Hepatitis B Virus YMDD Research Articles

SAT0140 SAFETY OF TOFACITINIB THERAPY IN HBSAG CARRIERS WITH RHEUMATOID ARTHRITIS: A PROSPECTIVE STUDY

Background:Targeted synthetic DMARDs (ts-DMARDs) are becoming more available and affordable in developing countries, where the prevalence of hepatitis B virus (HBV) infection is still an important public health issue. The safety of ts-DMARDs therapy in terms of the reactivation of hepatitis B virus (HBV) infection need more concern. Rare data from a prospective study focus on the use of ts-DMARDs in patients with concurrent rheumatoid arthritis (RA) and HBV infection were available by now.Objectives:To evaluate the influence of tofacitinib on reactivation of HBV infection in HBsAg carriers with RA.Methods:In this 52 weeks observation, HBsAg carriers with active RA (DAS28>5.1) despite failed combined treatment with MTX and other non-biological DMARDs were enrolled. Patients must have normal liver function prior to study. All patients received therapy with tofacitinib (5mg twice daily) and concomitant MTX (10-12.5mg/w). Entecavir was prescribed preventively for patients who had a baseline HBV load >2000 copy/ml (group 1), and Lamivudin for patients with HBV load ≤ 2000 copy/ml (group 2). Liver enzymes (AST/ALT) and HBV viral load were monitored every 4 weeks. Increased viral load and abnormal liver function were managed according to expert opinion.Results:Thirteen patients (10 female) were recruited. Nine patients had a baseline viral load >2000 copy/ml (group 1, with preventive Entecavir), and the other 4 patients had a viral load ≤ 2000 copy/ml (group 2, with preventive Lamivudin). Two patients from group 1 discontinued tofacitinib at week 12 due to ineffectiveness, and both continued taking Entecavir for another 3 months after the discontinuation of tofacitinib.No reactivation of hepatitis B was observed in patients from group 1. One patients (female, 54 years old) from group 2 underwent a mild increase of both ALT and AST (67 and 56 IU/L, respectively) at week 16. An elevated viral load (4.9e6 copies/ml, baseline 1.4e3) and a HBV YMDD mutant was also found. The tofacitinib treatment continued. After prescription of Adefovir (combined with the pre-existing Lamivudin), both liver enzyme and viral load decreased to normal range in 8 weeks and remained normal throughout the study.Conclusion:An aggressive Tofacitinib + MTX therapy may be a safe option for HBsAg carriers with cs-DMARDs refractory RA. More active and effective prophylaxis strategy may be recommended to reduce the risk of HBV reactivation during the treatment.

Highly sensitive and specific detection of hepatitis B virus DNA and drug resistance mutations utilizing the PCR-based CRISPR-Cas13a system

ObjectivesUndetectable or low-level hepatitis B virus (HBV) DNA and drug resistance mutations in patients may increase the risk of HBV transmission or cause active viral replication and other clinical problems. Here, we established a highly sensitive and practical method for HBV and drug resistance detection using a polymerase chain reaction (PCR) -based CRISPR-Cas13a detection system (referred to as PCR-CRISPR) and evaluated its detection capability using clinical samples. MethodsSpecific CRISPR RNAs (crRNAs) are designed for HBV DNA detection and YMDD (tyrosine-methionine-aspartate-aspartate) variant identification. The HBV DNA was detected in 312 serum samples for HBV diagnosis using quantification PCR (qPCR) and PCR-CRISPR. Additionally, 424 serum samples for YMDD testing were detected by qPCR, direct sequencing, and our assay. ResultsUsing PCR-CRISPR, one copy per test of HBV DNA was detected with HBV-1 crRNA in 15 min after PCR amplification. Consistent results with qPCR were observed for 302 samples, while the remaining 10 samples with low-level HBV DNA were detectable by PCR-CRISPR and droplet digital PCR but not by qPCR. PCR-CRISPR diagnosed all 412 drug-resistant samples detected by the YMDD detection qPCR kit and direct sequencing, as well as the other 12 drug-resistant samples with low-level HBV DNA undetectable by qPCR and direct sequencing. ConclusionsWe developed a novel PCR-CRISPR method for highly sensitive and specific detection of HBV DNA and drug resistance mutations. One copy per test for HBV DNA and YMDD drug resistance mutations could be detected. This method has wide application prospects for the early detection of HBV infection, drug resistance monitoring and treatment guidance.

Clinical significance of periodic detection of hepatitis B virus YVDD mutation by ultrasensitive real-time amplification refractory mutation system quantitative PCR during lamivudine treatment in patients with chronic hepatitis B.

Monitoring hepatitis B virus (HBV) mutants periodically during nucleoside analogue treatment is of great clinical significance, particularly in persistently HBV DNA-positive patients. However, few studies have investigated the dynamic changes of HBV YMDD (Tyr-Met-Asp-Asp) and YVDD (Tyr-Val-Asp-Asp) populations in chronic hepatitis B (CHB) patients whilst undergoing lamivudine (LMV) treatment. In this study, we sought to investigate the dynamic changes of HBV YMDD and YVDD variants by ultrasensitive real-time amplification refractory mutation system quantitative PCR (RT-ARMS-qPCR) and evaluate its significance for changes in the treatment of CHB patients. RT-ARMS-qPCR was established and evaluated with standard recombinant plasmids. Fifteen CHB patients receiving LMV (100 mg daily) were consecutively recruited and followed up for 60 weeks. Serum samples were obtained from each patient at baseline and every 12 weeks. The total HBV DNA, HBV YMDD DNA and YVDD DNA levels were measured using RT-ARMS-qPCR at all given time points after treatment. Routine liver biochemistry parameters, including aspartate aminotransferase and alanine aminotransferase, were also measured every 12 weeks. The linear range of the assay was between 1×10(12) and 1×10(5) copies ml(-1). The low detection limit was 1×10(4) copies ml(-1). After 60 weeks of LMV treatment, nine patients experienced virological breakthrough. The YVDD variant could be detected 12-48 weeks before virological breakthrough. The YVDD variant was detected as the predominant population (range 69.4-100 %) in patients by the time virological breakthrough appeared. We concluded that RT-ARMS-qPCR was sensitive for the detection and quantification of low levels of HBV mutation. Periodic detection of HBV YM(V)DD every 12 weeks during LMV treatment is helpful for therapeutic decision making.

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.

THU0217 Safety of Infliximab Therapy in HbsAg Carriers with Rheumatoid Arthritis: A Prospective Study

BackgroundPrevalence rate of hepatitis B virus (HBV) infection in China (13%) and many other developing countries are much higher than that in developed countries. When anti-TNF agents are more available...

Relationship between hepatocellular carcinoma and hepatitis B virus genotype with spontaneous YMDD mutations

To investigate the relationship between hepatitis B virus (HBV) genotype with spontaneous YMDD mutations and hepatocellular carcinoma (HCC) in HBV-related cirrhosis. We investigated 264 liver cirrhosis patients who were not treated with antiviral drugs, including 81 patients with HCC. YMDD mutations were detected by fluorescent hybridization bioprobe polymerase chain reaction (PCR) and melting curve assay using the Diagnosis Kit for HBV YMDD Mutation. Serum HBV genotypes were detected by real-time PCR using genotype-specific TaqMan probes. Statistical analysis was performed according to data type using the t test, χ(2) test and unconditional logistic regression analysis. In the HCC group, genotype C strains, spontaneous YMDD mutations, and genotype C strains with YMDD mutations were detected in 33 (40.74%), 13 (16.05%) and 11 (13.58%) patients, respectively. In the liver cirrhosis (LC) group, HBV genotype C strains, spontaneous YMDD mutations, and genotype C strains with YMDD mutations were detected in 33 (18.03%), 7 (3.83%) and 2 (1.09%) patients, respectively. The differences in genotype C strains, spontaneous YMDD mutations, and genotype C strains with YMDD mutations between the two groups were statistically significant (χ(2) = 15.441, P = 0.000; χ(2) = 11.983, P = 0.001; P = 0.000). In the HCC and LC groups, there were seven patients infected by genotype B strains with YMDD mutations and 13 by genotype C strains with YMDD mutations. Further research revealed that HCC occurred in 2 patients infected by genotype B strains with YMDD mutations and 11 infected by genotype C strains with YMDD mutations. The difference was statistically significant (P = 0.000). Unconditional logistic regression analysis revealed that patients infected by genotype C strains with spontaneous YMDD mutations had a 7.775-fold higher risk for the development of HBV-related HCC than patients infected by other type HBV strains (P = 0.013, 95%CI: 1.540-39.264). Genotype C strains with spontaneous YMDD mutations are an independent risk factor for HCC in LC patients and are important for early warning of HCC.

HBV genotypes distribution and YMDD spontaneous mutation in Qingdao population

To investigate the distribution of hepatitis B virus (HBV) genotypes in Qingdao, and the relationship of HBV genotypes with the serum HBV-DNA levels and HBV YMDD spontaneous mutation of patients, then to discuss the clinical significance. Hepatitis B virus genotypes and YMDD spontaneous mutation of 144 patients were detected by real time PCR (Taqman probe), then the results were analyzed by statistical method. Of the 144 patients, 130 (90.3%) were genotype C, 12 (8.3%) were genotype B, and 2 (1.4%) were neither genotype B nor genotype C; 33 (22.9%) were detected to have YMDD mutation, and 25 (75.5%) were YVDD positive, 3 (9.1%) were YIDD positive, 5 (15.2%) were YVDD and YIDD positive. There were no significant differences between clinical diagnosis, serum HBV-DNA levels, YMDD spontaneous mutation and HBV genotypes (P > 0.05). Genotype C is the dominant position for HBV genotype in Qingdao. Untreated patients with chronic hepatitis B have YMDD spontaneous mutation. HBV genotypes have no association with YMDD spontaneous mutation and the development of diseases.

Variants and quasispecies of reverse transcriptase region in polymerase gene of hepatitis B virus during lamivudine treatment

To investigate the variants and quasispecies of reverse transcriptase region in polymerase gene of hepatitis B virus (HBV) during lamivudine treatment and their relationship with genotypes and viral loads. HBV DNA of 117 chronic hepatitis B patients treated with lamivudine were amplified by using PCR. The PCR products including the YMDD motif were sequenced by DNA sequencer, of which, HBV DNA viral loads of 99 patients were determined by real-time PCR and 64 samples were sequenced by Pyrosequencing. In HBV YMDD variant group and no variant group, the HBV genotypes were 79.6% and 86.7% of type C, 18.5% and 12.7% of type B, 1.9% of A/B recombinant type and 2.6% of type D, respectively. The viral loads (log 10) were 6.5699 and 6.6165, respectively. There was no significant difference in HBV genotypes and viral loads between these two groups. The rtL180M variant was found in association with the rtM204I/V variant, HBV variants and wild-type in YMDD motif all existed together in these two groups. HBV variants (quasispecies) in YMDD motif could be quantified by pyrosequencing, which would be a feasible measure during nucleoside or nucleotide analogue therapy against chronic HBV infection.

Prevention and Risk Factors of the HBV Recurrence After Orthotopic Liver Transplantation: 160 Cases Follow-Up Study

The aim of this study was to analyze the combination with long-term, low-dose hepatitis B immunoglobulin (HBIG) and nucleos(t)ide analogs as prophylaxis for hepatitis B virus (HBV) recurrence and to assess the risk factors of HBV recurrence after orthotopic liver transplantation (OLT). One hundred sixty patients undergoing OLT with HBV-related liver disease make up the cohort studied. Long-term, low dosage of HBIG in combination with nucleos(t)ide analogs were used as prophylaxis for HBV recurrence after OLT. Patient preoperative data were collected by a retrospective method, and the rate and risk factors of HBV recurrence post-OLT after a long-term follow-up were analyzed. Nineteen patients developed hepatitis B recurrence for a rate of recurrence of 11.88% (19/160). There was no significant correlation between HBV recurrence after OLT and the level of HBV DNA, HBeAg state pre-OLT, or the use of nucleoside analog drug therapy pre-OLT (P>0.05). Of 19 patients with HBV recurrence, 17 patients used Lamivudine, and HBV YMDD mutants were detected in nine cases. The HBV-YMDD mutation was the major reason for recurrence of HBV in our study (P<0.001). Long-term use of combination prophylaxis with nucleoside analogs and low-dose HBIG can effectively prevent hepatitis B recurrence after OLT, and that a positive preoperative serum HBV DNA status did not affect the recurrence rate of HBV post-OLT. Preoperative nucleoside analogs therapy is unlikely to be obligatory if the patients received effective combination prophylaxis postoperatively. HBV YMDD mutation is the primary reason for HBV recurrence in patients treated with Lamivudine after OLT.

Establishment of a PCR-product direct sequencing for the detection of HBV YMDD mutation

Objective To develop an assay of PCR-produet direct sequencing to detect hepatitis B virus (HBV) YMDD mutation, and compare the results gained by the sequencing and traditional real-time fluorescent PCR assays. Methods Serum samples were collected from 103 patients with chronic hepatitis B. HBV DNA were extracted from sers. YMDD mutation was detected by a commercial real-time PCR assay. Meanwhile, HBV reverse transcriptase-encoding gene was amplified by a nested PCR assay. The PCR products were directly subjected to sequencing at two directions, and the sequencing results were analyzed by NTI program. Using Kappa test, comparison was made between the results of rtM204-site mutations obtained by the direct sequencing and YMDD mutations by the real-time fluorescent PCR. Results The direct sequencing assay proved to be highly effective with bread range of detection in viral load from 500 to 1010copies/ml. And it may simultaneously avoid inhibitory effect caused by high viral load. The coincidence rates between two assays were 100% for YIDD, 97. 1% for YVDD, 76. 2% for YIDD/YVDD coexistence (Kappa = 0. 853, P < 0. 01). Conclusions The direct sequencing assay for HBV drug-resistant mutation detection is highly sensitive with broad dynamic range. It has high coincidence rate with real-time fluorescent PCR assay with advantage of detecting YMDD, YIDD and YVDD mutations simultaneously. Key words: Hepatitis B virus; Sequence analysis, DNA; Polymerase chain reaction; Mutation

Prevention and treatment of hepatitis B recurrence after liver transplantation

To investigate the effect of the regimen of lamivudine (LAM) combined with hepatitis B immunoglobulin (HBIG) in prevention and treatment of re-infection of hepatitis B virus (HBV) and recurrence of hepatitis B after orthotopic liver transplantation (OLT) for HBV related end stage liver disease. The clinical data of 183 adult liver transplantation patients who lived more than 6 months and were followed up for 14.6 months with complete data were studied retrospectively. According to the HBV prevention strategy, these recipients were divided into two groups: group of pure LAM (n = 106) and group of LAM plus intramuscular injection of low dose HBIG (n = 77). The rate of HBsAg negative conversion 1 week after OLT of the LAM group was 82.10% (87/106), significantly lower than that of the LAM + HBIG group [94.81% (73/77), P = 0.010]. The rates of HBV reinfection, HB recurrence, and YMDD mutation of the lamivudine group were 16.98% (18/106), 11.32% (12/106), and 8.49% (9/106) respectively, all significantly higher than those of the LAM + HBIG group [6.49% (5/77), 2.60% (2/77), and 1.30% (1/77) respectively, P = 0.035, 0.028, and 0.035 respectively]. All the patients with YMDD mutation were treated with adefovir (ADF) with improvement. Analysis showed no obvious difference in the effect of LAM given intramuscularly or intravenously. The protocol of combination of LAM and HBIG is highly effective, safe, and cost-effective in preventing the recurrence of HBV after OLT. YMDD mutation can be treated by ADF with satisfactory results.

The long-term effects of lamivudine treatment in patients with HBeAg-negative liver cirrhosis

In hepatitis B virus (HBV)-related liver cirrhosis, patients with HBV replication show a higher mortality rate than those without. We aimed to investigate the long-term effects of lamivudine on HBV DNA suppression, Child-Pugh score, and survival in patients with hepatitis Be antigen (HBeAg)-negative liver cirrhosis. Sixty-eight patients (51 male, 17 female) diagnosed with HBV-positive liver cirrhosis, who were monitored by the hepatology and liver transplantation outpatient clinics of our hospital between June 1999 and May 2007, were included in the study. Lamivudine (100 mg/day) was administered orally. Follow-up visits were scheduled monthly during the first 3 months, and every 3 months thereafter. Complete blood count, haemostasis, biochemistry (aspartate aminotransferase [AST], alanine aminotransferase [ALT], amylase, urea, creatinine, total bilirubin, direct bilirubin, total protein, albumin), and alpha-foetoprotein were recorded every 3 months. HBV DNA levels, abdominal ultrasound and the Child-Pugh score were evaluated every 6 months. Sixty-eight patients (mean age, 52.05+/-12.6 years) were monitored for 49.51+/-18.51 months. Basal ALT, HBV DNA levels and Child-Pugh scores were 103.9+/-73.9 IU/ml, 4133+/-121,94 IU/ml, and 7.6+/-2.4, respectively. The ALT normalisation was 59.7% during the first year, 68.2% during the second year and 44.4% during the fifth year. There was a significant decrease in Child-Pugh scores in the first 3 follow-up years when compared with the baseline score (P<0.05). During the treatment, HBV DNA positivity and YMDD mutations were determined in 20 of 68 (29.4%) patients at 46+/-17.9 months. Nine patients (13.2%) developed hepatocellular carcinoma at 44.8+/-21.5 months. Thirteen patients (19.1%) died during the treatment due to liver failure or variceal bleeding. Lamivudine is beneficial in patients with HBeAg-negative liver cirrhosis in terms of improvement in liver function and enhancement of survival and quality of life. An HBV DNA suppressive effect and improvement in Child-Pugh score were seen especially in the first years. It is important to be aware of YMDD mutation early, as addition of new antivirals is necessary to overcome unwanted results of the mutation.

Significance of Viral Load, Core Promoter/Precore Mutations and Specific Sequences of Polymerase Gene in Hbv-Infected Patients on 3-Year Lamivudine Treatment

Comprehensive study on viral factors predicting treatment responsiveness to lamivudine is lacking. To define the significance of various viral factors and changes of viral population with lamivudine treatment. Hepatitis B virus (HBV) DNA levels at baseline, week 24, 52 and year 3 were measured in 80 patients on continuous lamivudine therapy for 3 years. Genotypes, core promoter/precore mutations, YMDD mutations, polymorphic sequence of polymerase gene (rt91 I/L, rt256S/C) were determined at baseline, week 12, 24 and 52. YMDD mutations were also determined at year 3. High alanine aminotransferase levels and presence of core promoter/ precore mutations at baseline were associated with higher chance of achieving HBV DNA <1,000 copies/ml (good response) and higher rate of hepatitis Be antigen (HBeAg) seroconversion at week 52. Achieving HBV DNA levels <1,000 copies/mi at week 24 as well as baseline core promoter/precore mutations were associated with higher chance of achieving good response, higher rate of HBeAg seroconversion and lower rate of YMDD mutations at year 3. Lamivudine reversed core promoter mutations to wild type in 25% of patients. All 5 patients with rt256C had poor HBV DNA response, persistent HBeAg and YMDD mutations by year 3. There was no difference in treatment response between patients with genotype B and C. Achieving HBV DNA levels <1,000 copies/ml at 24 week is the best target for short- and long-term treatment efficacy. Core promoter and precore mutations were associated with better treatment outcome, and rt256C polymorphism in the polymerase gene with poor response.

Rapid and Accurate Genotyping of YMDD Motif Variants in the Hepatitis B Virus Genome by an Improved Reverse Dot Blot Method

By using the "flow-through hybridization" principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.

Character of HBV (hepatitis B virus) polymerase gene rtM204V/I and rtL180M mutation in patients with lamivudine resistance

To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance. One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment. One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL180M/M204V mutation (41.28%), 28 patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations. HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.

Relationship between B virus hepatitis genotypes and therapeutic efficacy in early treatment for chronic hepatitis B by using lamivudine

To investigate the relationship between hepatitis B virus (HBV) genotype and therapeutic efficacy during the early phase of lamivudine treatment. Totally 595 patients with chronic hepatitis B were treated with lamivudine 100 mg/day for 12 months. HBV genotypes, contents of HBV DNA, HBeAg/anti-HBe and YMDD mutation after lamivudine treatment for 12 months were determined. The data were analyzed with SPSS software. In 595 patients, 8 (1.4%) were genotype A; 53 (8.9%) genotype B; 360 (60.5%) genotype C; 112 (18.8%) were coinfection of genotype B and C; 14 (2.4%) of A and C; 15 (2.5%) A and B; 6 (1.0%) of A, B, and C, and remaining 27 (4.5%) were unspecified. Patients were treated with lamivudine 100 mg/day for 12 months. Genotype B with HBV DNA levels turned to be negative (HBV DNA < 0.1 ng/L) was 87.2%, genotype C was 89.51%, coinfection of genotype B and C was 93.04% (P > 0.05). HBeAg seroconversion of genotype B was 11.65%, of genotype C was 20.64%, and of coinfection of genotype B and C was 18.57% (P > 0.05). All 69 strains of YMDD mutation were detected after lamivudine treatment for 12 months, in which genotype B was in 16.98%, genotype C in 15.38%, and coinfection of genotype B and C was in 13.86% (P > 0.05). There was no difference in HBV genotypes and the rate of development of YMDD mutations, HBeAg seroconversion, descending of HBV DNA level in Chinese patients with chronic hepatitis B.

The Clinical Impact of Early Detection of the Ymdd Mutant on the Outcomes of Long-Term Lamivudine Therapy in Patients with Chronic Hepatitis B

The early emergence of lamivudine (3TC)-resistant tyrosine-methionine-aspartate-aspartate (YMDD) mutants has been reported during 3TC therapy in patients with chronic hepatitis B (CHB) in hepatitis B virus (HBV)-endemic areas; however, its clinical impact during long-term 3TC therapy is unknown. This study was performed to investigate the impact of the early emergence of YMDD mutants 3 months after the initiation of treatment on the outcomes of long-term 3TC therapy in HBV e antigen (HBeAg)-positive CHB. We analysed YMDD genotypes in consecutive samples from 30 patients with HBeAg positive CHB throughout 3TC treatment using both restriction fragment length polymorphism and mass spectrometric assays. Long-term outcome was compared between patients who had YMDD mutations detected at 3 months and those who had no mutations. YMDD mutation was detected in 16 (53.3%) out of 30 patients at 3 months and only the rtM2041 mutation was found. Cumulative HBeAg loss rates at 3 years was 12.5% and 57.4% in patients who had the rtM2041 mutant and wild-type virus at 3 months, respectively (P=0.010). Cumulative viral breakthrough rates at 3 years was 75.0% and 14.3% in patients who had the rtM204I mutant and wild-type virus at 3 months, respectively (P=0.002). Logistic regression revealed that YMDD mutation at 3 months was significantly related to viral breakthrough within 24 months (P=0.003). In conclusion, early detection for HBV YMDD mutation at 3 months may be useful to predict the long-term outcomes of 3TC therapy in patients with HBeAg-positive CHB in HBV-endemic areas.

Long-term follow-up study of Chinese patients with YMDD mutations: significance of hepatitis B virus genotypes and characteristics of biochemical flares.

We sought to examine the role of hepatitis B virus (HBV) genotypes in virological breakthroughs and biochemical flares in patients with YMDD mutations during lamivudine therapy. Virologic breakthroughs (i.e., the reappearance of HBV DNA as determined by bDNA assay) and biochemical flares (mild flares = alanine aminotransferase [ALT] between 2 and 10 times the upper limit of normal [ULN]; severe flares = ALT >10 times ULN) were monitored in 154 hepatitis B e antigen-positive patients receiving long-term lamivudine. The HBV genotypes and YMDD mutations were determined. Forty-three patients had virological breakthroughs with YMDD mutations (median follow-up of 29.6 months [range, 22.3 to 61.4]). Twenty patients (47%) patients had mild biochemical flares; seven (16%) had severe flares. Two patients showed an elevation of bilirubin level that is >2 times the ULN. All patients recovered spontaneously. The cumulative risks for biochemical flares were 28, 47, and 58% for the first 3 years, respectively. Patients with biochemical flares compared to those without flares had a significantly higher median pretreatment ALT level (61 U/liter versus 34.5 U/liter [P = 0.012]). There were no differences in the cumulative risk of virological breakthroughs, risk, and severity of biochemical flares between patients with genotypes B (n = 11) and C (n = 32). There was an increase in the percentage of patients with single YMDD mutant at last follow-up compared to that at the time of virological breakthroughs (74% [n = 32] versus 47% [n = 20], respectively; P = 0.015). The chances of YMDD mutations with virological breakthroughs and biochemical flares were similar in patients with genotypes B and C. Biochemical flares were common, with 16% being severe in nature. High pretreatment ALT levels were associated with a higher chance of biochemical flares.

Occult HBV infection—both hidden and mysterious

Occult HBV infection—both hidden and mysterious

Evolution of wild type and mutants of the YMDD motif of hepatitis B virus polymerase during lamivudine therapy

Long-term lamivudine treatment for chronic hepatitis B virus (HBV) infection induces the emergence of lamivudine resistant HBV YMDD mutant strains. The aim of the present study was to observe the clone evolution of YMDD wild type and mutant strains in pretreatment and post-treatment samples during lamivudine therapy and analyze their clinical significance. Ten serum samples (five before and five after 48 weeks of therapy) obtained from five patients chronically infected with HBV and treated with lamivudine were studied. Part of the HBV polymerase gene flanking the YMDD motif was sequenced after polymerase chain reaction (PCR) amplification. Meanwhile, 20-24 clones were selected at random from each sample and YMDD wild type and mutant strains were detected by real-time fluorimetry PCR established in our laboratory. The YMDD mutants were not detectable in all five patients before treatment and were found in four patients after 48 weeks of therapy by sequencing directly on PCR products. Analysis of individual clones showed that the ratios of mutant strains in each of the five patients were 0, 9.5, 0, 4.5 and 5.6%, respectively, before therapy and 100, 100, 65, 100 and 0%, respectively, after 48 weeks of therapy. M552I was detected before therapy in one patient but M552V became the domain strain after therapy. Until 52 weeks of therapy, serum HBV DNA and alanine aminotransferase (ALT) breakthrough were found in two of the four patients with YMDD mutations. The fifth patient experienced breakthrough of ALT but HBV DNA remained undetectable. The mutant strains of YMDD motif of HBV polymerase could be found in patients before lamivudine treatment, indicating that antiviral therapy allows the rapid selection of resistant strains. The replication ability of the M552V mutant strain might be stronger than that of the M552I mutant strain.