Real Time-PCR HBV-DNA Analysis: Significance and First Experience in Armed Forces

Background

HBV DNA quantitation is used extensively world wide for the diagnosis and monitoring of treatment of Hepatitis B virus (HBV) infection. However, it has still to be popular in India. The aim of this study was to quantitate HBV – DNA by Real time – PCR method in Hepatitis B and in immuno-compromised patients, to compare the results with HBeAg detection and to monitor the response to therapy of chronic Hepatitis B patients to antivirals.

Methods

Ninety one serum samples of Hepatitis group of patients (all HBsAg positive), 41 samples from immuno-compromised patients (all HBsAg negative) and 49 patients of Chronic Hepatitis B group (all HBsAg positive) were the subjects of this first ever study in Armed Forces. Twenty serum samples from healthy volunteers and non-hepatitis B patients served as negative controls. The amplification detection was carried out in a Rotor-Gene 2000-sequence detector

Results

Amongst Hepatitis B group, 33% (30/91) of the samples were positive for HBV-DNA and 26% (24/91) of samples were positive for HBeAg. In the immuno-compromised group of patients 14.6% (6/11) of samples were positive for HIV-DNA and 9.7% (4/41) were positive for HBeAg. Of the Chronic Hepatitis B patients on treatment, all (100%) were positive by HBV-DNA, whereas 29/49 (59.2%) were positive by HBeAg before treatment. After treatment with antivirals, 06/49 (12.2%) were positive by both tests and 11/49 (22.5%) were positive only by HBV-DNA. 32/49 (65.3%) patients became negative serologically after therapy.

Conclusion

HBeAg status did not necessarily reflect HBV-DNA level in the serum, as 10/91 (11%) in the Hepatitis B group, 2/41 (4.9%) in the immuno compromised group and 20/49 (40.8%) patients in the Chronic Hepatitis B group were positive for HBV-DNA but negative for HBeAg. HBV-DNA was not found to be positive amongst any of the negative controls. Real time – PCR is a sensitive and reproducible assay for HBV-DNA quantitation and may be started in Armed Forces referral centers in the near future.Key Words: Real time – PCR, Chronic Hepatitis B, HBV – DNA, Antivirals     

Distribution of hepatitis B virus genotype and cancer predicting precore and basal core promoter mutations

Background

Chronic hepatitis B (CHB) infection which is associated with an increased risk of developing liver disease including cirrhosis and hepatocellular carcinoma. Viral factors that may increase the risk for HCC development include HBV DNA level, genotypes, and naturally occurring mutations such as hepatitis B virus precore (PC) (G1896A) and basal core promoter (BCP) A1762T/G1764A double mutations. HBV genotypes and subgenotypes can significantly influence HBeAg seroconversion rates, viremia levels, mutational patterns that could significantly influence the heterogeneity in clinical manifestations and even response to antiviral therapy.

Method

94 CHB infected individuals with detectable serum HBV DNA levels were studied. HBsAg, HBeAg, anti-HBc IgM antibody estimations were done by ELISA. HBV DNA estimation was done. The HBV genotypes were determined by TSP-PCR and 10 samples randomly selected for DNA sequencing. PC and BCP mutations were determined by DNA sequence analysis of core region.

Result

Of 94 study participant samples with detectable serum HBV DNA levels, 75 were successfully genotyped and sequenced for BCP/PC region. 30/75 (40%) harbored PC and BCP mutations. The total Double mutations of BCP at A1762T/G1764A nucleotide positions, and PC mutation at G1896A nucleotide position were seen in 29.3% and 21.3%, respectively. All 75 isolates were subtype D using TSP-PCR. However, by sequencing 2/10 were subtype A, while 8 were subtype D.

Conclusion

Our study reinforces that D is the predominant genotype in Indian population. It reveals that Indian CHB subjects have increased prevalence of BCP & PC mutations, which possibly may lead to development of HCC.     

Hepatitis B Immunisation in Health Care Workers

Background

Hepatitis B virus (HBV) infection is an important occupational risk in health care workers (HCW). In spite of HBV vaccine availability in Armed Forces, the high prevalence of HBV infection in HCW continues to be a problem. The study was undertaken to study the HBV vaccine-compliance among HCW.

Methods

A cross-sectional study was conducted at a tertiary care hospital. HCW were requested to fill up the pre set questionnaire to assess the HBV vaccination coverage.

Result

Amongst 254 HCW, only 57.7% were vaccinated against HBV. The vaccine compliance was lowest among housekeeping professionals. The mean age at vaccination was high (30.5 years). Amongst the vaccine non-compliant subjects, 34.3% were above 30 years of age. 32.2% HCW completed primary vaccination after spending more than 10 years in the profession. Accessibility of HBV vaccine, knowledge and perception of HBV risk were important factors in vaccine non-compliance.

Conclusion

Due to low and delayed HBV vaccine-compliance, HCW continue to be at the risk of occupational HBV. Health education highlighting occupational risk of HBV, accessibility of vaccine and mandatory vaccination of HCW is recommended to increase HBV vaccine compliance among HCW.Key Words: Health care workers, Hepatitis B virus, Occupational risk, Hepatitis B vaccine     

Spectrum of Acute Viral Hepatitis in Southern India

Background

The incidence of hepatitis-A among adults in India is on the decline as majority develops protective immunity to it by late adolescence. Most of these studies are from northern India. Clinical spectrum of sporadic acute viral hepatitis from southern India has not been well documented.

Methods

A prospective hospital based study was conducted in a large military hospital in southern India. 224 consecutive patients with acute viral hepatitis were studied for their presentation, etiology and clinical features.

Result

Hepatitis-E was detected in 102 (45.4%), hepatitis A in 74 (33%) and hepatitis B in 28 (12.5%) patients. Acute hepatitis C was detected in two patients. 15 patients had a mixed infection. Hepatitis A constituted 41.2% and 31.3% of all cases in the age groups 11-20 and 21-30 years respectively. Cholestasis was present in 68 (30.4%) patients with hepatitis E accounting for most (61.8%) cases. There were four (1.8%) cases of acute liver failure. Two cases were due to hepatitis E and one case each was due to hepatitis A and hepatitis B. A relapsing course was seen in four cases due to hepatitis-A.

Conclusion

Hepatitis A remains a significant cause of sporadic acute viral hepatitis in young adults in southern India.Key Words: Viral hepatitis, Hepatitis A     

Baseline Hepatitis B Virus DNA Level is a Promising Factor for Predicting the 3rd Month Virological Response to Entecavir Therapy: A Study of Strict Defined Hepatitis B virus Induced Cirrhosis

Background:Cirrhosis is a common complication of chronic hepatitis B.It remains unclear if viral and biochemical parameters at baseline affect virological response to entecavir and therefore warrant in...     

Hepatitis B e antigen negative chronic hepatitis in Indian patients : A reality

Background: Hepatitis B e antigen negative chronic hepatitis (e⁻ CHB) with detectable levels of hepatitis B virus DNA (HBV DNA) in serum has been reported in cases from Asia. This study was undertaken to find out prevalence e⁻CHB and to correlate its presence with the clinical status and severity of the illness in cases of chronic liver disease in India.     

Antibody to Hepatitis B Surface Antigen in Vaccinated Health Care Workers

Background

Health care workers (HCWs) in Armed Forces are immunised against Hepatitis B virus (HBV), however they are not subjected to anti-HBs (antibody to Hepatitis B surface antigen) assessment after primary vaccination. The present study was undertaken to determine the protection offered by HBV vaccine in HCW.

Methods

Cross-sectional study was carried out at tertiary care hospital. A total 146 HBV vaccine compliant HCW were evaluated for quantitative anti-HBs by enzyme immune assay.

Result

129 (88.4%) subjects had protective levels of anti-HBs. Higher age at vaccination was an important risk factor in low vaccine response. Decline in anti-HBs with time was evident. Anti-HBs levels were more than 10mIU/ml in subjects even after 11 years of primary vaccination. There was no difference in protection in booster and non booster groups.

Conclusion

Age is the most important factor in HBV vaccine response. Booster dose of HBV vaccine is not necessary in healthy HCW for atleast ten years after primary vaccination. The study recommends early primary vaccination of HCW and ‘initial’ anti-HBs assay for confirmation of vaccine response.Key Words: Anti-Hepatitis B surface antigen, Health care workers, Hepatitis B virus vaccine     

Efficacy and Safety of Bortezomib in Multiple Myeloma Patients with Hepatitis B: A Multicenter Retrospective Study

Background:

The efficacy and safety evidence of bortezomib in multiple myeloma (MM) patients with hepatitis B is vacant. This study aimed to investigate the efficacy and safety of bortezomib in MM patients with hepatitis B in China.

Methods:

From 2006 to 2011, 739 newly diagnosed MM patients were screened for serum hepatitis B virus (HBV) biomarkers. HBV-infected patients were followed for HBV reactivation by monitoring of serum alanine transaminase (ALT) and HBV DNA load. The pattern of HBV reactivation in relation to bortezomib was evaluated. Seven hundred thirty-nine MM patients were included in this study.

Results:

The prevalence of MM patients infected with HBV was 3.4% (n = 25), of which 17 cases were treated with bortezomib. Bortezomib had no significant influence on liver function (ALT before and after treatment: 36.69 ± 8.90 U/L vs. 11.31 ± 2.74 U/L, P = 0.19) and HBV DNA of MM patients with HBV (detectable HBV DNA percentage: 5.9% vs. 11.8%, P = 0.12).

Conclusions:

Bortezomib can be used safely and effectively in MM patients with hepatitis B. HBV prophylaxis and surveillance are recommended during the MM treatment.     

Incidence of Hepatitis E Virus Infection in Recipients of Blood or Blood Products Transfusion

Background

Hepatitis E, generally known to be transmitted faeco-orally, has been shown to have significant transmission by blood borne route. Paucity of data on asymptomatic viremia in blood donors and higher incidence of Hepatitis E in haemodialysis patients and thalassemics mandate a prospective study of blood recipients to elucidate the exact incidence and natural history of post transfusion Hepatitis E.

Methods

A total of 2000 recipients of blood or blood products transfusion were followed up for two months to detect development of post transfusion Hepatitis E, by clinical examination, transaminases and immunoglobulin M anti hepatitis E virus (IgM anti HEV). Estimation of hepatitis E virus ribonucleic acid (HEV RNA) was done in patients with elevated levels of transaminases.

Result

Out of 2000 patients, 5(0.25%) were positive for IgM anti HEV at the time of transfusion and were excluded from the study. Rest of 1995 patients were followed up for two months post transfusion. A total of 1303 (65.3%) patients were followed up for two months and 1636 (82.0%) patients at least once in two visits. None of the followed up patients reported development of jaundice or had clinically evident hepatitis, although 62 patients had raised transaminases detected at either one or both the visits.

Conclusion

All followed up patients were tested for IgM anti HEV at both the visits and none were found to be positive. Patients with raised transaminases were subjected to HEV RNA and all were found to be negative.Key Words: Hepatitis E, Post transfusion, Parenteral transmission     

Outbreak of Viral Hepatitis E in a Regimental Training Centre

Background

An outbreak of viral hepatitis occurred in a regimental centre with 265 cases occurring during a 3 months period.

Methods

190 serum samples were tested for IgM antibodies against viral hepatitis E by Enzyme Immuno Assay (EIA) and for antibodies against Hepatitis A and Hepatitis B viruses. Epidemiological investigation comprised review of surveillance data, filling up epidemiological case sheet, sanitary survey, inspection of water supplies and bacteriological examination of water for coliforms.

Result

97.4% of the serum samples were positive for IgM antibodies against Hepatitis E virus. Two leaks were detected in water pipelines, which were passing through contaminated areas around improperly functioning septic tanks and soak pits. The attack rate among recruits being supplied water through leaking pipelines was 11.1% whereas it was 2.89% in those not directly exposed. This difference was statistically significant (p<0.001). Bacteriological examination of water showed a high coliform count.

Conclusion

The outbreak of viral hepatitis E occurred due to sewage contamination of water pipelines.Key Words: Hepatitis E, Outbreak     

低水平乙型肝炎病毒DNA慢性乙型肝炎患者血清乙型肝炎病毒RNA水平及其影响因素研究

背景　导致乙型肝炎病毒（HBV）持续性感染的根本原因是共价闭合环状DNA（cccDNA）的持续性存在,现有的血清学指标难以准确反应肝细胞内cccDNA的存在状态,血清HBV RNA为cccDNA的直接转录体,能精确地反映肝细胞中cccDNA的存在状态。目的　观察低水平HBV DNA慢性乙型肝炎（CHB）患者血清HBV RNA水平,并分析其影响因素。方法　回顾性选取2018年5—7月于湖南师范大学附属第一医院确诊且符合研究标准的CHB患者,患者均处于HBV DNA低水平状态。依据患者乙肝e抗原（HBeAg）情况将患者分为HBeAg阳性患者和HBeAg阴性患者,比较其年龄、性别、血清丙氨酸氨基转移酶（ALT）水平、血清天冬氨酸氨基转移酶（AST）水平、血清乙肝表面抗原定量（qHBsAg）水平、血清HBV DNA水平、血清HBV RNA水平、血清HBV RNA水平低于检测下限发生率,分析CHB患者血清HBV RNA水平及其低于检测下限的影响因素,以及血清HBV RNA水平与ALT、AST、qHBsAg、HBeAg、HBV DNA的相关性。结果　72例患者中HBeAg阳性12例,HBeAg阴性60例。HBeAg阳性患者年龄小于HBeAg阴性患者,血清AST、qHBsAg、HBV RNA水平高于HBeAg阴性患者,血清HBV DNA水平、血清HBV RNA水平低于检测下限发生率低于HBeAg阴性患者（P<0.05）。多因素线性回归分析结果显示,HBeAg（B=0.837,t=2.634,P=0.010）是CHB患者血清HBV RNA水平的影响因素（P<0.05）。多因素Logistic回归分析结果显示,qHBsAg〔OR=1.682,95%CI（1.055,2.681）,P=0.029〕、HBeAg〔OR=3.85,95%CI（1.068,13.880）,P=0.039〕是CHB患者血清HBV RNA水平低于检测下限的影响因素。CHB患者血清HBV RNA水平与qHBsAg（rs=0.322,P=0.006）、HBeAg（rs=0.235,P=0.047）相关。在HBeAg阳性患者中,血清HBV RNA水平与qHBsAg（rs=0.848,P<0.001）、HBeAg（rs=0.725,P=0.008）相关。结论　不同HBeAg情况的低水平HBV DNA CHB患者血清HBV RNA水平存在较大差异,HBeAg是CHB患者血清HBV RNA水平的影响因素,qHBsAg、HBeAg是CHB患者血清HBV RNA水平低于检测下限的影响因素。     

Efficacy and Safety of Tenofovir and Lamivudine in Combination with Efavirenz in Patients Co-infected with Human Immunodeficiency Virus and Hepatitis B Virus in China

Background:

The prevalence of hepatitis B virus (HBV) infection is high among individuals infected with human immunodeficiency virus (HIV) in China. Both HIV and HBV can be treated with tenofovir disoproxil fumarate (TDF) and lamivudine (3TC), so we evaluated the safety and efficacy of combination antiretroviral therapy (ART) that included TDF, 3TC, and efavirenz (EFV) among ART-naive individuals who were co-infected with HIV and HBV.

Methods:

One hundred HIV/HBV co-infected ARV-naive individuals were started on the regimen of TDF, 3TC, and EFV, and the levels of plasma HBV DNA, HIV RNA, and biochemical evaluation related to the function of liver and kidney were analyzed.

Results:

Concerning efficacy, this study found that by week 48, the vast majority co-infected participants receiving this ART regimen had undetectable HBV DNA levels (71%) and/or HIV RNA levels (90%). Concerning safety, this study found that the median estimated glomerular filtration rate of participants decreased from baseline (109 ml·min⁻¹·1.73 m⁻²) to week 12 (104 ml·min⁻¹·1.73 m⁻²) but was almost back to baseline at week 48 (111 ml·min⁻¹·1.73 m⁻²).

Conclusion:

This combination ART regimen is safe and effective for patients with HIV/HBV co-infection.

Trial Registration:

ClinicalTrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT01751555","term_id":"NCT01751555"}}NCT01751555; https://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT01751555","term_id":"NCT01751555"}}NCT01751555.     

慢性乙型肝炎肝损伤患者临床特征与e抗原的关系

目的：对轻度及中度肝损伤的e抗原（Hepatitis Be antigen,HBeAg）阳性慢性乙型肝炎（chronic hepatitis B,CHB）和e抗原阴性CHB患者的临床特征进行比较。方法：对44例HBeAg阳性CHB和36例HBeAg阴性CHB进行病史询问,检测肝功能、血常规、血清乙型肝炎病毒（HBV）免疫学标志和HBVDNA,并进行腹部B超检查。结果：HBeAg阳性CHB组和HBeAg阴性CHB组在性别构成比例、HBV感染阳性家族史和肝脾肿大方而差异尤显著意义（P〉0．05）。HBeAg阳性CHB组和HBeAg阴性CHB组平均年龄分别为（29．23±8．246）岁和（41．53±13．146）岁（P〈0．001）;HBV感染年限≥5年者分别占36．36％和63．89％（P〈0．05）：HBVDNA滴度对数值平均为8．27±1．328和6．02±1．536（P〈0．01）。结论：在2组患者肝功能、血常规等临床特征一致的情况下,HBeAg阴性CHB患者有年龄偏大、感染年限较长和HBVDNA滴度较低的特点。     

Oral Oxymatrine Preparation for Chronic Hepatitis B: A Systematic Review of Randomized Controlled Trials

Objective

To evaluate the efficacy and safety of oral oxymatrine preparation for the treatment of chronic hepatitis B (CHB).

Methods

Randomized controlled trials (RCTs) on oral oxymatrine preparation in treating patients with CHB were retrieved until October 2013 by searching PubMed, the Cochrane Library, Embase and four Chinese databases, irrespective of language and publication status. Data extraction and data analyses were conducted according to the Cochrane standards. The risk of bias for each included trials and the quality of evidence on pre-specified outcomes were assessed. The RevMan software was used for statistical analyses.

Results

Totally 52 RCTs enrolling 5,227 participants were included, of which 51 RCTs were included in meta-analyses. Oral oxymatrine preparation including oxymatrine capsule and oxymatrine tablet were associated with statistically significant effect on the clearance of hepatitis B virus (HBV) DNA, HBV surface antigen and HBV e antigen, and were beneficial to the normalization of serum alanine aminotransferase and aspartate aminotransferase. Nevertheless, the overall methodological quality and the quality of evidence in the included trials were poor. In addition, safety of oral oxymatrine preparation was not confirmed.

Conclusions

Oral oxymatrine preparation showed some potential benefits for patients with CHB. However, the overall quality of evidence was limited and the safety of oral oxymatrine preparation for CHB patients was still unproven. More high quality evidence from rigorously designed RCTs is warranted to support the clinical use of oral oxymatrine preparation for patients with CHB.

     

慢性乙型肝炎患者HBV基因表型与血清学测定的临床意义

目的 分析慢性乙型肝炎患者HBV基因表型和血清学特征,以探讨二者的临床意义。方法 检测210例慢性乙型肝炎患者HBV DNA载量和HBV 基因亚型、HBV血清标志物HBsAg和HBeAg以及ALT水平,分析免疫耐受期、免疫清除期、低水平复制期和复发期4个时相中HBV基因亚型与血清标志物水平、HBsAg效价与HBV DNA载量、HBeAg及ALT水平的相关性。结果 慢性乙型肝炎患者HBV基因型主要为HBV DNA高载量的B2和中等载量的C2亚型,阳性率分别为59.0%和25.2%。B2亚型与HBsAg、HBeAg和ALT水平均呈显著正相关(P<0.01),C2亚型与各指标水平均无显著相关性(P<0.05)。乙型肝炎不同时相的基因亚型阳性率之间差异无统计学意义(P>0.05),但HBsAg水平与HBV DNA量、HBeAg和ALT水平分别存在不同的相关性。结论 HBV基因分型和血清学检测可早期预测慢性乙型肝炎患者长期疾病进展结果。     

Correlations between serum hepatitis B surface antigen and hepatitis B core antibody titers and liver fibrosis in treatment-naïve CHB patients

Background

Previous studies have revealed that quantitative hepatitis B surface antigen (HBsAg) or hepatitis B core antibody (qAnti-HBc) levels can be used as predictors of treatment response in both interferon-α and nucleoside analogue therapies. Few data have been published regarding the relationship between quantitative HBsAg or Anti-HBc levels and liver fibrosis stages in patients with chronic hepatitis B (CHB).

Prevalence of occult hepatitis B amongst Indian human immunodeficiency virus type 1 infected individuals—a pilot study

Background

The diagnosis of hepatitis B is routinely based on the detection of hepatitis B surface antigen (HBsAg) only. However, occult hepatitis B virus (HBV) infection (OBI), which is defined as infection with positive hepatitis B core antibody (anti-HBc) antibodies, positive DNA (deoxyribonucleic acid) PCR (polymerase chain reaction), and undetectable HBsAg, as well as anti-HBs antibodies in serum or plasma of HBV infected individuals, will remain undetected using this screening diagnostic approach of detecting HBsAg. The current study aims in studying the prevalence of the OBI amongst human immunodeficiency virus type 1 (HIV-1) infected individuals who have not been exposed to anti-retroviral therapy.

Method

Estimation of HBsAg, anti-HBs, and anti-HBc total antibody status amongst 100 HIV-1 infected study participants was carried out using enzyme-linked immunosorbent assay (ELISA) kits. Detection of HBV-DNA was carried out by in-house qualitative PCR. CD4 + T lymphocyte counts were analysed using Becton Dickinson''s (BD) FACSCount™ system.

Results

The median age of the HIV-1 infected study population was 35 years (range: 22–67), with the gender distribution being 53 males and 47 females. The mean CD4 T lymphocyte count of the study participants was 210/mm³. Overall, serological evidence of HBV infection was observed in 28% of the HIV-1 infected study participants. There was 5% seropositivity for HBsAg, of which 2% were additionally positive for HBV-DNA-PCR. “Anti-HBc alone” status was seen in 18% of study participants, this being statistically higher in those with CD4 T lymphocyte counts < 200/mm³. While there was a single specimen with co-positivity for anti-HBc total antibodies and HBV-DNA, 5% of the in the study population exhibited anti-HBs antibodies positivity, with one sample exhibiting dual positivity for HBsAg and anti-HBs antibodies.

Conclusion

Occult HBV infections may contribute to chronic liver damage, and associ-ated reactivation amongst immunocompromised individuals, HIV-1 in-fected being a subset of them. “Anti-HBc” testing followed by HBV-DNA detection by PCR can be utilised for such populations to detect OBIs. Early detection of hepatitis B viraemia will be important for deciding the antiviral therapeutic protocol so as to avoid evolution of antiviral resistance in the circulating HBV strains in HIV-1 infected individuals harbouring OBIs.Key Words: human immunodeficiency virus, occult hepatitis B     

慢性乙型肝炎患者血清HBVDNA载量与肝功能指标关系研究

目的研究慢性乙型肝炎患者不同临床分型与HBVDNA载量及肝功能之间的关系。方法分别采用实时荧光定量PCR法及速率法对482例慢性乙型肝炎(CHB)进行血清HBVDNA定量和ALT、AST检测,根据其临床分型分为e--CHB、e+-CHB,分别观察其与HBVDNA载量、ALT、AST水平关系。结果 e--CHB与e+-CHB相比,前者年龄远大于后者(P<0.01),血清HBVDNA载量低于后者(P<0.01),血清ALT、AST水平高于后者(P<0.01)。e--CHB组随血清HBVDNA载量升高,其ALT、AST水平进行性升高(P<0.05),e+-CHB组血清ALT、AST水平与HBVDNA载量无关。结论 CHB不同临床分型在病毒学、生化学存在明显不同,对于e+-CHB患者HBeAg阳性是判断HBV复制的良好指标,其体内HBVDNA载量与肝脏炎症程度无关。e--CHB患者体内HBVDNA载量与肝内炎症程度相关,应重视对e--CHB患者的随访和治疗。