synthesis. BCP variants at nucleotides 1762 (A1762T) and 1764 (G1764A) likewise impair the production of HBeAg but at the transcriptional rather than the translational level. The impact of mutations in the precore and basal core promoter regions of the HBV virus on the course of chronic liver disease is not well established.

A comparison of hepatitis B viral markers of patients in different clinical stages of chronic infection.

PURPOSE: Hepatitis B viral markers may be useful for predicting outcomes such as liver-related deaths or development of hepatocellular carcinoma. We determined the frequency of these markers in different clinical stages of chronic hepatitis B infection. METHODS: We compared baseline hepatitis B viral markers in 317 patients who were enrolled in a prospective study and identified the frequency of these tests in immune-tolerant (IT) patients, in inactive carriers, and in patients with either hepatitis B e antigen (HBeAg)-positive or HBeAg-negative chronic hepatitis or cirrhosis. RESULTS: IT patients were youngest (median age 27 years) and HBeAg-negative patients with cirrhosis were oldest (median age 58 years) (p = 0.03 to <0.0001). The male to female ratio was similar both in IT patients and in inactive carriers, but there was a male preponderance both in patients with chronic hepatitis and in patients with cirrhosis (p < 0.0001). The A1896 precore mutants were most prevalent in inactive carriers (36.4%) and HBeAg-negative patients with chronic hepatitis (38.8%; p < 0.0001), and the T1762/A1764 basal core promoter mutants were most often detected in HBeAg-negative patients with cirrhosis (65.1%; p = 0.02). Genotype A was detected only in 5.3% of IT patients, and genotype B was least often detected in both HBeAg-Positive patients with chronic hepatitis and cirrhosis (p = 0.03). The hepatitis B viral DNA levels were lowest in inactive carriers (2.69 log(10) IU/mL) and highest in IT patients (6.80 log(10) IU/mL; p = 0.02 to <0.0001). At follow-up, HBeAg-positive and HBeAg-negative patients with cirrhosis accounted for 57 of 64 (89.1%) liver-related deaths (p < 0.0001). CONCLUSION: Differences in baseline hepatitis B viral markers were detected in patients in various clinical stages of hepatitis B virus infection. HBeAg-positive and HBeAg-negative patients with cirrhosis accounted for the majority of the liver-related fatalities.

Replicative efficiency and pathogenicity of hepatitis B virus e-minus precore variant.

To study the replicative efficiency and pathogenicity of hepatitis B virus precore variant (A1896), anti-hepatitis B virus e antigen (HBe) titre was studied in naturally occurring wild-type virus infection, A1896 variant infection and dual infection. Higher titre of anti-HBe was found in patients with no virus replication and in patients coinfected with the wild-type virus and A1896 variant, which suggest that anti-HBe may either act as an inhibitor of virus replication or as selective pressure for the A1896 variant. Three site-directed mutants were constructed in the duck hepatitis B virus (DHBV) precore region. A frame shift in the encapsidation signal region abolished replication of DHBV; mutation in the initiation codon of the precore and mutation to generate a termination codon at the distal region of the precore resulted in decreased replication in the duck model. More significant pathological changes were found in the liver tissues of ducks infected with the mutant which mimicked the HBV A1896 variant.

Interferon Treatment on HBeAg Positive Chronic Hepatitis B with HBV Precore Mutant in Liver Tissue / 대한내과학회지

BACKGROUND: This study investigates the prevalance of HBV precore mutant in chronic B hepatitis patients and whether HBV precore mutants affect hepatic inflammation and response to interferon alfa. METHODS: HBV DNA in liver tissue from 48 chronic hepatitis patients was amplified by polymerase chain reaction. The HBV precore mutants were detected by direct sequencing of amplified PCR products. Thirty-three HBeAg-positive patients (Group 1: wild- type, Group 2: mixed) were received 3-6 MU INF three times a week for 4-6 months. We did follow-ups for at least six months(mean : Group 1-11.3, Group 2- 13.7 months). A complete responder was defined as persistent(>6 months) normalization of transaminase and loss of HBeAg and/or seroconversion. RESULTS: The HBV precore mutants were found in 15 cases(31.2%) among 48 patients: 7 cases(21.2%) in 33 HBeAg-positive patients and 8 cases(53.3%) in 15 HBeAg-negative patients. The HBV precore mutants were more frequently found in HBeAg-negative patients(p= 0.043). Differences in severity of hepatic pathology were not observed in the wild-type versus mutant-type chronic hepatitis B patients(p =1.00). Initial response rate was not significantly different between two Groups(p= 0.228), but complete response rate had a lower tendency in Group 2 (p=0.073). CONCLUSION: There is a tendency for HBV precore mutants to be less responsive to INF therapy than wild type. Therefore the patients with chronic hepatitis B should be treated as early as possible in natural history of their liver disease before the emergence of HBV precore mutants.

A Study on HBV Precore Mutant in Liver Tissues of Chronic Hepatitis B Patiets / 대한간학회지

BACKGROUND/AIMS: In order to determine the relationship between the HBV precore mutant and the severity of liver disease in Korea, we performed liver biopsies in patients with HBV related chronic liver disease and compared the types of mutations and histologic findings in the same liver tissue simultaneously. METHODS: HBV DNA in liver tissues was amplified by polymerase chain reaction (PCR). The precore mutants were detected by PCR-SSCP(single strand conformation polymorphism), cloning the amplified PCR products and direct sequencing for them. RESULTS: 1. HBV DNA was detected in liver tissues of 28 cases among 30 patients with PCR. And with SSCP, the most cases were mixed type infections. 2. The HBV precore mutants were found in 12 cases among the total number of 28 cases(42.9%) and all mutations were G to A change at nucleotide 1896, creating a stop codon at codon 28. However, 10 cases among 12 mutants were associated with simultaneous another mutation at different positions or regions;9 cases at core gene region, 2 cases at nucleotide 1856(C to T change at codon 15), one case at core promoter, and one case with double mutations at nucleotide 1837 and 1846 respectively. Also, all HBV precore mutants were combined with wild type HBV sequence. 3. The relationship between HBV precore mutants and HBeAg status revealed that 4 cases from 13 HBeAg positive(30.8%) and 8 from 15 HBeAg negative or Anti-Hbe positive(53.3 %) were mutants. 4. In analysis of the types of mutants and histopathological findings of liver diseases, 6 among 15 chronic active hepatitis(40.0%), all 3 cases with hepatocellular carcinoma(100,0 %), 2 among 4 asymptomatic carriers with minimal histopathologic changes(50.0%) and a case with chronic lobular heaptitis(100.0%) showed precore region mutation. CONCLUSION: The patterns of HBV precore mutants in Korea could be summarized as followings. Firstly, most of the mutations are composed of G to A change at nucleotide 1896. Secondly, the most of the mutants at nuclmtide 1896 have been associated with simultaneous mutations at core promoter, core gene, and rarely at other positions, and manifested usua'ly mixed type viremic conditions. Thirdly, although precore mutation could be occurred in asymptomatic carrier, this type of mutation might be closely related with chronic or severe liver disease. However, it needs further investigations hereafter.