Detection of YMDD mutation using mutant-specific primers in chronic hepatitis B patients before and after lamivudine treatment

INTRODUCTIONHepatitis B virus (HBV) is one of the most common infectious diseases in the world. More than 300 million people worldwide are estimated to have chronic HBV infection. Ten percent of these patients will die as a direct consequence of persistent viral infection[1]. Nucleoside analogue therapy allows safe, long-term suppression of HBV and is a major milestone in the treatment of chronic hepatitis B. Lamivudine, the f irst of these agents approved worldwide, effectively supp…     

Low rate of YMDD motif mutations in polymerase gene of hepatitis B virus in chronically infected patients not treated with lamivudine

Background Lamivudine is used for the treatment of chronic hepatitis B (CH-B), and exhibits excellent antiviral activity. However, longterm administration increases the likelihood of the emergence of resistant viruses, with an accompanying relapse of hepatitis. However, recent studies have reported lamivudine-resistant viruses in patients with CH-B before such treatment. The aim of this study was to investigate whether YMDD mutants occur in nature.Methods The existence of lamivudine-resistant viruses was examined in 20 asymptomatic carriers of hepatitis B virus (ASC), 10 patients who lost hepatitis B surface antigen (HBsAg) during follow-up and in 20 lamivudine-treated patients with and without breakthrough hepatitis. Both polymerase chain reaction (PCR) restriction fragment length polymorphism and SMITEST hepatitis B virus (HBV)-YMDD mutation detection methods were used to detect resistant viruses.Results No YMDD mutants were detected in the sera of the 20 ASC at the initial and final medical examinations, nor were YMDD mutants detected in sera collected at the initial medical examination, about 6 months before, or immediately after the loss of HBsAg in the 10 patients. In the 20 patients treated with lamivudine, YMDD mutants were not detected in any of them before treatment, whereas mutants were detected in the sera of 10 patients during treatment.Conclusions Our results suggest that lamivudine-resistant YMDD mutant viruses were present in a few patients with HBV infection who before they have been treated with lamivudine.     

Emergence of YMDD motif mutant of hepatitis B virus during short-term lamivudine therapy in South Korea

BACKGROUND/AIMS: The emergence of a YMDD mutant resistant to lamivudine therapy has been reported in patients with hepatitis B treated with long-term lamivudine therapy. However, it is not well known whether the YMDD mutant could be detected early in lamivudine therapy in hepatitis B virus (HBV) endemic areas. The aim of this study was to investigate the emergence of the YMDD mutant during short-term lamivudine therapy in South Korea. METHODS: We prospectively investigated the emergence of the YMDD mutant by the nested PCR assay using restriction fragment length polymorphism in 28 patients with chronic hepatitis B who were treated with 100 mg of lamivudine daily for 12 weeks. RESULTS: The YMDD mutant was detected in 17 (60.7%) out of 28 patients at week 12, and the only type of mutation found was the YIDD mutation. When we carried out the nested PCR serially in five patients, YIDD mutants were detected as early as 2 weeks by the nested PCR assay. The nested PCR results were in concordance with DNA sequencing in one patient's serial samples. CONCLUSIONS: YMDD mutants in HBV were detected within a few weeks during lamivudine therapy in South Korea, which suggests that the YMDD mutant may exist even before lamivudine therapy in HBV endemic areas.     

乙型肝炎病毒YMDD联合前C变异及其临床意义

目的：研究拉米夫定治疗过程中乙型肝炎病毒（HBV）前C区，P区YMDD基序变异及其对疗效的影响。方法：对5例拉米夫定（100mg/d)治疗过程中HBeAg血清转换而HBV DNA仍阳性的慢性乙型肝炎患者，采用聚合酶链反应（PCR）产物直接测序方法分析HBV前C、P区的基因序列，结果：5例患者前C区均发现有G1896A变异，其中3例HBeAg血清转换前未发现此变异，2例已有此变异，同时5例患者在拉米夫定治疗后检测出YMDD变异，变异类型均为M5521，其中有1例在出现M5521变异40周后变为M552V变异，有2例M5521联合L528M变异，5例患者中有1例治疗无反应，另4例在治疗过程中HBV DNA突发，结论：拉米夫定治疗过程中YMDD变异的出现可导致HBV DNA突发，而前C区G1896A变异可致HBeAgbe阴转，因此在治疗过程中出现HBeAg血清转换后需结合HBV DNA检测，排除前C区变异的可能。     

拉米夫定治疗中乙型肝炎病毒YMDD野毒株和变异株的变化

目的 观察慢性乙型病毒性肝炎(CHB)患者应用拉米夫定治疗前后,YMDD野毒株及变异株的动态变化,并分析其临床意义。方法 取5例CHB患者治疗前与治疗48周的10份血清标本,先用PCR法扩增包括YMDD基序的乙型肝炎病毒(HBV)部分核苷酸序列,然后进行DNA序列测定,同时分别进行克隆,每份标本随机挑选20～24株单克隆,用实时荧光PCR法检测YMDD野毒株及其变异株。结果 5例CHB患者治疗前PCR产物直接测序结果未检出YMDD变异,治疗48周时有4例检出YMDD变异,但对每株克隆的分别检测显示：治疗前YMDD变异株(YIDD／YvDD)所占比率分别为0％、9．5％、0％、4．5％、5．6％;治疗48周时所占比率分别为100％、100％、65％、100％、0％。其中1例患者治疗前检出YIDD变异株,而治疗48周时YVDD变异株则变成优势株。治疗52周时,4例YMDD变异患者中2例HBVDNA和血清丙氨酸转氨酶(ALT)突破,1例患者ALT突破,但HBVDNA为阴性。结论 YMDD变异株在拉米夫定治疗前的血清中已存在,在服用拉米夫定后,由于选择性抑制了野毒株,使YMDD变异株由弱势株变成优势株,部分患者可导致拉米夫定临床耐药。YVDD变异株可能比YIDD变异株的复制能力强。     

Detection of YMDD mutants using universal template real-time PCR

AIM: To establish a rapid and accurate method for the detection of lamivudine-resistant mutations in hepatitis B virus and monitor of lamivudine resistance during lamivudine treatment in patients with chronic hepatitis B virus infection. METHODS: We established a real-time PCR method using a universal template and TaqMan probe to detect YMDD mutants. Variants of YVDD and YIDD were tested by individual reactions (reaction V and reaction I) and total hepatitis B viruses were detected in another reaction for control (reaction C). Results were determined by deltaCt < 3.5 (deltaCt = Ct of reaction V or I - Ct of reaction C). Clones of the HBV polymerase gene containing different YMDD mutations were tested. Serum samples from 163 lamivudine-treated patients with chronic hepatitis B virus infection were detected using this method and the results were confirmed by DNA sequencing. RESULTS: As many as 1000 copies per milliliter of wide-type plasmid were detected and nonspecific priming was excluded. In the 163 samples from patients treated with lamivudine, lamivudine-resistant mutations were detected in 51 samples. CONCLUSION: This universal real-time PCR is a rapid and accurate method for quantification of YMDD mutants of HBV virus in lamivudine-treated patients and can be used to monitor lamivudine-resistant mutations before and during lamivudine therapy.     

Detection of YMDD motif mutations in some lamivudine-untreated asymptomatic hepatitis B virus carriers

BACKGROUND/AIM: It is well documented that long-term lamivudine treatment induces emergence of lamivudine-resistant hepatitis B virus (HBV), namely, YMDD motif mutation in some patients chronically infected with HBV. We previously reported that there were no YMDD mutant viruses in patients with chronic hepatitis B who were not treated with lamivudine. In this series, we examined mutations in the YMDD motif gene in asymptomatic carriers who maintained normal ALT values for 1 year or more. METHODS: Serum samples obtained from 18 patients chronically infected with HBV who consulted our university were used. None of these patients had any experience of using antiviral agents. For detection of mutant viruses, a kit developed in our laboratory was used. RESULTS: Mutations were detected in five of 18 samples: YMDD+YIDD in three samples and YMDD+YVDD+YIDD in two samples. All of these five samples were positive for anit-HBe. In five samples in which mutations were observed, sequencing was carried out following subcloning. CONCLUSION: The present study demonstrates that YMDD mutant viruses are present in lamivudine-untreated asymptomatic hepatitis B virus carriers as well.     

Treatment of chronic hepatitis B patients with tyrosinemethionine-aspartate-aspartate mutations

Lamivudine is an antiviral used for the treatment of chronic hepatitis B. Several studies have reported various mutations that are induced by lamivudine therapy. These mutations in the tyrosine-methionineaspartate-aspartate(YMDD) motif are necessary and sufficient to confer high-level lamivudine resistance. During treatment with lamivudine, mutations develop in the YMDD motif of the hepatitis B virus(HBV) polymerase gene and lamivudine cannot prevent the replication of the mutant form. The virulence strain of developed mutation in the polymerase gene is lower than the original virus and they are susceptible to treatment with some other nucleoside analogs except lamivudine. Entecavir and tenofovir are potent HBV inhibitors and they can be confidently used as first line monotherapies. We read the article written by Tan et al that lamivudine therapy improved the clinical course in HBV patients with natural YMDD mutations. We think that lamivudine use for this patient group is not appropriate. These patients should use YMDD mutant form-effective drugs such as adefovir, tenofovir.     

Successful orthotopic liver transplantation for lamivudine-associated YMDD mutant hepatitis B virus

YMDD hepatitis B virus (HBV) mutants emerge in more than one third of patients treated with lamivudine. The development of escape mutants potentially limits the efficacy of this new therapy for chronic HBV infection. Transplant centers may be hesitant to perform orthotopic liver transplantation (OLT) in patients with progressive liver disease who develop the YMDD mutant because of concerns of graft reinfection. We describe a 56-year-old man with chronic liver disease caused by HBV who successfully underwent OLT after the emergence of a YMDD mutant and progressive liver disease. Perioperatively, he received high-dose intravenous hepatitis B immune globulin (HBIG) treatment with continued lamivudine treatment. Thirty-two months after OLT, there was no evidence of HBV reinfection with the use of lamivudine and high-dose HBIG. Our experience suggests that the emergence of a YMDD HBV mutant is not a contraindication to OLT. The combination of lamivudine and high-dose HBIG can protect against reinfection of the hepatic allograft with the YMDD HBV escape mutant.     

Quantitation of hepatitis B lamivudine resistant mutants by real-time amplification refractory mutation system PCR

BACKGROUND/AIMS: Lamivudine is an antiviral drug that is used to treat hepatitis B virus (HBV) infection. Long-term therapy does not completely suppress viral replication, and resistant mutants emerge. Resistance is mediated by changes in the tyrosine-methionine-apartate-aspartate (YMDD) motif in the catalytic site of the HBV polymerase gene. We describe a method to detect and quantify mutant viral populations using amplification refractory mutation system (ARMS) PCR. METHODS: We developed a real-time ARMS-PCR to detect point mutations in the polymerase gene. Using real-time PCR (LightCycler) with a ResonSense probe, PCRs were performed using clones of the HBV polymerase gene containing the different YMDD mutations. Dilution series of the templates were made and tested against each of the primer pairs. This method was applied to quantify mutant virus in patient serum samples. RESULTS: As little as 0.01% mutant DNA in 10(5)-10(9) copies wild-type DNA were detected. The method is more sensitive than amplicon sequencing, which is the current method of mutant determination in the YMDD motif. CONCLUSIONS: This study demonstrates a rapid, highly sensitive and reproducible method of quantifying mutant HBV virus in lamivudine treated patients. It can be used to monitor patients before and during lamivudine therapy.     

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

BACKGROUND AND AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Early detection and quantification of lamivudine-resistant hepatitis B virus mutants by fluorescent biprobe hybridization assay in lamivudine-treated patients

Background Long-term lamivudine treatment induces the emergence of lamivudine-resistant hepatitis B virus (HBV). The objective of this study was to develop a fluorescent biprobe hybridization (FBH) assay for the detection and quantification of HBV mutants in the clinical course of lamivudine-treated patients and to evaluate its clinical usefulness. Methods We developed an FBH assay to detect mutations in the HBV DNA polymerase gene. The assay's detection sensitivity was determined using a dilution series of wild-type/mutant plasmid DNA. Blood samples obtained from 27 lamivudine-treated patients were analyzed. Results Mutant DNA levels as low as 10% of total HBV DNA were detected (sensitivity = 100%, specificity = 80%). HBV mutants were detected in five of the 27 patients during an average follow-up of 20 months after lamivudine administration. In one of the five patients, the YIDD mutant was detected at the initiation of lamivudine treatment, while the remaining four patients were identified as having YIDD mutants within 3 months after beginning lamivudine administration. Of the five patients with an HBV mutant, four developed breakthrough hepatitis more than 10 months after the detection of HBV mutants, following the reappearance or a re-increase of HBV DNA, characterized by a predominance of the mutant. The YIDD mutant was detected in one patient, even when the titer of the serum HBV DNA was below the detection limit of commercially available quantitative polymerase chain reaction. Conclusions The FBH assay is an efficient method for detecting and quantifying HBV mutants, as early as 3 months after lamivudine administration.     

Changes in serum hepatitis B e antigen (HBeAg) levels associated with the emergence of YMDD mutants in HBeAg non-seroconverted patients during lamivudine therapy.

Background: To elucidate the associations between the changing patterns of hepatitis B e antigen (HBeAg) levels and the emergence of tyrosine–methionine–aspartate–aspartate (YMDD) mutants in HBeAg non‐seroconverted patients undergoing lamivudine therapy. Methods: This study analysed 76 HBeAg‐positive naïve chronic hepatitis B patients treated with lamivudine. The median duration of therapy was 52 weeks. The YMDD mutants were detected in 35 patients. The changing patterns of HBeAg levels were categorized into three groups: Descending, Descending–Ascending and Fluctuation. HBeAg breakthrough was defined as progressive HBeAg decreasing to <10% of pretreatment levels, followed by increases exceeding 50 S/Co [the ratio of the sample (S) to the cut‐off (Co)] above nadir levels. Results: Of 76 patients, the sensitivity and specificity for predicting YMDD mutants by the Descending–Ascending pattern were 66 and 100% respectively. Of 17 patients with YMDD mutants in the Descending–Ascending group, hepatitis B virus (HBV) DNA first increased, followed by increased HBeAg levels and finally by biochemical breakthrough. The median intervals between virological breakthrough and HBeAg breakthrough, between HBeAg breakthrough and biochemical breakthrough and between virological breakthrough and biochemical breakthrough were 4, 24 and 33 weeks respectively. Conclusions: Serial HBeAg levels are useful in predicting YMDD mutant emergence in HBeAg non‐seroconverted patients during lamivudine therapy.     

乙型肝炎病毒YMDD及e抗原相关多重变异及其临床意义

目的 研究拉米夫定治疗慢性乙型肝炎期间HBVYMDD基序、影响HBeAg分泌的多重变异情况与临床的关系。方法 采用基因芯片技术对拉米夫定治疗9～30个月的慢性乙型肝炎患者进行YMDD基序、G1896A、A1814C、A1762T和G1764A(BCP双突变)单碱基变异检测。结果 102例慢性乙型肝炎患者拉米夫定平均治疗18个月时,22例发生YMDD变异,其中8例发生多重变异,包括G1896A3例、A1814C2例、G1896A A1814C、BCP双突变、BCP双突变 G1896A多重变异各1例,单纯YMDD变异和前5例联合变异均为HBeAg阳性,而后3例多重变异则为HBeAg阴性,其中1例多重变异继续治疗3个月后转变为单纯YMDD野生株阳性,同时伴有HBeAg的复阳。结论 拉米夫定治疗过程中存在YMDD及HBeAg相关多重变异的优势病毒株可能是HBVDNA复阳、同时伴有HBeAg阴转的原因之一,拉米夫定治疗过程中,HBeAg阴性时应监测其可能的相关变异。     

不同实验方法在监测拉米夫定耐药突变中的应用价值

目的 通过对三种检测乙型肝炎病毒(HBV)YMDD突变方法的比较,评价其在监测拉米夫定抗HBV治疗中发生耐药突变的价值。方法 用熔解曲线法、聚合酶链反应微板核酸杂交-酶联免疫吸附法(PCRmnh-ELISA)和错配聚合酶链反应限制性片段长度多态性分析法(mPCR-RFLP)对拉米夫定治疗过程中出现HBV DNA由阴转阳的44例慢性乙型肝炎患者的血清进行YMDD突变检测,比较它们的敏感性,并与直接测序法比较它们的特异性。结果 mPCR-RFLP法、PCRmnh-ELISA法和熔解曲线法检出HBV DNA的灵敏度分别为10~4、10~5和10~6拷贝/ml。44例患者血清中,用mPCR-RFLP法检出26例为YMDD突变株,18例为野生株。在26例突变株中,熔解曲线法检出16例,PCRmnh-ELISA法检出18例;而在18例野生株中,熔解曲线法检出2例突变株,PCRmnh-ELISA法检出13例突变株。将上述三种方法检测结果不一致的16例标本进行测序,mPCR-RFLP法、熔解曲线法和PCRmnh-ELISA与测序的符合率分别为93.8%(15/16)、43.8%(7/16)、18.8%(3/16),差异有极显著性(x~2=18.7,P<0.01)。结论 mPCR-RFLP法检测YMDD突变株具有较好的可靠性,是监测拉米夫定耐药株的一种非常有效的方法;熔解曲线法和PCRmnh-ELISA法需要进一步完善以提高敏感性和特异性。     

Absence of mutations in the YMDD motif/B region of the hepatitis B virus polymerase in famciclovir therapy failure.

BACKGROUND/AIMS: Nucleoside analogues such as lamivudine and famciclovir are potent drugs for treatment of chronic hepatitis B virus infection. Breakthrough infections during lamivudine therapy are associated with mutations in the YMDD motif and putative B region of the HBV polymerase. This study investigated whether failure of famciclovir therapy is also associated with presence or emergence of particular mutations in the HBV polymerase. METHODS: We analyzed longitudinally the sequence of the priming and polymerase domain in seven patients with primary non-response to therapy and two patients with a breakthrough during therapy. Two patients who responded to therapy served as a control. RESULTS: The YMDD motif and the B region were conserved in all isolates. V-->I changes at position 555 just downstream of the YMDD motif were observed before and during therapy in a virus subpopulation of two patients with a primary non-response. In patients with a breakthrough, 378-V-->I and 424-N-->D mutations emerged in the N terminal part of the polymerase domain during follow-up. Lamivudine rescue therapy initiated in four patients, including a patient infected with YMDD(555-V-->I) variants, efficiently reduced viremia. CONCLUSIONS: These data indicate that failure of famciclovir therapy can occur independently of mutations in the YMDD motif or B region of the HBV polymerase and provide a rationale for rescue therapy with lamivudine.     

乙型肝炎病毒多聚酶变异与拉米夫定治疗反应

目的 探讨乙型肝炎病毒（HBV）多聚酶酪氨酸－蛋氨酸－天氧氨酸－天冬氨酸（YMDD）基序变异对拉米夫定抗病毒疗效的影响。方法 对19例拉米夫定治疗（100mg/d）48周时血清病毒核酸阳性的慢性乙型肝炎患者,采用聚合酶链反应（ PCR）产物直接测序技术,检测其血清中HBV多取和酶（YMDD）变异情况,并观察其血清HBV DNA和丙氨酸转氨酶（ALT）水平。结果 在检出YMDD变异株的10 例患者（4例为YV^550DD型,均伴L^526→M突变,6例为YI^550DD型）,至52周为止,共5例出现HBV DNA突发（分枝DNA信号扩增法）,其中2例伴ALT再高。追踪其治疗前血清均未发生上述变异。而在未检出YMDD变异的9例患者中,1例出现DNA突发后又阴转,而另1例治疗过程中HBV DNA未阴转、ALT始终波动于正常值上下的患者,发现多聚酶其他部位的变异^473→R,D^477→N,D^480→N,L^491→M)治疗前后相同。结论 乙型肝炎病毒多聚酶（ YMDD）变异与药物诱导相关,发生变异者较无变异者疗效降低;在拉米夫定治疗过程中,该变异的检测将有助于其疗效监测。     

Course of virologic breakthroughs under long-term lamivudine in HBeAg-negative precore mutant HBV liver disease

We studied the course of virologic breakthroughs detected by a quantitative polymerase chain reaction (PCR) assay in 32 of 78 patients with hepatitis B e antigen (HBeAg)-negative precore mutant hepatitis B virus (HBV) chronic liver disease under long-term lamivudine monotherapy. Serum HBV DNA levels were measured every 3 months and on every biochemical breakthrough. YMDD mutants were detected in 30 of the 32 patients with virologic breakthroughs. Among these 32 patients, biochemical remission rate was 44% at 6 months, 21% at 12 months, and 0% at 24 months after the onset of virologic breakthrough. Development of biochemical breakthroughs was associated with a significant increase of serum HBV DNA levels, which exceeded 100,000 copies/mL in 19 of 20 patients (95%) with biochemical breakthroughs and in only 1 of 8 patients (12.5%) remaining in biochemical remission for at least 6 months after the onset of virologic breakthrough (P <.001). Alanine aminotransferase (ALT) level peaked within 0 to 3 months after the onset of biochemical breakthrough and decreased at 6 months but remained abnormal in all but 2 patients. Follow-up liver histologic lesions in patients with biochemical breakthroughs did not differ from baseline findings, although they were significantly improved in patients remaining in virologic and biochemical remission. In conclusion, the frequent emergence of viral resistance under long-term lamivudine monotherapy in HBeAg-negative precore mutant HBV chronic liver disease is followed by increasing viremia levels culminating in the development of biochemical breakthroughs in most cases. ALT activity peaks close to the onset of biochemical breakthrough, decreasing thereafter but remaining persistently abnormal with fluctuating levels.     

Favorable outcome of orthotopic liver transplantation in a patient with subacute liver failure due to the emergence of a hepatitis B YMDD escape mutant virus.

BACKGROUND/AIMS: Patients with end-stage liver disease due to chronic hepatitis B infection in whom a YMDD escape hepatitis B virus (HBV) mutant has emerged under lamivudine treatment are generally denied liver transplantation (OLT). METHODS: We report the case of a male patient who was started on prophylactic treatment with lamivudine in the context of recurrent episodes of HBV reactivation during high dose immunosuppressive therapy for relapsing severe pulmonary sarcoidosis. RESULTS: Following the emergence of a YMDD escape mutant virus under lamivudine treatment, he developed subacute liver failure requiring liver transplantation. The patient was treated with a combination of intravenous hepatitis B immune globulin (HBIG) which was started perioperatively and also continued lamivudine after OLT. Twelve months after OLT, there was no evidence of HBV reinfection of the liver graft with the use of HBIG and lamivudine. CONCLUSIONS: This observation suggests that emergence of the YMDD mutation is not a contra-indication to OLT, providing adequate immunoprophylaxis using HBIG and lamivudine combination therapy.