Epstein-Barr viral load measurement as a marker of EBV-related disease.

Epstein-Barr virus (EBV) is associated with a wide variety of benign and neoplastic diseases. EBV viral load assays that may prove useful in rapid assessment of disease status are now available. The two most common approaches to viral load measurement are quantitative, competitive PCR, and real-time PCR. Laboratory studies have shown that these assays are sensitive and specific for measuring EBV DNA in blood samples. Clinical investigations suggest a role for viral load measurement in predicting and monitoring EBV-associated tumors, including nasopharyngeal carcinoma, post-transplant lymphoproliferative disorder, Hodgkin's disease, and AIDS-related lymphoma. These new laboratory tools show promise in improving clinical management of affected patients.     

Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrow transplant patients

BACKGROUND: In bone marrow and stem cell transplant patients, the widespread use of preemptive cytomegalovirus (CMV) antiviral therapy necessitates faster, more precise, and more sensitive quantitative laboratory methods for serial viral load monitoring. METHODS: We developed a novel CMV viral load assay using real-time PCR of plasma DNA prepared by an automated robotic workstation. Fluorescent hybridization probes directed at the glycoprotein B (gB) gene (or EcoRI D region) of CMV were used to detect and quantify PCR products. The beta-globin gene was amplified in parallel to control for the efficiency of the extraction and PCR steps. RESULTS: The assay was linear (R = 0.999) from a lower detection limit of 125 copies/mL to 5 x 10(9) copies/mL with a PCR efficiency of 1.975 (gB) or 2.02 (EcoRI D). The viral loads determined by PCRs directed at these two different viral targets were no different (n = 53; R = 0.928). The interassay CV was 3.5%, and the intraassay CV was 1-4%. Compared with a commercially available quantitative competitive PCR assay (Roche MONITOR; R = 0.59), the mean CMV viral load by real-time PCR was 3.1 times higher (mean ratio; P = 0.002). The diagnostic sensitivity and specificity of the real-time assay were 96% and 100%, respectively (n = 147), compared with 74% and 98% for a qualitative PCR assay (Roche AMPLICOR). On a subset of samples, the diagnostic sensitivity of viral culture was no greater than 50% (n = 44). Of 1115 clinical referral samples from 252 patients, 10% of the samples and 18% of the patients had low-level CMV viremia (median, 500 copies/mL). In this predominantly (85%) bone marrow transplant testing cohort, serial CMV viral load results were the predominant clinical trigger for the initiation, monitoring, and cessation of preemptive antiviral therapy. CONCLUSIONS: The combination of automated DNA preparation and semiautomated real-time fluorescent PCR detection allows for a sensitive, precise, and accurate high-throughput assay of CMV viral load that can be used as the laboratory trigger for preemptive antiviral therapy.     

Evaluation of the Epstein-Barr virus R-gene quantification kit in whole blood with different extraction methods and PCR platforms

Reliable real-time quantitative PCR assays to measure Epstein-Barr virus (EBV) DNA load (EBV) are useful for monitoring EBV-associated diseases. We evaluated a new commercial kit, EBV R-gene Quantification kit (Argene, Varilhes, France) to quantify EBV DNA load in whole blood. Assay performance was assessed with two PCR platforms (LightCycler 2.0 and SmartCycler 2.0) and three commercial DNA extraction methods. The assay was compared with our in-house real-time EBV PCR using samples from the Quality Control for Molecular Diagnostics 2006 EBV proficiency program and using 167 whole-blood specimens from individuals with infectious mononucleosis, from transplanted or HIV-infected patients, and from EBV-seropositive healthy carriers. The EBV R-gene assay was sensitive to 500 copies of EBV DNA per milliliter of whole blood with the two PCR platforms and the three extraction methods and was linear across 4 orders of magnitude. Intra- and interassay coefficients of variations were less than 20%. Nine of 10 samples tested with the EBV R-gene were in agreement with the expected qualitative results of the Quality Control for Molecular Diagnostics 2006 EBV proficiency program, and 7 of 10 samples were within +/-0.5 log units of the expected quantitative values, with discrepant results mostly observed for low viral load (ie, <1000 copies/ml). In the clinical specimens, the correlation between the R-gene assay and the in-house PCR was high (r=0.92). In conclusion, the EBV R-gene assay accurately assesses the EBV DNA load in whole blood of patients with various forms of EBV infections.     

Prevalence of Cytomegalovirus DNA in Cord Blood and Voided Urine Obtained from Pregnant Women at the End of Pregnancy

Objective

This study was undertaken to determine the prevalence of congenital cytomegalovirus (CMV) infection in pregnant women at the end of pregnancy in Kuwait using cord blood and maternal urine.

Subjects and Methods

Urine samples were collected prior to childbirth, and cord blood was collected immediately after delivery from 983 women. Anti-CMV IgG and IgM antibodies were determined using ELISA; CMV DNA was detected using nested PCR, and viral load was calculated using real-time PCR. CMV concentration in samples was categorized as low when the viral load ≤10³ copies/µl, intermediate when the viral load = 10³−10⁴ copies/µl, and high when the viral load >10⁴ copies/µl. The cord blood serology outcome was compared to cord blood PCR, cord blood viral load, maternal urine PCR and viral load analyses.

Results

Serology showed that of the 983 cord blood samples, 89 (9%) were positive for anti-CMV IgM antibodies; PCR test showed 44 (4.5%) contained CMV DNA, and there was a high viral load in all. Maternal urine PCR showed that 9 (10.11%) women had CMV DNA, and there was a high viral load in 7 (78%). The kappa test for measures of agreement showed a reasonable agreement (0.45) between cord blood PCR and urine PCR.

Conclusion

This study showed that CMV infection in the cord blood sera of pregnant women is common in Kuwait and highlights the need for more clinically based studies to follow up newborns with congenital CMV infection.Key Words: Active cytomegalovirus infection, Cord blood, Maternal urine, Polymerase chain reaction, Viral load     

Significance of qualitative polymerase chain reaction combined with quantitation of viral load in the diagnosis and follow-up of cytomegalovirus infection after solid-organ transplantation

Quantitative PCR was evaluated in the monitoring of patients with ongoing posttransplantation cytomegalovirus (CMV) infection and antiviral therapy, compared to leukoDNAemia and serology. From January 1998 until May 1999, 61 patients were followed up weekly during 3 months after transplantation by a qualitative PCR. The quantitative PCR was performed on plasma samples from 21 selected patients, of whom 12 had a primary infection and 9 a reactivation or reinfection. Analysis of the viral load differences showed that the viral loads in patients with a primary infection were significantly higher than viral loads in patients with a reactivation (p < 0.01). Based on the results of our study, we can state that qualitative PCR is a good marker for initiating preemptive therapy. In addition, viral quantitation is clinically useful for accurate diagnosis of established CMV disease, and monitoring of antiviral therapy.     

Quantification of epstein-barr virus DNA is helpful for evaluation of chronic active epstein-barr virus infection

Chronic active Epstein-Barr virus infection (CAEBV) presents with chronic or recurrent infectious mononucleosis-like symptoms, such as low-grade fever, liver dysfunction, lymphadenopathy, and hepatosplenomegaly. Immunological methods are useful for the diagnosis of viral infections. However, CAEBV patients do not necessarily have high titers of Epstein-Barr virus (EBV)-specific antibodies. Hosts that are immunocompromised after hematopoietic stem cell transplantations sometimes suffer from systemic EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and EBV-positive lymphoma. Patients with EBV-associated diseases are often diagnosed by analyses of bone marrow. Cytomegalovirus (CMV) can cause serious pneumonia or retinitis in immunocompromised hosts. In order to noninvasively understand the clinical status of patients with EBV-associated diseases, we conducted real-time polymerase chain reaction (PCR) methods in their peripheral blood in order to quantify EBV and CMV DNA levels, which reflect viral activity. Here, we describe a 30-year-old Japanese female patient with CAEBV. The patient had repeated fever, fatigue, and liver dysfunction. The histopathological results of liver biopsies were positive for EBV-encoded RNA-1. Acute hepatitis was associated with the EBV infection. The whole-blood EBV DNA levels were high and above 1.0 × 10⁷ copies/mL. After immunosuppressive and antiviral therapies, EBV DNA levels lowered. However, she had to receive bone marrow transplantation because of her EBV-HLH. As the number of lymphocytes increased in the post-transplantation period, EBV DNA levels gradually increased again. The simultaneous detection of CMV DNA was more sensitive than the CMV antigenemia test that is often used to diagnose CMV infections. Unfortunately, the patient died due to a fungal infection. Observing EBV DNA levels closely with real-time quantitative PCR methods is helpful for evaluating the changes in the clinical course.     

Comparison of quantitative competitive polymerase chain reaction-enzyme-linked immunosorbent assay with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation

Development of highly sensitive quantitative assays for cytomegalovirus (CMV) DNA detection is crucial for identification of immunodeficient patients at high risk of CMV disease. We designed 2 internally controlled competitive quantitative assays, enzyme-linked immunosorbent assay (ELISA)-based and real-time polymerase chain reaction (PCR) tests, using amplification of the same segment of the CMV genome. The aim of this study was to compare sensitivity, specificity, and laboratory performance characteristics of these assays. In both assays, a 159-bp segment of UL83 gene was amplified. External and internal controls were constructed by cloning the amplification product and heterogenous DNA segment flanked by target sequences for CMV-derived primers into bacterial plasmids, respectively. Real-time PCR was performed on LightCycler (Roche Diagnostics, Mannheim, Germany), and amplicons were detected using fluorescence resonance energy transfer probes. Alternatively, PCR products were labeled by digoxigenin, hybridized to immobilized probes, and detected by ELISA. The assays were tested on genomic DNA isolated from laboratory strains of CMV, QCMD control panel, and CMV DNA-positive peripheral blood DNA samples from hematopoietic stem cell transplant recipients, previously characterized by pp65 antigenemia and qualitative nested PCR. Real-time and ELISA-based PCR assays showed a linear course of 1-10(8) and 10-10(5) copies of CMV DNA per reaction, respectively. When compared with ELISA-based PCR, real-time PCR showed superiority in inter- and intra-assay reproducibility. Both assays were highly specific in detecting CMV DNA. No difference in amplification efficiency of internal or external standards and wild-type CMV DNA was found. The assays exhibited 83% concordance in CMV DNA detection from clinical samples, all discrepant samples having low CMV DNA copy numbers. There was a good correlation between viral DNA loads measured by the 2 assays. Statistically significant correlation was observed between the numbers of CMV DNA copies and pp65-positive leukocytes in the samples tested. Both variants of competitive PCR are adequately sensitive to be used for CMV DNA quantitation in clinical samples. LightCycler PCR, having superior performance characteristics and being less time-consuming, seems to be more suitable for routine diagnosis.     

Rapid detection of cytomegalovirus infection in transplant patients

Human cytomegalovirus (CMV) is a well-known cause of morbidity and mortality in transplantation patients. Monitoring of CMV reactivation from latency is critical for these patients. The key to efficient and effective management of CMV infection is a test capable of rapidly monitoring and quantifying the presence of CMV in the blood. This is essential for the identification of subjects at high risk of developing CMV disease, for example, patients receiving steroid or immunosuppressive compounds for accelerated graft-versus-host disease, transplant rejection and also for the application and monitoring of pre-emptive antiviral therapeutic strategies. The assays presently available and frequently used in this setting include conventional and shell vial culture, the CMV antigenemia assay, PCR for CMV DNA, hybrid capture assay for CMV DNA and detection of CMV RNA by nucleic acid sequence-based amplification. The low sensitivity and low reproducibility of conventional cell culture and shell vial assays limit their role in the management of CMV infection to one of disease diagnosis. Diagnostic assays, such as the pp65 antigenemia and other molecular assays, have improved the ability to diagnose CMV disease quickly and accurately. These methods fulfill the requirements for a good diagnostic assay: they have high sensitivity, most can quantify viral load and they are rapid and reproducible. Their characteristics allow these assays to be used to predict the development of CMV disease and monitor response to therapy.     

Molecular diagnosis of animal diseases: some experiences over the past decade

The experiences of a veterinary research and diagnostic laboratory are summarized on the development and application of the PCR to diagnose a wide range of viral diseases in animals. The group started the routine diagnostic application of the PCR as early as 1988 and today a total of 35 nested PCR assays are in routine use for the detection of 15 DNA and 20 RNA viruses. Special tools and laboratory practice were applied to avoid false-positive results, while false-negatives are avoided by internal controls (mimics). At present, the classical nested PCR methods are being replaced by real-time TaqMan and molecular beacon assays and the multiplex real-time PCR detection of viruses is also under development. By direct sequencing of the PCR products, phylogeny studies are performed and molecular epizootiology results are provided for rapid and exact identification of virus variants. Molecular epizootiology also contributes to trace the routes of virus spreading on large geographic areas. Recently, large efforts have been made to follow the recommendations of Office International des Epizooties for the standardization and international harmonization of the molecular diagnostic assays.     

Early detection of plasma cytomegalovirus DNA by real-time PCR after allogeneic hematopoietic stem cell transplantation

Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Therefore, preemptive ganciclovir therapy based on early detection of CMV reactivation is widely used to prevent CMV disease. Real-time polymerase chain reaction (PCR) has been widely used for monitoring CMV reactivation as well as the antigenemia assay that detects CMV structural phosphoprotein with a molecular weight of 65,000 (pp65). We developed a real-time PCR assay system for CMV based on a double-stranded DNA-specific dye, SYBR Green I, and quantified DNA, which was extracted automatically from plasma. This real-time PCR assay and the pp65 antigenemia assay were compared in parallel with 357 blood samples obtained from 64 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Real-time PCR assay results correlated with those of the pp65 antigenemia assay (p < 0.0001). It is noteworthy that the detection of CMV DNA by PCR preceded the first positive antigenemia by 14 days. In this study, 10 of 64 patients developed CMV disease. The antigenemia assay detected CMV reactivation earlier than the development of CMV disease only in four of 10 patients. In contrast, our real-time PCR detected CMV-DNA before the development of CMV diseases in eight of 10 patients. The real-time PCR with SYBR Green I as a detection signal is simple and readily performed, and may be a useful system for early detection of CMV reactivation after allo-HSCT.     

基于ddPCR技术分析EB病毒载量特征及与qPCR技术的比较研究

目的解决临床工作中应用实时荧光定量PCR(qPCR)方法检测EB病毒(EBV)载量与临床诊断不符的问题,探讨微滴式数字PCR(ddPCR)和qPCR方法检测EBV载量的能力并为临床提供可能的解决方案。方法收集510例疑似EBV感染相关疾病患者血浆标本,采用ddPCR和qPCR两种方法测定同一血浆标本的EBV-DNA载量。结果 EBV感染人群中,EBV-DNA载量较其他地区低,载量中位数仅360copies/mL,其中初诊未治鼻咽癌患者中位病毒载量为4 590copies/mL,治疗后鼻咽癌患者中位病毒载量下降为430copies/mL,免疫力低下者中位病毒载量为130copies/mL,而淋巴瘤患者中位病毒载量为840copies/mL;qPCR检测EBV感染以400copies/mL为界值,高于400copies/mL时,ddPCR与qPCR的EBV-DNA测定水平值呈中度相关(r=0.533,P<0.05),低于400copies/mL时,ddPCR与qPCR的EBV-DNA测定水平值呈弱相关(r=0.299 5,P<0.05);以ddPCR为标准,qPCR检测EBV-DNA的灵敏度仅为0.317,以ddPCR检测结果为标准,构建qPCR的受试者工作特征曲线下面积为0.871,此时临界值(qPCR)为10copies/mL,灵敏度为0.824,特异度为0.780。结论采用ddPCR方法或优化qPCR的临界值去检测EBV-DNA载量更能为临床诊断EBV感染提供有利支持。     

Quantitation of CMV by real-time PCR in transfusable RBC units

BACKGROUND: CMV is one of the most significant pathogens infecting immunocompromised individuals. CMV is transmissible through transfusion of blood components. The goal of this study was to measure CMV levels in RBC units using a sensitive and quantitative DNA amplification assay. STUDY DESIGN AND METHODS: An assay to measure CMV load was developed by using real-time PCR to target the major immediate early viral gene. A probe (TaqMan, Applied Biosystems) was used to confirm product specificity and to permit quantitation of CMV in blood samples on a sequence detection system (ABI Prism 7700, Applied Biosystems). RESULTS: The assay was shown to be accurate, linear, and sensitive to as few as five copies of CMV DNA per PCR. The assay was applied to aliquots of RBC units from 203 healthy donors, 110 of whom were seropositive for CMV. CMV DNA was not detected in any of the 203 RBC samples. CONCLUSION: The findings statistically imply that at least 98.5 percent of RBC units have a CMV load of less than 250 copies per mL. Future clinical studies on larger numbers of units are required to determine the utility of real-time PCR in evaluating the risk of CMV transmission and in confirming the efficacy of WBC reduction.     

Predictive value of quantitative PCR-based viral burden analysis for eight human herpesviruses in pediatric solid organ transplant patients

Human herpesviruses can cause significant morbidity and mortality in pediatric solid organ transplant recipients. It was hypothesized that viral burden quantification by polymerase chain reaction using an internal calibration standard could aid in distinguishing between viral disease and latency. Here we report the results of a 2-year prospective study of 27 pediatric solid organ (liver, kidney, or heart) transplant recipients in which multiple samples were analyzed for levels of all eight human herpesviruses by internal calibration standard-polymerase chain reaction. Herpes simplex viruses 1 and 2, varicella-zoster virus, and Kaposi’s sarcoma-associated herpesvirus were not detected in any of these samples. Human herpesvirus types 6 and 7 were detected in half of the patients, but were present at low levels, similar to those found in reference populations. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were detected in 89% and 56% of the patients, respectively. Viral burden analysis suggested distinct patient populations for CMV, with a natural cutoff of 10,000 viral targets/ml blood strongly associated with disease. In some cases, a dramatic increase in CMV levels preceded clinical evidence of disease by several weeks. EBV viral burden was relatively high in the only patient presenting with an EBV syndrome. However, two other patients without evidence of EBV disease had single samples with high EBV burden. Rapid reduction in both EBV and CMV burden occurred with antiviral treatment. These data suggest that viral burden analysis using internal calibration standard-polymerase chain reaction for CMV, and possibly other herpesviruses, is an effective method for monitoring pediatric transplant patients for significant herpesvirus infection and response to therapy.     

荧光定量PCR检测传染性单核细胞增多症不同血样本EBV的研究

[目的]寻找荧光定量PCR检测传染性单核细胞增多症（IM）EBV DNA拷贝量的最佳样本。[方法]采用荧光定量PCR分别检测了IM病人的外周血单个核细胞、全血基因组DNA及血浆的EBV DNA拷贝量,并比较各种方法的阳性率及拷贝量。[结果]三种不同来源的样品荧光定量PCR方法的阳性率分别为76％、69％、23％。全基因组及单个核细胞组阳性率比较经x^2检验,差异无显著性,且两组拷贝量比较差异亦无显著性。血浆组阳性率及拷贝量与前两种方法比较差异均有显著性。[结论]外周血单个核细胞是荧光定量PCR检测IM的EBV DNA量的最佳样本。     

Visual Format for Detection of Mycobacterium tuberculosis and M. bovis in Clinical Samples Using Molecular Beacons

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete.For effective treatment of tuberculosis (TB), rapid and accurate diagnosis is essential. Conventional polymerase chain reaction (PCR)-based assays designed to detect Mycobacterium tuberculosis that involve electrophoresis based analysis of amplicons are relatively fast but are laborious and the potential risk of inadvertent dispersal of amplicons leading to contamination of untested samples exists. To overcome these difficulties a number of probes, including TaqMan probes, molecular beacons, and side-by-side probes, that can report the amplification of the correct amplicon in sealed tubes have been developed.^1,2,3 Several different instruments that measure increase in fluorescence of these probes while simultaneously performing amplification have also become available. Although such real-time PCR assays are more quantitative than conventional ones, the instruments used are not commonly available in resource-poor locations. Since such localities are often where tuberculosis is more prevalent, it is important to develop sealed tube assays that can be implemented on conventional PCR machines but have the simplicity and accuracy of probe-based detection.In the previously developed tests different coding and intergenic regions from the M. tuberculosis genome, such as devR, IS6110, IS986, RNA polymerase gene, and ribosomal RNA gene have been used as targets for amplification using molecular beacons or fluorescent probes.^4,5,6,7 One of the limitations of these tests is that they have exclusively focused on M. tuberculosis. However, other closely related bacteria such as M. bovis have been often associated clinically with human and bovine samples in practice.^8,9,10 Therefore an ideal test should distinguish between M. tuberculosis and M. bovis. We used the mce3 operon that has a differential organization in the M. tuberculosis and M. bovis genome as a suitable target for the assay.¹¹To develop a simple sealed tube test that can reliably detect M. tuberculosis in clinical samples using conventional PCR instrument for amplification and a common lamp for transillumination, we used molecular beacons as probes. However, the signal intensity in PCRs performed with molecular beacons is often limited because the two product strands of the amplicon bind to each other excluding the probe from the target strand. We improved the fluorescence intensity of these reactions by performing asymmetric PCR that produced more of the molecular beacon target strand than of the opposite strand. The optimizations were performed using a real-time instrument. We show that our assay is robust, specific, sensitive, and can be completed in less than 3 hours. The presence of mycobacteria in the sample can be conclusively established by visualizing the fluorescent amplicon in a blue light transilluminator by the naked eye. We also compared and correlated our endpoint visual format assay with acid-fast bacilli (AFB) smear microscopy; a gel-based conventional multiplex PCR (CM-PCR) assay, culture, and clinical diagnosis. The simplified visual format assay was found to be an accurate predictor of the presence of M. tuberculosis and M. bovis in clinical samples.     

Fast detection of DNA of hepatitis B virus by real time PCR

90 blood plasma samples from patients with suspected chronic viral hepatitis B (CVHB) were analyzed by real-time polymerized chain reaction (PCR). The findings were compared with the results obtained by 2 PCR electrophoresis-based test-systems; the sensitivity limit for the quantification of DNA of hepatitis B virus (HBV) was determined; in the present case, the limit corresponded to 30 replications of HBV DNA to reaction (600 GE/ml). The positive result of real-time PCR was registered in 53.3% of cases. The quantity of HBV DNA replications in blood plasma samples varied from 30 to 3.9 x 10(6) per reaction (600-7.8 x 10(7) GE/ml). Serological profiles were analyzed in 18 patients with the verified diagnosis of CVHB. HBV DNA was detected in blood of 65% of HBsAg(+)-patients. The markers of HBeAg replication were noted in 35.5% of patients; it is noteworthy, that HBeAg(+)-samples were characterized by a higher level of viral loads (> or = 10(6) GE/ml) versus HBeAg(-)-samples (> or = 6 x 10(3) GE/ml). An analysis of blood-plasma samples dynamically obtained from one patient with chronic renal insufficiency and CVHB showed a decreased level of viral load from 1.7 x 10(7) GE/ml to a negative finding of real-time PCR registered after a therapy course by zeffix. Hence, the automated and standardized real-time PCR, when used at a multi-field patient-care facility, ensures a better diagnosis of viral hepatitis B.