Lamivudine-resistant Mutants Research Articles

Real-time PCR detection of multiple lamivudine-resistant mutations with displacing probes in a single tube

Background Detection of lamivudine-resistant hepatitis B virus (HBV) is essential to clinical diagnosis and treatment. Objectives To establish a single tube, real-time PCR assay for simultaneous detection of multiple lamivudine-resistant mutations in serum samples. Study Design By using four sequence-specific displacing probes labeled with different fluorophores, a single real-time PCR reaction can tell whether a sample contains any of the following HBV variants: wild-type, rtM204 mutant; mixtures of wild-type and rtM204 mutant; mixtures of rtM204 and rtL180 mutant; mixtures of wild-type, rtM204 mutant and rtL180 mutant. The assay was evaluated with 50 HBV mutation(s)-containing samples and 36 HBeAg-positive samples. Results The results of the real-time PCR assay were consistent with the DNA sequencing, but with much higher sensitivity for detecting a mixture of quasispecies. As few as 10 2–10 3 copies/ml HBV of all four sequences in pure population and as little as 5% mutant DNA in the presence of wild-type DNA can be detected. Conclusions Application of this high throughput assay into clinical use should enable earlier diagnosis and better treatment of lamivudine-resistant HBV.

Navigating the Maze of Hepatitis B Treatments

Navigating the Maze of Hepatitis B Treatments

Comparison of hepatitis B virus subgenotypes in patients with acute and chronic hepatitis B and absence of lamivudine‐resistant strains in acute hepatitis B in Japan

Hepatitis B virus (HBV) has been classified into eight genotypes and can be further divided into several subgenotypes that have different geographic distributions. Because of increased human migration, the prevalence of rare subgenotypes is increasing in Japanese patients with acute hepatitis B. Lamivudine-resistant strains of HBV have begun to emerge in association with chronic hepatitis B. The aim of this study was to investigate the distribution of HBV subgenotypes and lamivudine-resistant strains in patients in Japan with acute hepatitis B. One hundred twenty-three patients with acute hepatitis B and 123 with chronic hepatitis B were studied. HBV subgenotypes and lamivudine-resistance mutations were determined by direct sequencing of the preS and polymerase region, respectively. HBV subgenotypes Aa (n=3), Ae (n=23), Ba (n=7), Bj (n=3), Cs (n=7), Ce (n=76), D (n=2), and H (n=2) were detected in patients with acute hepatitis. In patients with chronic hepatitis, HBV subgenotypes Ae (n=4), Ba (n=1), Bj (n=18), and Ce (n=100) were found. Non-common Japanese subgenotypes, that is, non-Bj and non-Ce, were detected more frequently in patients with acute hepatitis (35.8%) than in patients with chronic hepatitis (4.1%) (Odds ratio, 0.076; 95%CI, 0.029-0.200; P<0.0001). Lamivudine-resistance mutations were detected in chronic hepatitis patients with breakthrough hepatitis but not in other patients. In conclusion, the prevalence of uncommon Japanese HBV subgenotypes is expected to increase, although lamivudine-resistant strains have not yet been found in patients with acute hepatitis B.

Low risk of hepatitis B virus recurrence after withdrawal of long-term hepatitis B immunoglobulin in patients receiving maintenance nucleos(t)ide analogue therapy

Hepatitis B virus (HBV) recurrence rates of 0-16% had been reported in patients maintained on nucleoside analogues (NA) after hepatitis B immunoglobulin (HBIG) discontinuation after orthotopic liver transplantation (OLT). However, follow-up in most studies was short. We aimed to determine the long-term risk of HBV recurrence using this strategy. All HBV patients who received > or =7 doses of intravenous HBIG after OLT, with no HBV recurrence while receiving HBIG, and who eventually discontinued HBIG and were maintained on NA, were included. HBV recurrence was defined as HBsAg-positive or HBV DNA > or =5 log copies/mL on 2 consecutive occasions. Twenty-one patients met the inclusion criteria. Immediate post-OLT prophylaxis was combination HBIG and NA in 15 patients, whereas 6 patients received HBIG monotherapy for 62-109 months before NA was added. HBIG was discontinued a median of 26 (range, 0.2-121) months after OLT. Median follow-up post-HBIG discontinuation was 40 (range, 5-51) months. Only 1 patient, who had 12 months of HBIG and was noncompliant to NA therapy, had HBV recurrence, 34 months after HBIG discontinuation. One patient had HBV DNA of 3.3 log copies/mL 47 and 48 months after HBIG discontinuation but remained HBsAg-negative. Lamivudine-resistant mutations were detected in both patients. Probability of HBV recurrence was 0% and 9% at 2 and 4 years after HBIG discontinuation. Three patients had 1-2 episodes of transiently detectable HBV DNA. All were HBV DNA and HBsAg negative on repeated tests over a period of 2-36 months. Maintenance therapy with NA after discontinuation of long-term HBIG therapy is associated with a low risk of HBV recurrence after OLT in compliant HBV patients.

Viral blips during long-term treatment with standard or double dose lamivudine in HBe antigen negative chronic hepatitis B

To evaluate safety and effect on hepatitis B virus (HBV) suppression of a long-term treatment with lamivudine (LAM) at standard (100 mg/d) or double (200 mg/d) dose in chronic hepatitis B. This was a case study with matched controls (1:3) in patients with chronic hepatitis B with anti-HBe antibodies. Twelve patients received LAM 200 mg/d and 35 LAM 100 mg/d, for a median of 28 mo. A primary response (PR; i.e. negative HBV-DNA with Amplicor assay) was achieved in 100% of LAM-200 patients and 83% of LAM-100 patients. A virological breakthrough occurred in 16.7 and 24.7%, respectively, of the PR-patients, with the appearance of typical LAM resistance mutations in all but one patient. Viremia blips (i.e. transient HBV-DNA below 80 IU/mL in patients who tested negative at Amplicor assay) were detected using a real time polymerase chain reaction (PCR) and occurred in seven out of nine patients with subsequent BT and in four out of 32 patients with end-of-study response (77.7% vs 12.5%; P = 0.001) at chi-square test). At the end of the study, 51.4% of LAM-100 patients and 83.3% of LAM-200 patients had remained stably HBV-DNA negative. Double-dose LAM was well tolerated. Long-term treatment of anti-HBe positive chronic hepatitis B with double dose lamivudine causes a more profound and stable viral suppression as compared to conventional treatment.

The High Incidence of the Emergence of Entecavir-Resistant Mutants among Patients Infected with Lamivudine-Resistant Hepatitis B Virus

Hepatitis B virus (HBV) infection remains to be one of the most prevailing infection in the world, causing chronic liver diseases. Although lamivudine has been effective to suppress HBV replication, longer durations of administration can lead to the emergence of drug-resistant mutant viruses, followed by reactivation of hepatic inflammation (breakthrough hepatitis). Moreover, the optimal period of administration as well as the effects of anti-viral nucleot(s)ide such as lamivudine, adefovir, and entecavir, has not been established. To evaluate the efficacy of the anti-viral effects of entecavir for lamivudine-resistant HBV, we administered entecavir sequentially in four patients with chronic HBV infection, who demonstrated the emergence of lamivudine-resistant HBV and histological active hepatitis. The antiviral effects were evaluated by the serum viral loads and biochemical laboratory data. After follow-up periods of more than 36 months, we found high incidence in the emergence of entecavir resistant mutants (3/4, i.e., 75%). An additional mutation at the 184th amino acid, different from the previously reported lamivudine-resistant mutations (80th, 180th, and 204th), seemed to have a close relationship with the induction of entecavir-resistant mutants at least for Japanese HBV genotype C. Our observation draws attention to the possibility that the usage of entecavir for lamivudine-resistant HBV could promptly induce entecavir-resistant mutations in addition to lamivudine-resistance.

Researching on the relation between lamivudine-resistant mutation and HBV genotypes

目的 探讨乙型肝炎病毒(HBV)基因型与拉米夫定耐药性变异类型间的关系.方法 选取95例来自本院肝炎门诊和病房的YMDD变异阳性血清样品,应用限制性内切酶MboⅠ和EarⅠ对PCR产物进行酶切分析及分型,并作遗传进化树图分析验证.结果 95例样品中检出C型75例,占79%;B型20例,占21%,进化树图分析证明酶切分型法确切可靠;B型病毒的YVDD变异、YIDD变异及YMDD+YVDD+YIDD联合变异的例数分别为11例、5例和4例,而C型中则分别为35例、27例和13例,对B、C基因型与YMDD变异类型间作卡方检验,得出差异无统计学意义(χ2＝0.856,P=0.710).结论 使用MboⅠ和EarⅠ酶切分型操作简单,结果可靠.HBV B型和C型病毒感染对发生不同YMDD变异类型无影响。

Precore mutant HBV-infection in a cirrhotic patient experiencing lamivudine-resistance and adefovir-nonresponse: no histological and immuno-histochemical evidence of re-infection treating with tenofovir plus lamivudine over 1 post-transplant year

Aims: Antiviral-drug-resistance is a common phenomenon in chronic hepatitis B therapy. Lamivudine-resistant mutants are mostly sensitive to the nucleoside analogue adefovir dipivoxil. Adefovir-resistant mutants are rare and retain sensitive to lamivudine. Patients with cirrhosis and precore mutant infection have an increased risk for exacerbation of liver disease due to drug-resistance. Viral resistance against both drugs is an indication for switching treatment to experimental antiviral regimes. Main therapeutic need is to prevent virological failure as well as to reach sufficient viral suppression before liver transplantation.

Emergence of a Novel Lamivudine-Resistant Hepatitis B Virus Variant with a Substitution Outside the YMDD Motif

Lamivudine is a major drug approved for treatment of chronic hepatitis B virus (HBV) infection. Emergence of drug-resistant mutants with amino acid substitutions in the YMDD motif is a well-documented problem during long-term lamivudine therapy. Here we report a novel lamivudine-resistant strain of HBV with an intact YMDD motif, which included an amino acid substitution, rtA181T, in the reverse transcriptase (RT) domain of HBV polymerase. The substitution also induced a unique amino acid substitution (W172L) in the overlapping hepatitis B surface (HBs) protein. The YMDD mutant strains were not detected even by using the sensitive peptide nucleic acid-mediated PCR clamping method. The detected nucleotide substitution was accompanied by the emergence of an additional nucleotide substitution that induced amino acid change (S331C) in the spacer domain. The rtA181T mutant strain displayed a threefold decrease in susceptibility to lamivudine in in vitro experiments in comparison with the wild type. In vivo analysis using human hepatocyte-chimeric mice confirmed the resistance of this mutant strain to lamivudine. We developed a method to detect this novel rtA181T mutation and a previously reported rtA181T mutation with the HBs stop codon using restriction fragment length polymorphism PCR and identified one patient with the latter pattern among 40 patients with lamivudine resistance. In conclusion, although the incidence is not high, we have to be careful regarding the emergence of lamivudine-resistant mutant strains with intact YMDD motif.

Systematic review: lamivudine prophylaxis for chemotherapy‐induced reactivation of chronic hepatitis B virus infection

Reactivation of hepatitis B virus infection in asymptomatic hepatitis B surface antigen carriers undergoing chemotherapy or immunosuppressive therapy is a well-documented and potentially fatal complication. Data supporting the use of lamivudine for primary prophylaxis have emerged, but its use remains controversial and is not standardized. To review current randomized-controlled trials, randomized trials and prospective case series to provide a clinically applicable, evidence-based recommendation. The published literature was identified using a MEDLINE/PubMed search with secondary review of cited publications, and inclusion of all prospective studies. In nine prospective trials and one randomized-controlled trial, the rate of hepatitis among subjects receiving lamivudine prophylaxis ranged from 0% to 20% (16 of 173, 9.2%), compared with 33-67% among controls. Of patients receiving prophylaxis, 0-24% (15 of 173, 8.7%) developed hepatitis B virus reactivation, compared with 29-56% of controls. Three reactivation-related mortalities were reported (one receiving prophylaxis, two controls). No patients withdrew secondary to toxicity or development of lamivudine-resistant mutations. The available data show a four- to sevenfold decrease in the rate of hepatitis and hepatitis B virus reactivation in patients who receive lamivudine prophylaxis. It is thus recommended that all hepatitis B surface antigen carriers receive lamivudine, or a comparable anti-viral agent, as prophylaxis from the initiation of chemotherapy until at least 1 year following its completion.

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.

Sequential treatment with lamivudine and interferon- monotherapies in hepatitis B e antigen-negative Chinese patients and its suppression of lamivudine-resistant mutations

To assess the efficacy of sequential treatment with lamivudine and interferon-alpha monotherapies in Chinese patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B. One hundred and sixty-two patients with HBeAg-negative chronic hepatitis B were included in this study. Ninety-eight were treated with lamivudine alone (100 mg per day) for 48 weeks (group B). Sixty-four were treated with lamivudine alone (100 mg per day) for 20 weeks, then combined with interferon-alpha-2b (5 million units three times per week) for 4 weeks and then treated for another 24 weeks with interferon-alpha-2b alone (5 million units three times per week) (group A). All patients were followed for an additional 24 weeks. After 48 weeks of treatment, the percentage of patients with normalization of alanine aminotransferase (ALT) levels or hepatitis B virus (HBV) DNA levels below 1000 copies/mL was not significantly different between the lamivudine monotherapy group (55.10% and 55.10%, respectively) and the sequential treatment group (59.36% and 56.25%, respectively). The percentage of patients with normalized ALT levels was significantly higher in group A (53%) than in group B (36%) at week 72 (P<0.05). The percentage of patients with lamivudine-resistant mutations was significantly higher with lamivudine monotherapy (22.45%) than with sequential therapy (P<0.05). Sequential treatment of chronic hepatitis B with lamivudine and interferon-alpha monotherapies is as effective as lamivudine-alone treatment in Chinese patients. However, sequential treatment can significantly suppress the emergence of lamivudine-resistant mutations.

Patterns of hepatitis B virus infection in Brazilian human immunodeficiency virus infected patients: high prevalence of occult infection and low frequency of lamivudine resistant mutations

Hepatitis B virus (HBV) molecular profiles were determined for 44 patients who were infected with human immunodeficiency virus (HIV) type 1 and had antibodies to the hepatitis B core antigen (anti-HBc), with and without other HBV serological markers. In this population, 70% of the patients were under lamivudine treatment as a component of antiretroviral therapy. HBV DNA was detected in 14 (32%) patients. Eight out of 12 (67%) HBsAg positive samples, 3/10 (30%) anti-HBc only samples, and 3/22 (14%) anti-HBs positive samples were HBV DNA positive. HBV DNA loads, measured by real time polymerase chain reaction, were much higher in the HBsAg positive patients (mean, 2.5 x 10(9) copies/ml) than in the negative ones (HBV occult infection; mean, 2.7 x 10(5) copies/ml). Nine out of the 14 HBV DNA positive patients were under lamivudine treatment. Lamivudine resistant mutations in the polymerase gene were detected in only three patients, all of them belonging to the subgroup of five HBsAg positive, HBV DNA positive patients. A low mean HBV load (2.7 x 10(5) copies/ml) and an absence of lamivudine resistant mutations were observed among the cases of HBV occult infection.

Liver transplantation for hepatitis B-related cirrhosis: recent advances

In the absence of preventative therapy, deceased-donor liver transplantation indicated for hepatitis B virus (HBV) cirrhosis results in dismal graft and patient survival due to HBV infection of the liver graft. Major advances in the management of HBV-infected recipients during the past 15 years have reduced the rate of graft infection, resulting in improved outcomes, comparable to those for patients transplanted for non-HBV indications. Long-term use of hepatitis B immunoglobulin for passive immunotherapy is effective in preventing re-infection. Combination therapy with hepatitis B immunoglobulin and lamivudine after liver transplantation reduces HBV recurrence. Adefovir dipoxil is a safe and effective alternative oral antiviral treatment for lamivudine-resistant mutant HBV. The high cost of hepatitis B immunoglobulin remains a problem that must be overcome by the development of HBV vaccines and potent adjuvants.

Lamivudine resistance in patients with chronic hepatitis B: Role of clinical and virological factors

Lamivudine resistance is associated with long-term monotherapy for chronic hepatitis B and can lead to potentially serious clinical consequences. Scant information exists regarding the influence of hepatitis B virus variants in the development of resistance. The present study was designed to identify factors predictive of lamivudine resistance, with a particular focus on the role of precore and basal core promoter variants in the setting of hepatitis B e antigen-negative disease. Eighty-five patients, representing four major genotypes, were followed prospectively on lamivudine therapy. Resistance was defined as an increase in viral load, with polymerase gene sequencing confirming a lamivudine resistance mutation. Median follow up was 19 months (6-54 months). The Cox proportional hazards model was used to determine variables independently predicting for the early onset of lamivudine resistance. The rate of lamivudine resistance was 6%, 31% and 51% at 12, 24 and 48 months, respectively. Multivariate analysis identified the precore variant, high baseline alanine aminotransferase (ALT), and persistent viremia (at 6 months) as independent predictors of the early development of lamivudine resistance, with rate ratios of 4.93 (95% confidence interval [CI]: 1.32-18.5), 1.22 (95%CI: 1.08-1.49), and 4.73 (95%CI: 1.49-15.0), respectively (P < 0.05). Female sex predicted early resistance (rate ratio 5.27 [95%CI: 1.23-22.5, P < 0.05]) although numbers were small (n = 12). Genotype did not influence treatment response nor time to onset of resistance. Patients with precore variant hepatitis B virus are likely to develop lamivudine resistance early and should be considered for alternate first-line monotherapy. In the future, combination antiviral therapy may limit the development of resistance.

Response to Lamivudine-Resistant Hepatitis B Virus Infection Postliver Transplantation from a Hepatitis B Core Antibody Donor

To the Editor: We would like to respectfully respond to the comments of Dr. Hans Tillmann. We appreciate his interest in our case report and review. We would like to clarify that we do not believe that the liver initially harbored a lamivudine-resistant mutant, but that the mutation occurred during prolonged lamivudine exposure leading to de novo breakthrough hepatitis B virus infection. There is no reason to suspect the donor already had a lamivudine-resistant mutation, otherwise the recipient should have developed acute hepatitis B at a much earlier period due to the lack of protection by lamivudine and loss of anti-HBs after 6 months. Hepatitis B virus infection from a source other than the graft would also be very unlikely. Retrospective analysis of our cohort of 402 recipients (including the reported case), receiving 467 liver transplants between March 1998 and December 2001, revealed three cases of de novo Hepatitis B (0.6%) after a median follow up of 4.5 years (1Bonatti H Machicao V Dickson RC Mendez J Results of liver transplantation in HBV core antibody positive individuals and recipients of allografts from HBV core antibody positive individuals..Eur Surg. 2005; 37: 15Google Scholar). One case was acquired during unprotected intercourse. The second was an anti-HBc and anti-HBs negative recipient who developed acute hepatitis B 4 months after liver transplantation. Hepatitis B donor status was unknown at the time of transplantation and therefore no lamivudine prophylaxis was administered. The donor was subsequently found to be anti-HBc positive after retrospective analysis of stored serum. The third was the patient presented in the case report who developed HBV more than 3 years posttransplant while receiving continuous lamivudine prophylaxis. He was a heterosexual, elderly male without other viral exposures (HIV and HCV negative) or other viral risk factors pre- or posttransplantation. This data strongly support that the HBV infection was acquired from the graft. The development of lamivudine-resistant mutation after prolonged lamivudine therapy is not surprising. HBV DNA has been shown to persist after liver transplantation despite successful antiviral prophylaxis. Terrault et al. demonstrated HBV DNA in 8/9 patients a mean of 27 (range 13–33) months after liver transplantation for HBV despite successful HBIg prophylaxis (2Terrault NA Zhou S Combs C et al.Prophylaxis in liver transplant recipients using a fixed dosing schedule of hepatitis B immunoglobulin..Hepatology. 1996; 24: 1327-1333Crossref PubMed Google Scholar). In addition, the frequency of lamivudine resistance is known to increase with time in patients receiving lamivudine monotherapy for chronic Hepatitis B. Lai et al. described rates of 53% and 70% after 3 and 5 years of therapy (3Lai CL Dienstag J Schiff E et al.Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B..Clin Infect Dis. 2003; 36: 687-696Crossref PubMed Scopus (558) Google Scholar). The other patient in our series who developed de novo HBV from an anti-HBC graft also developed a lamiuvidine-resistant mutation after 1 year on lamivudine treatment. We agree with Dr. Tilman that the proof of our hypothesis might be possible if specimens from the liver and later on consecutive serum samples would be available for retesting. Unfortunately, we cannot provide these data. Four lessons should be learned form this case. First, donors must be tested for anti-HBc antibodies and these organs ideally should be allocated to Hepatitis B-exposed individuals. Second, serum and tissue from donors and recipients should be stored for retrospective studies. Third, a significant follow-up period is required to make any definitive statements about recurrent or de novo hepatitis B after liver transplant. Fourth, the risk for development of resistant mutations posttransplant must not be neglected.

Rapid quantitation of lamivudine-resistant mutants in lamivudine treated and untreated patients with chronic hepatitis B virus infection

BackgroundLong-term lamivudine treatment induces emergence of lamivudine-resistant hepatitis B virus (HBV) in a significant number of patients with chronic HBV infection. Rapid and quantitative methods to determine the percentage of lamivudine-resistant mutants in total HBV are important during lamivudine therapy. MethodsWe established a quantitative real-time PCR method with selective primers and TaqMan probe to detect the percentage of lamivudine-resistant mutants in total HBV without the need of external DNA standards. This percentage was calculated as the PCR efficiency raised to the differences between threshold cycle number (ΔCt) of mutant and control reactions. Clones of the HBV polymerase gene containing the different YMDD variants were diluted in series and tested. Serum samples from 145 lamivudine-treated and 98 untreated patients with chronic hepatitis B virus infection were analyzed using this method and compared with DNA sequencing. ResultsAs little as 0.1% mutant plasmids in 106–109 copies/ml of wild-type plasmids were detected. Among the 145 patients treated with lamivudine, 42 of them had mutants with percentages of 5–100%. In six discordant results between real-time PCR and DNA sequencing, real-time PCR detected mutants with percentages of 5–20%, which were concordant with subclone sequencing. Five of 98 lamividine-untreated patients had mutants of 10–20% in wild-type virus populations. Compared to DNA sequencing, real-time PCR was fast and cost-effective. ConclusionThis real-time PCR is a rapid, sensitive and cost-effective method for relative quantitation of YMDD mutants of HBV.

Effect of Various Pyrimidines Possessing the 1-[(2-Hydroxy-1-(hydroxymethyl)ethoxy)methyl] Moiety, Able To Mimic Natural 2‘-Deoxyribose, on Wild-type and Mutant Hepatitis B Virus Replication

Hepatitis B virus (HBV) is the most common cause of chronic liver disease worldwide. Development of drug resistance against clinical anti-HBV drug lamivudine due to long-term use and rebound of viral DNA after cessation of treatment has been a major setback of the current therapy. We have synthesized a series of pyrimidine nucleosides possessing a variety of substituents at the C-5 position, and a 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl] flexible acyclic glycosyl moiety at the N-1 position, that have the ability to mimic the natural 2'-deoxyribosyl moiety. Some of these potential antiviral compounds included variations at both C-5 and C-6 positions of the uracil base. Other variations of the uracil derivatives were the 6-aza congeners. 4-Amino and 4-methoxy pyrimidine derivatives were also made. Compounds in which the base moiety was substituted by 5-chloro- (25), 5-(2-bromovinyl)- (32), or 5-bromo-6-methyl- (37) groups possess significant activity against duck-HBV, wild-type human HBV (2.2.15 cells), and lamivudine-resistant HBV containing single and double mutations. No cytotoxicity was seen in host HepG2 and Vero cells, up to the highest concentration tested. The anti-HBV activity exhibited by compounds 25, 32, and 37 was superior for human HBV and comparable for DHBV to that of the corresponding purine nucleoside, ganciclovir. Further, they were only 10-15-fold less inhibitory against human HBV in 2.2.15 cells than the reference drug, lamivudine. Other compounds in the series were moderately inhibitory against DHBV and wild-type human HBV. The size of the halogen and the electronegativity of the substituents at the 5- and 6-positions are important for antiviral activity toward HBV. These compounds were also evaluated for their antiviral activity for West Nile virus, respiratory syncytial virus, SARS-coronavirus, and hepatitis C virus. They were generally inactive in these antiviral assay systems (at concentrations up to 100 microg/mL). 1-[(2-Hydroxy-1-(hydroxymethyl) ethoxy)methyl]-5-fluorocytosine (34) showed some inhibitory activity against hepatitis C virus. Taken together, these data support our previous observations that the 5-substituted pyrimidine nucleosides containing acyclic glycosyl moieties have potential to serve as a new generation of potent, selective, and nontoxic anti-HBV agents for wild-type and lamivudine-resistant mutant HBV.

High rates of progressive hepatic functional deterioration whether lamivudine therapy is continued or discontinued after emergence of a lamivudine-resistant mutant: a prospective randomized controlled study

The management of patients with lamivudine-resistant mutants remains challenging, and no clear evidence has been presented concerning the discontinuation of lamivudine. Seventy-four patients with lamivudine-resistant mutants were prospectively enrolled and randomized; 37 patients continued (group A) and 37 patients discontinued lamivudine therapy (group B). The median follow-up was 20 months. Serum albumin levels were reduced and prothrombin time was prolonged in both groups versus baseline (P = 0.015 and 0.045, respectively). Four patients in group A (10.8%) and six in group B (16.2%) experienced hepatitis flare, but the difference was not significant (P > 0.05). Multivariate analyses identified a younger age as a risk factor for hepatitis flare (P = 0.021). Seven (18.9%) decompensations occurred in group A and five (13.5%) in group B, which was not a significant difference (P > 0.05). Multivariate analyses revealed higher alanine aminotransferase and a lower platelet count as risk factors for hepatic decompensation (P = 0.001 and 0.001, respectively). The patients whose platelet count was <65 000/microl experienced hepatic decompensations more frequently (50%) than those with platelet counts >65 000/microl (13.2%) during follow-up (P = 0.05). The clinical course of group B was not significantly different from that of group A. Therefore, the discontinuation of lamivudine may be a feasible option when other antiviral agents active against lamivudine-resistant mutants are unavailable.

Characteristics of drug resistant HBV in an international collaborative study of HIV-HBV-infected individuals on extended lamivudine therapy

Little is known about the prevalence and pattern of hepatitis B virus (HBV) mutations in HIV/HBV co-infected individuals on long-term lamivudine (3TC) therapy. HBV polymerase/envelope/basal core promoter/pre-core sequences from 81 HIV-HBV co-infected persons who received at least 6 months 3TC were compared to HBV reference sequences. Host and viral characteristics associated with HBV mutations were determined. HBV viraemia was detected in 53 persons (65%) and was associated with lower CD4 cell count nadir and higher HIV RNA at the time of testing but not with 3TC duration. Known 3TC-resistant mutations occurred in 50% and 94% of viremic patients with < 2 years and > 4 years 3TC, respectively. The CD4 cell count at testing was significantly higher in those with 3TC-resistant mutations. The triple polymerase mutant (rtL173V, rtL180M, rtM204V), which behaves as a vaccine escape mutant in vitro, occurred in 17% of viraemic patients. Polymerase mutations that may confer resistance to other anti-HBV agents were also detected. In HIV-HBV co-infected patients, greater immunocompromise is associated with continued HBV viraemia while on 3TC, and development of 3TC-resistant mutations are inevitable with prolonged 3TC use. These mutant viruses may limit future therapeutic options due to cross-resistance or may produce HBV vaccine escape mutants. Thus, timing and selection of antiretroviral therapy is critical in this population.