Quantitative Detection of the M204V Hepatitis B Virus Minor Variants by Amplification Refractory Mutation System Real-Time PCR Combined with Molecular Beacon Technology

Mutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif are frequently associated with resistance to antivirals and represent a major concern in the treatment of hepatitis B virus (HBV) infection. Conventional methods fail to detect minority populations of drug-resistant viral quasispecies if they represent less than 25% of the total sample virus population. The amplification refractory mutation system real-time PCR (ARMS RT-PCR) was combined with molecular beacon technology using the LightCycler system. The samples from HBV patients selected for assay evaluation included (i) 57 samples from treatment-naïve patients for biological discriminatory ability (cutoff) estimation, (ii) 12 samples from patients with treatment failure that were M204V positive by sequencing, and (iii) 13 samples from patients with treatment failure that were negative for mutation at codon 204 by sequencing. The discriminatory ability of the assay was 0.25% when tested with laboratory-synthesized DNA target sequences. The median mutant-to-wild-type ratio for samples from naive patients tested positive for the wild type and for mutant variants was 0.01% (5th and 95th percentiles = 0.0001 and 0.04%, respectively). A value of 0.04% was selected as the biological cutoff of the assay of clinical samples. In all samples M204V positive by sequencing (12/12), the mutant variant was detected as the predominant population (range, 82.76 to 99.43%). Interestingly, in 5 (38%) of 13 samples negative by sequencing, the M204V variant was detected at a ratio above the biological cutoff (0.05 to 28%). The assay represents an efficient technique for the early detection and quantification of M204V variants before mutant strains emerge to dominate the population.Nucleoside analogs inhibit hepadnavirus replication by terminating viral DNA synthesis and have been used in the treatment of patients with hepatitis B virus (HBV) infection. Antiviral treatment has been shown to rapidly reduce serum HBV DNA to levels below the detection limit of standard commercial assays and to be well tolerated without major adverse events (23). However, as with other antivirals, resistance may occur at a rate mainly dependent on the nucleoside/nucleotide analogue used. Taking lamivudine as an example, resistance increases with longer durations of treatment, from 24% at year 1 to 65 to 70% of treated patients at year 5 (17).Resistance to lamivudine has been frequently associated with mutations that reside in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif of the C domain (M204I, M204V) and secondarily with mutations residing outside the C domain but mainly in the B domain (V173L, L180M) of the HBV polymerase region (Pol/RT). The M204V mutation has also been associated with resistance to other antivirals such as entecavir and emtricitabine; selection of M204I/V mutants affects future treatment options (4, 16). In vitro studies have demonstrated that mutant (Mut) viral variants exhibit a diminished replication capacity compared with that of wild-type (WT) strains (21). However, under the selective pressure of antiviral drug treatment that prevents replication of the WT variant, Mut variants are able to outcompete the WT virus, as they appear to be sufficiently fit (8).In general, the development of HBV resistance is detected by nucleotide sequencing of PCR products. This genotypic technique is unable to detect and quantify minorities of viral quasispecies below 25% (5, 19, 22). Clinical observations suggest that minor viral variants can frequently emerge as the major viral population. Due to the limitations of the currently used techniques, it has been difficult to establish the exact time and mode of emergence of the Mut virus.The aim of our study was to develop a sensitive, reproducible, and accurate method for the quantification of viral populations with the M204V mutation, even at concentrations below the currently used thresholds, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) assay with molecular beacon biotechnology.     

Evaluation of the Abbott HBV RUO Sequencing Assay Combined with Laboratory-Modified Interpretive Software

The Abbott HBV RUO Sequencing assay (Abbott Molecular Inc., Des Plaines, IL), which combines automated sample processing, real-time PCR, and bidirectional DNA sequencing, was evaluated for detection of nucleos(t)ide analogue (NA) resistance-associated mutations located in the hepatitis B virus (HBV) polymerase (Pol) gene. Interpretive software from the assay manufacturer was modified to allow interrogation of the overlapping HBV surface (S) gene sequence for HBV genotype determination and detection of immune escape mutations. Analytical sensitivity (detection and sequencing) of the assay was determined to be 103.9 IU/ml (95% confidence interval [CI], 80.0 to 173.3) for HBV genotype A. Testing of commercially available HBV genotype panels consisting of 23 individual members yielded complete agreement between expected results and results obtained from the laboratory-developed HBV genotype library. Excellent specificity was observed among clinical specimens with serologic or molecular markers for various unrelated blood-borne viruses (n = 6) and sera obtained from healthy, HBV-negative blood donors (n = 20). Retrospectively selected clinical specimens tested by a commercial reference laboratory HBV sequencing assay (n = 54) or the Trugene HBV Genotyping kit (n = 7) and the Abbott HBV RUO Sequencing assay showed minor differences in detection and reporting of NA resistance-associated mutations in 7 of 61 (11.5%) specimens but complete agreement of genotype results. The Abbott HBV RUO Sequencing assay provided a convenient and efficient assay workflow suitable for routine clinical laboratory use, with the flexibility to be modified for customized detection of NA resistance-associated mutations, HBV genotype determination, and detection of immune escape mutations from a single contiguous HBV sequence.     

Hepatitis B and liver transplantation: 2008 update

The ultimate goal of treatment is suppression of viral replication to undetectable HBV-DNA levels prior to and after liver transplantation (LT) to prevent infection of the newly transplanted liver. Most published data are available from therapy with lamivudine (LAM) in pre- and post-transplant HBV patients. Add-on therapy with adefovir dipivoxil (ADV) in pre-transplant LAM-resistant patients has been shown to represent an effective antiviral strategy leading to hepatic recompensation in many cases and, eventually, removal from the waiting list. Newer nucleos(t)ide analogues such as entecavir, tenofovir and telbivudine have shown lower resistance rates than LAM and more antiviral potency in studies in the non-transplant setting. Combined hepatitis B immune globulin (HBIG) and nucleos(t)ide analogue therapy have been widely adopted as the most effective treatment strategy against recurrent HBV disease after LT. Many programs have evaluated lower doses or a shorter duration of HBIG and intramuscular versus intravenous routes of administration. Active immunisation using recombinant HBV vaccines, including the S, pre-S1 and pre-S2 regions, and those with immunostimulatory adjuvants, seem to be more immunogenic than the currently available vaccines and have been used in studies to replace HBIG. Furthermore, it has been shown that immune memory against HBV can be adoptively transferred from organ donors to transplant recipients. Nucleos(t)ide analogue combination therapies might provide an alternative to the current treatment paradigm with costly HBIG; however, experience with this new treatment regimen is very limited and controlled clinical studies are urgently warranted to investigate its safety and efficacy and to determine which nucleos(t)ide analogue combinations will be the most promising in the long term after LT.     

Hepatitis B virus: From diagnosis to treatment

During the next few decades, vaccination against hepatitis B virus (HBV) will dramatically change the epidemiological profile of this worldwide infection especially when Heath Policies encourage including HBV vaccination program for the newborns. However, it is still estimated that more than 2000 millions living people have met HBV. Symptomatic hepatitis with jaundice is less frequent than asymptomatic infection; however, as much as 350 millions of individuals remain chronically infected by HBV. In these cases, the need for efficient antiviral therapy remains clear when a viral replication is observed to control the risk of progression and the need for liver transplantation, which represents the only end-stage treatment. Indeed, patients having chronic hepatitis B (CHB) can now be successfully treated using nucleos(t)ide analogs (NA) or pegylated interferon (PEG-IFN). Therefore, beside vaccination, prevention of the progression of the disease to cirrhosis and liver decompensation, leading to end-stage liver disease and/or to hepatocellular carcinoma, by inhibiting viral replication seems to represent the best approach to improve survival. At last but not least, co-morbidities and other viral infections, leading also to chronic liver cirrhosis or liver inflammation such as the specific satellite delta virus (HDV), human immunodeficency virus (HIV) and/or hepatitis C (HCV) virus, are able to accelerate the progression and have to be taken in account. Interestingly, in treated infection, the dogma of the irreversibility of the liver fibrosis, when the cirrhosis is constituted, is tumbling down. In this review, we will focus on the clinical, virological and therapeutic aspects of hepatitis B infection in order to expose the proposals to follow-up and treat HBV-infected patients and the prevention of drug-resistant HBV mutants that frequently arise, leading to treatment failure and progression to liver disease.     

Development of SYBR Green I-based real-time PCR assay for detection of drug resistance mutations in cytomegalovirus

Ganciclovir (GCV) is an antiviral drug that is used to treat cytomegalovirus (CMV) infection. However, long-term monotherapy does not commonly result in complete suppression of viral replication and is associated with the emergence of resistant mutants. In this study, a method for detecting CMV resistance mutations was carried out by real-time amplification refractory mutation system PCR (real-time ARMS PCR) using SYBR Green I fluorescent dye. Three recombinant plasmids were constructed by overlapping extension PCR to be used as standard mutation or wild-type models. Four pairs of primers were used to amplify the approximately 150 bp of the UL97 gene spanning codon 460, where mutations associated with resistance to GCV invariably occur. As little as 20% mutants DNA in 10(7)copies/ml mixture DNA were detected. Though this approach was not more sensitive than PCR-restriction fragment length polymorphism (RFLP) for the detection of the presence of mixtures, it was a high-throughput and automation method, and the specific mutation type can be deduced by the real-time ARMS PCR data. Overall, this study has demonstrated an approach that could be a sensitive and rapid method for the detection of GCV resistance-associated mutation in CMV.     

Real-time PCR assay using molecular beacon for quantitation of hepatitis B virus DNA

Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (ad and ay subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 10(1) to 10(7) copies per reaction (1.0 x 10(2) to 1.0 x 10(9) copies ml(-1)). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (r = 0.901; P < 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.     

Phylogenetic analysis of hepatitis B virus genotype F complete genome sequences from Chilean patients with chronic infection

Molecular epidemiological data concerning the hepatitis B virus (HBV) in Chile are not known completely. Since the HBV genotype F is the most prevalent in the country, the goal of this study was to obtain full HBV genome sequences from patients infected chronically in order to determine their subgenotypes and the occurrence of resistance-associated mutations. Twenty-one serum samples from antiviral drug-naive patients with chronic hepatitis B were subjected to full-length PCR amplification, and both strands of the whole genomes were fully sequenced. Phylogenetic analyses were performed along with reference sequences available from GenBank (n = 290). The sequences were aligned using Clustal X and edited in the SE-AL software. Bayesian phylogenetic analyses were conducted by Markov Chain Monte Carlo simulations (MCMC) for 10 million generations in order to obtain the substitution tree using BEAST. The sequences were also analyzed for the presence of primary drug resistance mutations using CodonCode Aligner Software. The phylogenetic analyses indicated that all sequences were found to be the HBV subgenotype F1b, clustered into four different groups, suggesting that diverse lineages of this subgenotype may be circulating within this population of Chilean patients.     

Development of a quantitative real-time detection assay for hepatitis B virus DNA and comparison with two commercial assays

A highly reproducible and sensitive real-time detection assay based on TaqMan technology was developed for the detection of hepatitis B virus (HBV) DNA and compared with two commercially available assays. The assay was validated with the Viral Quality Control panel, which also includes EUROHEP HBV DNA standards. This real-time PCR detection system had a dynamic range of 373 to 10(10) genome copies per ml and showed an excellent correlation with both the commercial HBV Digene Hybrid Capture II microplate assay (Digene Diagnostics) and the HBV MONITOR assay (Roche Diagnostics). To demonstrate its clinical utility, four chronically HBV-infected patients treated with lamuvidine were monitored using the three different assays. From the results we concluded that this assay is an excellent alternative for monitoring of HBV-infected patients in routine diagnostics and clinical practice, enabling the analysis of a large dynamic range of HBV DNA in a single, undiluted sample.     

Line probe assay for monitoring drug resistance in hepatitis B virus-infected patients during antiviral therapy

Since the introduction of antiviral compounds such as lamivudine and famciclovir in the treatment schedules of patients with chronic hepatitis B virus (HBV) infection, the accumulation of a variety of mutations in the HBV polymerase gene has been observed. The selection of these mutations is generally considered the cause of viral nonresponsiveness and treatment failure. Therefore, the detection of these mutations is of clinical importance. Previously genotyped HBV strains isolated from treated and untreated patients were amplified with primers specific for the HBV polymerase region from amino acids 465 to 562. Amplified products were cloned into plasmid vectors. The clones were used as reference strains. A set of 38 highly specific oligonucleotide probes covering three different codon positions, L528M, M552V/I, and V/L/M555I, were selected. These probes were applied as 19 different lines on a membrane strip. The strips were then hybridized with PCR fragments from the reference panel, revealing the amino acids at the three codon positions simultaneously for each clone. PCR products generated from two patients infected with HBV genotypes A and C, respectively, and treated with nucleoside analogs were analyzed on these strips. A gradual increase in genetic HBV polymerase complexity was observed in follow-up samples compared to that in pretreatment samples. Additional analysis of HBV polymerase DNA fragments in recombinant plasmid clones demonstrated the existence of (i) clones with double mutations, (ii) clones with single mutations at either codon 528, 552, or 555, and (iii) the simultaneous occurrence of two or more viral populations within one sample. This line probe assay detected the complex quasispecies nature of HBV and provided some insight into the dynamics of resistance mutations.     

A genotype-independent real-time PCR assay for quantification of hepatitis B virus DNA

Accurate quantification of hepatitis B virus (HBV) DNA levels is important for monitoring patients with chronic HBV infection and for assessing their responses to antiviral therapy. This study aimed to develop a real-time PCR assay that is sensitive and can accurately quantify a wide range of HBV DNA levels across the known HBV genotypes. An "in-house" real-time PCR assay using primers and a TaqMan probe in a highly conserved region of the HBV surface gene was designed. The assay was standardized against a WHO standard and validated against plasmids of HBV genotypes A through H. The linear quantification range was approximately 5 x 10(0) to 2.0 x 10(9) IU/ml. Results of samples from patients infected with HBV genotypes A through H tested using our real-time "in-house" PCR assay showed an excellent correlation with those of the Cobas Amplicor HBV Monitor (R2=0.9435) and the Cobas TaqMan HBV (R2=0.9873) tests. We have established a real-time PCR assay that is genotype independent and can accurately quantify a wide range of HBV DNA levels. Further studies of additional samples are ongoing to validate the genotype independence of our assay.     

竞争性聚合酶链反应法定量检测慢性乙型肝炎患者血清中乙型肝炎病毒前C区野毒株及变异株

设计了以内参照为基础的竞争性聚合酶链反应（ＰＣＲ）方法，同时定量检测ＨＢｅＡｇ阴性的慢性乙型肝炎患者血清中乙型肝炎病毒（ＨＢＶ）基因前Ｃ区野毒株及变异株，并观察了在应用α干扰素（ＩＦＮ）治疗以后的变化。结果发现干扰素治疗有效者中，病毒核酸量迅速下降至转阴；而干扰素治疗无效者中，病毒核酸量也可有所下降，但仍高于检测水平甚至反跳；另外，野毒株对ＩＦＮ的反应似乎优于突变株。该方法可用于研究乙型肝炎病毒感染过程及考核抗病毒药物疗效。     

竞争性聚合酶链反应法定量检测慢性乙型肝炎患者血…

设计了以内参照为基础的竞争性聚合酶链反应方法，同时定量检测ＨＢｅＡｇ阴性的慢性乙型肝炎患者血清中乙型肝炎病毒基因前Ｃ区野毒株及变异株，并观察了在应用α干扰素治疗以后的变化。结果发现干扰素治疗有效者中，病毒核酸量迅速下降至转阴；而干扰素治疗无效者中，病毒核酸量也可有所下降，但仍高于检测水平甚至反跳。     

Rapid, high-throughput detection of rifampin resistance and heteroresistance in Mycobacterium tuberculosis by use of sloppy molecular beacon melting temperature coding

Rifampin resistance in Mycobacterium tuberculosis is largely determined by mutations in an 80-bp rifampin resistance determining region (RRDR) of the rpoB gene. We developed a rapid single-well PCR assay to identify RRDR mutations. The assay uses sloppy molecular beacons to probe an asymmetric PCR of the M. tuberculosis RRDR by melting temperature (T(m)) analysis. A three-point T(m) code is generated which distinguishes wild-type from mutant RRDR DNA sequences in approximately 2 h. The assay was validated on synthetic oligonucleotide targets containing the 44 most common RRDR mutations. It was then tested on a panel of DNA extracted from 589 geographically diverse clinical M. tuberculosis cultures, including isolates with wild-type RRDR sequences and 25 different RRDR mutations. The assay detected 236/236 RRDR mutant sequences as mutant (sensitivity, 100%; 95% confidence interval [CI], 98 to 100%) and 353/353 RRDR wild-type sequences as wild type (specificity, 100%; 95% CI, 98.7 to 100%). The assay identified 222/225 rifampin-resistant isolates as rifampin resistant (sensitivity, 98.7%; 95% CI, 95.8 to 99.6%) and 335/336 rifampin-susceptible isolates as rifampin susceptible (specificity, 99.7%; 95% CI, 95.8 to 99.6%). All mutations were either individually identified or clustered into small mutation groups using the triple T(m) code. The assay accurately identified mixed (heteroresistant) samples and was shown analytically to detect RRDR mutations when present in at least 40% of the total M. tuberculosis DNA. This was at least as accurate as Sanger DNA sequencing. The assay was easy to use and well suited for high-throughput applications. This new sloppy molecular beacon assay should greatly simplify rifampin resistance testing in clinical laboratories.     

锁核酸结合分子信标技术检测乙型肝炎病毒DNA G1896A点突变研究

目的研究和评估乙型肝炎病毒（HBV）DNA G1896A点突变的实时荧光PCR检测方法。方法收集经测序验证HBV DNA G1896A未突变的野生型样本150例和已发生突变的突变型样本58例,在突变区域设计分子信标探针,点突变处进行锁核酸处理,利用荧光PCR方法检测HBV前C区G1896A点突变;再从临床标本中随机抽取18例、8例和19例荧光PCR结果分别显示为G1896A突变的标本、杂合的标本以及野生型的标本的PCR产物进行序列测定。结果突变型质粒和野生型质粒的检测灵敏度均可以达到100copies/ml;突变型探针检测高浓度的野生型质粒时无检测信号,野生型探针检测高浓度突变型质粒时无检测信号;突变型模板在总杂合模板中的比例达到5%时则都可以将突变型检测出来;序列测定的8例G1896A突变标本、6例杂合标本和19例野生型标本的PCR产物结果和荧光PCR检测结果完全吻合。结论实时荧光PCR检测方法可以快速、简便、准确地检测HBVDNAG1896A点突变,是检测点突变的重要方法,具有重要的临床应用价值。