INNO-LiPA Hepatitis B Virus DR Research Articles

Hepatitis B Virus Genotypes and Antiviral Resistance Mutations in Romanian HIV-HBV Co-Infected Patients.

Background and Objectives: Romania has one of the highest prevalence of hepatitis B virus (HBV) infection in human immunodeficiency virus (HIV) patients, mostly in those parenterally infected during childhood; nevertheless, there are scarce data on the virological profile of co-infection. The objective of this study was to assess the prevalence of HBV genotypes and antiviral resistance-associated mutations (RAMs) in these co-infected patients, in order to monitor the viral factors associated with the evolution of liver disease. Materials and Methods: HBV genotypes and RAMs were detected using nested PCR and line probe assays (INNO-LiPA HBV genotyping assay, and INNO-LiPA HBV DR v2, Innogenetics). Results: Out of 117 co-infected patients, 73.5% had detectable HBV-DNA, but only 38.5% presented an HBV viral load >1000 IU/mL. HBV genotype A was present in 66.7% of the cases and was dominant in patients parenterally infected during early childhood, who experienced multiple treatment regimens, with a mean therapy length of 15.25 years, and present numerous mutations associated with lamivudine (LAM) resistance, but very rarely active liver disease. HBV genotype D was detected in 33.3% of the cases, mostly in recently diagnosed injecting drug users who are treatment naïve, but, nevertheless, present RAMs in 63.5% of the cases, suggesting transmitted drug resistance, and display more frequently advanced liver fibrosis (36.1% vs. 12.3%; p = 0.033). The most frequently encountered RAMs are M204V/I: 48.8%, L180M: 33.3%, L80V: 28.8%, and V173L: 42.2%. There are no significant differences in the distribution of RAMs in patients infected with different HBV genotypes, except for the L80V and N236T mutations, which were more frequently found in HBV genotype A infections (p = 0.032 and p = 0.004, respectively). Conclusions: HBV genotypes A and D are the only genotypes present in HIV–HBV co-infected patients from Romania, with different distributions according to the infection route, and are frequently associated with multiple RAMs, conferring extensive resistance to LAM.

Mutations in HBV DNA Polymerase Associated With Nucleos(t)ide Resistance Are Rare in Treatment-naive Patients

Prior studies have detected hepatitis B virus (HBV) DNA polymerase mutations in treatment-naive patients. However, most of these studies used either direct polymerase chain reaction sequencing, which detects these mutations with low levels of sensitivity, or patient cohorts that were not well-characterized. We investigated the prevalence of HBV mutations in DNA polymerase by using a line probe assay. In a prospective, cross-sectional study, we enrolled 198 treatment-naive patients with chronic hepatitis B (52.5% male; mean age, 41 years) from February 2009 to May 2011 from 3 gastroenterology and liver clinics in Northern California. Exclusion criteria included infection with hepatitis C or D viruses or human immunodeficiency virus. All patients completed a questionnaire (to determine demographics, history of liver disease, prior treatments, family medical history, drug and alcohol use, and environmental risk factors for hepatitis) that was administered by a research coordinator; mutations in HBV DNA polymerase were detected by using the INNO-LiPA HBV DR v.3 assay. Most patients were Vietnamese (48.5%) or Chinese (36.4%) and were infected with HBV genotypes B (67.5%) or C (24.2%). Mutations in HBV DNA polymerase were found in 2 patients (1%), rtI233V (n = 1) and rtM250M/L (n = 1). In a multicenter prospective study of treatment-naive patients with chronic hepatitis B, we detected mutations in HBV DNA polymerase in only 1%. Because of the low prevalence of these mutations and the uncertain clinical significance of such quasispecies, routine HBV DNA polymerase mutation analysis cannot be recommended before initiation of antiviral therapy for treatment-naive patients with chronic hepatitis B. The analysis requires further molecular and clinical studies.

Role of the line probe assay INNO-LiPA HBV DR and ultradeep pyrosequencing in detecting resistance mutations to nucleoside/nucleotide analogues in viral samples isolated from chronic hepatitis B patients

Despite the effectiveness of nucleoside/nucleotide analogues in the treatment of chronic hepatitis B (CHB), their long-term administration is associated with the emergence of resistant hepatitis B virus (HBV) mutants. In this study, mutations resulting in antiviral resistance in HBV DNA samples isolated from 23 CHB patients (nine treatment naïve and 14 treated previously) were studied using a line probe assay (INNO-LiPA HBV DR; Innogenetics) and ultradeep pyrosequencing (UDPS) methods. Whilst the INNO-LiPA HBV DR showed no resistance mutations in HBV DNA samples from treatment-naive patients, mutations mediating lamivudine resistance were detected in three samples by UDPS. Among patients who were treated previously, 19 mutations were detected in eight samples using the INNO-LiPA HBV DR and 29 mutations were detected in 12 samples using UDPS. All mutations detected by the INNO-LiPA HBV DR were also detected by UDPS. There were no mutations that could be detected by INNO-LiPA HBV DR but not by UDPS. A total of ten mutations were detected by UDPS but not by INNO-LiPA HBV DR, and the mean frequency of these mutations was 14.7 %. It was concluded that, although INNO-LiPA HBV DR is a sensitive and practical method commonly used for the detection of resistance mutations in HBV infection, UDPS may significantly increase the detection rate of genotypic resistance in HBV at an early stage.

High-Throughput Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry as an Alternative Approach to Monitoring Drug Resistance of Hepatitis B Virus

Long-term antiviral therapy of chronic hepatitis B virus (HBV) infection can lead to the selection of drug-resistant HBV variants and treatment failure. Moreover, these HBV strains are possibly present in treatment-naive patients. Currently available assays for the detection of HBV drug resistance can identify mutants that constitute ≥5% of the viral population. Furthermore, drug-resistant HBV variants can be detected when a viral load is >10(4) copies/ml (1,718 IU/ml). The aim of this study was to compare matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multitemperature single-strand conformation polymorphism (MSSCP) with commercially available assays for the detection of drug-resistant HBV strains. HBV DNA was extracted from 87 serum samples acquired from 45 chronic hepatitis B (CHB) patients. The 37 selected HBV variants were analyzed in 4 separate primer extension reactions on the MALDI-TOF MS. Moreover, MSSCP for identifying drug-resistant HBV YMDD variants was developed and turned out to be more sensitive than INNOLiPA HBV DR and direct sequencing. MALDI-TOF MS had the capability to detect mutant strains within a mixed viral population occurring with an allelic frequency of approximately 1% (with a specific value of ≥10(2) copies/ml, also expressed as ≥17.18 IU/ml). In our study, MSSCP detected 98% of the HBV YMDD variants among strains detected by the MALDI-TOF MS assay. The routine tests revealed results of 40% and 11%, respectively, for INNOLiPA and direct sequencing. The commonly available HBV tests are less sensitive than MALDI-TOF MS in the detection of HBV-resistant variants, including quasispecies.

Long-term add-on therapy with adefovir in lamivudine-resistant kidney graft recipients with chronic hepatitis B

To assess the long-term effectiveness and safety of adefovir (ADV) plus lamivudine (LMV) in LMV-resistant (R) kidney transplants with chronic hepatitis B, 11 such patients were treated with add-on ADV. Serum alanine aminotransferase, renal function and serum hepatitis B virus (HBV) DNA levels were assessed every 3 months; ADV mutations were searched for by INNO-LiPA HBV DR v2 assay. During 36 months (12-48), nine patients cleared serum HBV DNA with a 3-year cumulative virological response rate of 88%, without the emergence of ADV mutations. ADV dose was reduced in six patients (55%) showing a decline of creatinine clearance, in the absence of proximal tubulopathy. In LMV-R kidney graft recipients, long-term add-on therapy with ADV is efficacious and safe with timely adaptation of ADV dose.

Tenofovir Is Effective Alone or With Emtricitabine in Adefovir-Treated Patients With Chronic-Hepatitis B Virus Infection

We compared treatments for patients with chronic hepatitis B virus (HBV) infection who had an incomplete response to adefovir dipivoxil (ADV). We evaluated a combination of fixed-dose emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) from the start (early combination) versus TDF as monotherapy. Patients (n = 105) were randomly assigned to groups given TDF (n = 53) or FTC/TDF (n = 52). End points included HBV DNA suppression, biochemical and serologic response, and response by baseline or developed resistance mutations through 48 weeks of treatment. Patients given TDF monotherapy had the option to receive FTC, as fixed-dose FTC/TDF, if viremia persisted after week 24. At baseline, patients' mean HBV DNA level was 5.97 log(10) copies/mL, and 58% had received lamivudine (LAM); LAM- and ADV-associated mutations were detected in 13 and 10 patients, respectively, by population sequencing and in 14 and 18 patients, respectively, by reverse hybridization line probe assay (INNO-LiPA HBV DR). Through week 24 (direct comparison of blinded therapy), viral decay curves were identical between groups. At week 48, 81% of patients initially given TDF or TDF/FTC had HBV DNA levels below 400 copies/mL. The presence of baseline LAM- or ADV-associated mutations did not affect response. Adherence to therapy appeared to be the primary factor associated with HBV DNA levels below 400 copies/mL at week 48. TDF monotherapy and the combination of FTC and TDF had similar efficacy in patients with incomplete viral suppression after therapy with ADV; response was not influenced by the presence of baseline LAM- or ADV-associated mutations. Initial monotherapy followed by combination therapy was as effective as early combination therapy.

Monitoring of therapy in patients with chronic hepatitis B virus

The aims of this study were to evaluate therapy with lamivudine (LAM) and adefovir dipivoxil (ADV) monotherapy in chronic hepatitis B virus (HBV)-infected patients with frequent measurements of DNA levels, to characterize HBV genotypes, and to determine the emergence of nucleos(t)ide analogue mutants before and during the therapy by direct-sequencing the reverse transcriptase region and by INNO-LiPA HBV DR v3. A total of 15 chronic HBV patients were analysed: 11 were treated with ADV and four were treated with LAM. Viral genotype was determined, showing the presence of genotype D (73%) in 11 patients and genotype A (27%) in four patients. In the viral response to treatment, three patients developed substitutions at rtM204I associated with LAM resistance and one of these patients presented rtM204V/I plus rtL180M mutation. In contrast, of the 11 patients treated with ADV, three patients developed mutations (rtN236T; rtA181V; rtA181V plus rtN236T). With regard to this case, the same results were observed by INNO-LiPA HBV DR v3 and direct sequencing, but by direct sequencing we detected an extra mutation rtQ215S that was present in two patients: one patient who was on treatment with LAM had an rtQ215S mutation in addition to an rtM204I, and the second patient treated with ADV had rtA181V. Direct sequence analysis is an essential tool to optimize therapeutic management of HBV chronic infection in clinical practice to choose the appropriate nucleos(t)ide analogues and to detect extra mutations that are not included in the commercial kit.

Frequent detection of hepatitis B virus variants associated with lamivudine resistance in treated South African patients infected chronically with different HBV genotypes

This retrospective study investigated and characterized the YMDD motif of the hepatitis B virus (HBV) reverse transcriptase (RT) gene, in sequential samples of 17 South African patients with chronic hepatitis B infection on lamivudine treatment. The profile of HBV genotypes as well as the genetic variability of pre-core (pre-C) and basal core promoter regions (BCP) were also determined in these patients. Mutations within the RT gene were determined by direct sequencing using SpectruMedix SCE 2410 genetic analyzer and INNO-LiPA HBV DR (Innogenetics), while the genetic variability of the pre-C/BCP and surface gene were determined by direct sequencing only. HBV genotypes were determined by analysis of the surface, core and RT genes using a web-based genotyping tool (NCBI). HBV DNA was quantified using Cobas Amplicor HBV Monitor assay (Roche Diagnostics). Of the 17 patients, 13 (76.5%) carried YMDD mutations: 7 with rtM204I (2 HBeAg-positive and 5 HBeAg-negative) and 6 with rtM204V (4 HBeAg-positive and 2 HBeAg-negative). Of the 13 patients with resistant HBV strains, 8 (61.5%) carried genotype A, 3 (23%) genotype B, and 2 (15.3%) genotype C. Overall, only 5 of 13 (38%) patients with YMDD mutations experienced genotypic viral drug resistance and treatment failure. Of the 17 patients, 3 carried both pre-C (G1896A) and BCP (A1762T/G1764A) mutants, 1 pre-C only and 1 BCP only. This study demonstrated frequent detection of mutations associated with lamivudine-resistance in therapy-experienced South African patients infected chronically with different HBV genotypes, and confirmed that these mutations are not always accompanied by clinical relapse.

Rapid detection of the hepatitis B virus YMDD mutant using TaqMan-minor groove binder probes

Background TaqMan-minor groove binder (MGB) probes were used in a real-time PCR-based assay for the rapid and accurate detection of hepatitis B virus (HBV) YMDD mutants. Methods TaqMan-MGB probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The detection limit and sensitivity of the assay were determined using a dilution series of a mixture of wild-type and mutant plasmids. Serum samples collected from four patients with chronic mutant HBV infections during lamivudine therapy were analyzed using this method. Results The detection limit for YVDD and YIDD was 10 and 50 copies, respectively, whereas the sensitivity was 10% within a mixed virus population. In the clinical samples, mutant strains of HBV could be detected at levels < 2.6 log copies/ml of HBV DNA. While 15 of the 21 samples tested by this method were positive for the YMDD mutant, direct sequencing and a reverse hybridization line probe assay (INNO-LiPA HBV DR v2) detected the mutant strain in only 11 and 9 samples, respectively. Moreover, the data for 6 samples analyzed by TA cloning were fully consistent with our TaqMan PCR results. Conclusions We successfully established a sensitive and accurate assay for the YMDD mutant of HBV. This method may be useful for monitoring patients treated with lamivudine.

Hepatitis B virus genotypes and lamivudine resistance mutations in Jordan

To investigate and identify prevalent hepatitis B virus (HBV) genotypes and to explore lamivudine-resistant mutations among treated and untreated patients in Jordan. A total of 107 cases with chronic hepatitis B were recruited from different medical centers in Jordan. Serological tests were preformed for all cases using a microparticle enzyme immunoassay. HBV Genotyping was performed for 70 cases using Line probe genotyping assay. The YMDD mutations were explored for 20 cases (4 were lamivudine naive) using the INNO-LiPA HBV DR assay. Genotype D was the only detected genotype. A total of 6 YMDD mutations were detected in 5 treated patients (31%) while one mutation was detected in the naive patients. Seventeen percent of cases were positive for HBeAg and had statistically significant higher levels of serum aminotransferases. HBV genotype D appears to be the only circulating type in Jordanian patients. The YMDD mutations were detected in 31% of lamivudine-treated cases with similar patterns to those found in the literature. We also found a relatively low prevalence of HBeAg expression among examined cases (17%). Awareness of these serologic, genotypic and resistance patterns might help in the formulation of management plans and for predicting clinical outcomes. Further larger scale studies are needed to confirm our results and to examine possible associations among clinical, serologic, and genetic patterns of HBV infections in Jordan.

Low Resistance to Adefovir Combined With Lamivudine: A 3-Year Study of 145 Lamivudine-Resistant Hepatitis B Patients

Adefovir monotherapy is an established treatment modality for lamivudine-experienced patients with chronic hepatitis B, but it carries a significant risk of resistance in the long term. We assessed whether this risk could be overcome by adefovir-lamivudine combination therapy. A total of 145 lamivudine-resistant patients with chronic hepatitis B (73% cirrhotics, 86% hepatitis B e antigen negative, 92% genotype D) were treated with adefovir 10 mg in addition to lamivudine 100 mg. Liver function tests and hepatitis B virus (HBV) DNA (Versant 3.0) were assessed bimonthly, whereas adefovir-related mutations were searched by INNO-LiPA assay at baseline and at yearly intervals. During 42 months (range, 12-74), 116 patients (80%) cleared serum HBV DNA, 67 (84%) had normalized alanine aminotransferase levels, and 145 (100%) remained free of virologic and clinical breakthroughs, independently of the degree of HBV suppression. The rtA181V/T was the only adefovir-related mutation detected, which occurred in 6 patients at baseline (4%; 1 rtA181V and 5 rtA181T) and in an additional 3 patients (2%; all rtA181T) during treatment. In all these 9 patients, HBV DNA levels progressively declined during therapy to become undetectable in 7 (78%). The 1-, 2-, 3-, and 4-year cumulative rates of de novo rtA181T were 1%, 2%, 4%, and 4%, respectively. None of the cirrhotic patients clinically decompensated, but 11 (12%) developed hepatocellular carcinoma. Under prolonged adefovir-lamivudine therapy, patients with lamivudine-resistant hepatitis B were unlikely to develop genotypic resistance to adefovir and had durable prevention of virologic and clinical breakthrough.

A novel accurate ACRS-PCR method with a digestion internal control for identification of wild type and YMDD mutants of hepatitis B virus strains

As a consequence of the point mutation in the YMDD motif of the hepatitis B virus (HBV) polymerase gene, lamivudine-resistant mutants have been reported in chronic hepatitis B patients who underwent lamivudine therapy. The objective of the study was to develop a novel accurate artificially created restriction site (ACRS) method with a digestion internal control for identification of YMDD, YIDD and YVDD HBV strains. Three conserved, specific and diagnostic primers introducing NdeI, SspI and AleI cleavage sites were designed in order to identify YMDD, YIDD and YVDD strains, respectively; while, their reverse primers also modified with the above recognition sites in order to enzyme correctness monitoring and false outcome avoiding. Thirty-two chronic hepatitis B patients who had taken lamivudine for 1–3 years and checked by the Inno-LiPA HBV DR kit, were evaluated by the ACRS method and then compared to sequencing data. The results of the ACRS method revealed the YMDD mutant strain in 20 patients, YMDD plus YIDD pattern in 1 patient, YMDD plus YVDD in 4 patients, the YIDD in 4 patients and mixed infection with each three strains in 1 patient. The sequencing and Inno-LiPA results were in agreement with the ACRS results. The novel ACRS method is a reliable, rapid and a cost-effective technique for determination of HBV strains with the wild type and YMDD mutant patterns.

Detection of rtN236T and rtA181V/T Mutations Associated with Resistance to Adefovir Dipivoxil in Samples from Patients with Chronic Hepatitis B Virus Infection by the INNO-LiPA HBV DR Line Probe Assay (Version 2)

The nucleotide analog adefovir dipivoxil (ADV) is an effective antiviral treatment for chronic hepatitis B virus (HBV) infection, with resistance to ADV estimated to occur less frequently than resistance to lamivudine treatment. The detection of ADV resistance mutations is necessary during therapy to monitor and anticipate possible treatment failure. The INNO-LiPA HBV DR v2 (LiPA; Innogenetics, Ghent, Belgium) is a DNA hybridization line probe assay for the detection of HBV polymerase mutations associated with resistance to lamivudine and ADV. Evaluation of this assay to detect ADV resistance mutations was performed by analyzing 38 patients treated with ADV. Serial samples taken at 6-month intervals during treatment were available for most patients. A total of 124 samples were analyzed by both LiPA and sequencing. By LiPA analysis, 12 patients (31.5%) were found to have mutations associated with resistance to ADV (rtA181V/T and/or rtN236T). This contrasted with sequence analysis, which found nine patients (24%) with either or both mutations. Twice as many samples were rtN236T positive by LiPA (18 of 124) compared to sequence analysis (9 of 124). LiPA detected the rtN236T mutation at least 6 months earlier than its detection by sequencing in patients for whom consecutive serum samples were available. Although less sensitive, sequencing has the advantage of providing information on other polymerase mutations not represented on LiPA strips. The INNO-LiPA HBV DR v2 assay is a very sensitive and specific assay for the detection of the rtN236T mutation associated with resistance to ADV.

Performance of sequence analysis, INNO‐LiPA line probe assays and AFFIGENE assays in the detection of hepatitis B virus polymerase and precore/core promoter mutations

In this study, we compare results obtained by sequences analysis and commercial kits in the detection of hepatitis B virus (HBV) polymerase and precore (PC) and core promoter mutations. A total of 23 serum samples from lamivudine treated patients were tested for polymerase mutations by direct sequencing, INNO-LiPA HBV DR and AFFIGENE HBV DE/3TC. Full concordance among the three assays was observed in 63% of the total analysed codons. Concordant results were obtained between sequencing and LiPA in 80%, between sequencing and AFFIGENE in 73% and between LiPA and AFFIGENE in 74% of all tested codons. All discrepancies were observed in mixed population samples in which AFFIGENE and LiPA detected additional viral variants not revealed by sequence. In two patients, with serial samples, LiPA detected earlier than sequence and AFFIGENE an emerging mutate strain. PC and core promoter viral variants were detected in 28 serum samples collected from 14 HBV inactive carriers and from 14 hepatitis B patients with chronic liver disease. Direct sequencing, INNO-LiPA HBV PreCore and AFFIGENE HBV MUTANT VL 19 showed fully coincident results in 88% of tested positions. These findings showed that all assays evaluated were sensitive and accurate tools to analyse HBV genomic variability. Sequence analysis is essential to study new emerging mutations as LiPA and AFFIGENE assays are more easily useful in clinical laboratories to detect the appearance of well-characterized HBV variants.

Genotypic resistance to lamivudine among hepatitis B virus isolates in Mexico

Drug resistance of hepatitis B virus (HBV) is an increasing clinical problem. Resistance to lamivudine in HBV isolates in Mexico has been poorly explored. To characterize the mutation patterns associated with genotypic resistance to lamivudine and their prevalence among HBV isolates in Mexico. Thirty-nine Mexican HBV isolates were analysed by PCR and line probe assay for detection of genetic variants in the polymerase open reading frame domains B and C (INNO-LiPA HBV DR; INNOGENETICS N. V., Ghent, Belgium). This assay detects wild-type and mutations at codons 180, 204 and 207 of the HBV polymerase gene, and at codon positions 171, 172, 195, 196, 198 and 199 of the HBV surface antigen (HBsAg). HBV isolates were obtained from HBsAg-positive serum samples of 15 chronic hepatitis patients, two haemodialysis patients with chronic HBV carriage, 20 men found positive for HBsAg when seeking HIV testing and two AIDS patients with chronic HBV infection. None of the participants had received antiviral therapy. Overall, HBV wild-type was found in 37 (94.9%) out of the 39 isolates studied. Two (5.1%) out of the 39 isolates showed mixed wild-type and mutant populations. These mutations occurred in isolates from one hepatitis patient and one haemodialysis patient. The isolate from the hepatitis patient showed a double mutation at codon positions 180 (L180M) and 204 (M204V), thus a 2.6% prevalence of genotypic resistance to lamivudine was found. The isolate from the haemodialysis patient showed a single mutation at codon position 180 (L180M). The two HBV mutant isolates were further analysed for genotype and both isolates were genotype H. HBV genotypic resistance to lamivudine exists in Mexican isolates. The results highlight the importance of testing for HBV resistance before treatment and have implications for a more rational use of drugs.

Intrahepatic Hepatitis B Virus Covalently Closed Circular DNA Can Be a Predictor of Sustained Response to Therapy

This study aimed to determine whether intrahepatic hepatitis B virus (HBV) covalently closed circular (ccc) DNA and total HBV DNA levels at the end of therapy would predict sustained response to therapy. Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients receiving either lamivudine monotherapy or combination of peginterferon and lamivudine had liver biopsy at the end of 1 year therapy and were followed for 52 more weeks after cessation of therapy. Serum HBV DNA, intrahepatic HBV ccc DNA, and total HBV DNA levels were determined. Forty-seven patients, including 34 males and 13 females, were studied. Twenty-seven patients received combination therapy, and 20 patients received lamivudine monotherapy. Twenty-nine patients had end-of-treatment virologic response, and 15 patients had sustained response 52 weeks after therapy. At the end of treatment, log serum HBV DNA levels correlated well with log intrahepatic HBV cccDNA and log intrahepatic total HBV DNA levels. Log intrahepatic cccDNA and log intrahepatic total DNA levels were significantly lower among patients with sustained virologic response. The adjusted odds ratio for log cccDNA was 5.3 (95% CI: 1.5-18.2, P = .009) and, for log intrahepatic HBV DNA, was 4.4 (95% CI: 1.3-14.7, P = .015) to predict sustained virologic response. Using log cccDNA at -0.80 copies/genome equivalent as cutoff, the sensitivity, specificity, and positive and negative predictive values and accuracy of predicting sustained virologic response were 73%, 78%, 56%, 86%, and 77% respectively. Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.

Evaluation of Methods for Monitoring Drug Resistance in Chronic Hepatitis B Patients during Lamivudine Therapy Based on Mass Spectrometry and Reverse Hybridization

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based genotyping assay, termed restriction fragment mass polymorphism (RFMP) has been recently developed for detecting hepatitis B virus (HBV) mutants. The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutations that confer resistance to lamivudine. We compared the efficacy and usefulness of the RFMP assay with a commercial assay using a reverse hybridization line probe technology, namely INNO-LiPA HBV DR (referred to henceforth as the LiPA assay), for the detection of lamivudine-resistant HBV mutants. A total of 60 patient samples were analysed for the presence of mutations at rtL1 80M and rtM204I/V of HBV polymerase by the LiPA and RFMP assays. The ability to detect mutations at rtM204I/V was compared with defined mixtures of wild-type and mutant HBV cloned in plasmids at relative concentrations ranging from 1-25%. Concordance between methods was found to be 95.0% (57/60) when only the presence of resistance mutations was considered, regardless of quasispecies. In three cases, additional minor populations of resistant viruses were identified by RFMP. Defined mixtures were consistently successfully identified at a 1% relative concentration of mutant versus wild-type viruses by the RFMP assay and 4% by the LiPA assay. The RFMP assay proved to be an accurate and reliable tool for detection of lamivudine-resistant mutations and was more sensitive than the LiPA assay in detecting mixtures of mutant and wild-type viruses. The improved sensitivity of the RPMP assay can help monitor drug resistance as it develops, enabling early intervention and prevention.

Successful therapy of hepatitis B with tenofovir in HIV-infected patients failing previous adefovir and lamivudine treatment

Three HIV-infected patients with chronic hepatitis B (genotype A) were switched to adefovir therapy after unsuccessful lamivudine treatment. Surprisingly, adefovir therapy failed, although none of the virus isolates displayed mutations known to be associated with adefovir resistance (A181V, N236T). In two isolates we identified hepatitis B virus DNA polymerase mutation L217R, in one case we found multiple frameshifts in the same region. In all cases adefovir was replaced by tenofovir, resulting in a significant drop in the viral load.

Evaluation of the INNO-LiPA HBV genotyping assay for determination of hepatitis B virus genotype.

Specific genotypes of hepatitis B virus (HBV) are increasingly recognized for their clinical significance and association with particular viral mutations. Although many HBV genotyping methods exist, there has been no standardized or commercially available method for direct molecular typing of the HBV genome. A newly available line probe assay (INNO-LiPA HBV Genotyping assay; Innogenetics N.V., Ghent, Belgium) that allows the identification of HBV genotypes A to G was assessed by comparison with pre-S1/pre-S2 sequence analysis of the isolates in 188 serum specimens. All seven genotypes were detected by the line probe assay (LiPA), and complete concordance between LiPA and sequence analysis was observed for 152 specimens (81%). LiPA was able to detect 19 mixed genotype infections not detected by amplicon sequencing, which for the most part were confirmed by cloning and sequencing of the pre-S1/pre-S2 amplicon. Four specimens had discrepant results between the two methods, and five specimens had indeterminate results by LiPA. The HBV DNA in four specimens was unable to be amplified by the nested INNO-LiPA HBV DR amplification primers; however, the HBV DNA in six specimens unable to be genotyped by sequencing was clearly genotyped by LiPA. The INNO-LiPA HBV Genotyping assay appears to be useful for the rapid genotyping of HBV, particularly for the sensitive detection of mixed genotype infections.

Monitoring the Emergence of Hepatitis B Virus Polymerase Gene Variants during Lamivudine Therapy in Human Immunodeficiency Virus Coinfected Patients: Performance of Clip™ Sequencing and Line Probe Assay

Sera from 12 patients infected with human immunodeficiency virus and hepatitis B virus (HBV), on lamivudine as part of an antiretroviral therapy, were retrospectively analysed for the presence of HBV polymerase mutations by the line probe assay, INNO-LiPA HBV DR, and by the direct sequencing assay, TRUGENE HBV genotyping kit. Results at codons 180, 204 and 207 were compared for 44 samples. Full concordance was observed for 81.4% of the 129 analysed codons. Discordance involved only mixed populations: LiPA detected additional species in 19 codons and TRUGENE in five. Viral breakthrough occurred in seven patients, 12-33 months after lamivudine initiation. In five cases with close sampling available, both assays detected mutations before the rise in viral load, although earlier by LiPA for three patients. The time interval between the first mutant detection and viral escape ranged from 2 to 22 months. Mutations were detected in four of the five remaining patients: 1) at therapy initiation in a primary non-responder; 2) after 37 months, but replication became undetectable after tenofovir introduction; 3) transiently at 6 months by LiPA but treatment was ceased thereafter; 4) after 23 months but replication levels remained low during a 5-year follow-up. Interestingly, TRUGENE sequencing identified on late samples from three patients a variant carrying rtV173L plus rtL180M plus M204V mutations, having the in vitro characteristics of 'vaccine escape' mutants. Both assays appear to be valuable tools for the early detection of mutated HBV strains. The detection of genotypic therapeutic decision-making, although clinical or other virological factors may determine the rapidity of the viral breakthrough.