Lamivudine resistance mutations in patients infected with hepatitis B virus genotype D

AIM: To determine the distribution of viral genotypes for primary or acquired lamivudine resistance. METHODS: A total of 283 patients with chronic hepatitis B virus (HBV) infection (245 patients with chronic hepatitis B and 38 inactive hepatitis B surface antigen carriers) were included in the study. The HBV geno-type was determined by using quantitative real-time polymerase chain reaction and sequence analysis, and tyrosine-methionine-aspartate-aspartate (YMDD) motif mutations were determined using the reverse transcriptase hybridization method. RESULTS: Lamivudine resistance was determined in a total of 25 (10.7%) chronic hepatitis B patients. Eight subjects (4%) had primary resistance to lamivudine, and 17 (53.1%) had secondary resistance to lamivudine. Genotype D, which was isolated from 267 of the patients with chronic HBV infection, was the dominant genotype in Turkey. CONCLUSION: Identification of YMDD motif mutations should have a positive impact on the selection of proper antiviral medication for patients, even for those who are nucleoside nave.

Clinical implications of hepatitis B virus mutations:Recent advances

Hepatitis B virus(HBV)infection is a major cause of acute and chronic hepatitis,and of its long-term complications.It is the most variable among DNA viruses,mostly because of its unique life cycle which includes the activity of error-prone enzyme,reverse transcriptase,and the very high virion production per day.In last two decades,numerous research studies have shown that the speed of disease progression,reliability of diagnostic methods and the success of antiviral therapy and immunization are all influenced by genetic variability of this virus.It was shown that mutations in specific regions of HBV genome could be responsible for unwanted clinical outcomes or evasion of detection by diagnostic tools,thus making the monitoring for these mutations a necessity in proper evaluation of patients.The success of the vaccination programs has now been challenged by the discovery of mutant viruses showing amino acid substitutions in hepatitis B surface antigen(HBsAg),which may lead to evasion of vaccine-induced immunity.However,the emergence of these mutations has not yet raised concern since it was shown that they develop slowly.Investigations of HBV genetic variability and clinical implications of specific mutations have resulted in significant advances over the past decade,particularly in regard to management of resistance to antiviral drugs.In the era of drugs with high genetic barrier for resistance,on-going monitoring for possible resistance is still essential since prolonged therapy is often necessary.Understanding the frequencies and clinical implications of viral mutations may contribute to improvement of diagnostic procedures,more proper planning of immunization programs and creating the most efficient therapeutic protocols.

In vitro resistance to interferon of hepatitis B virus with precore mutation

AIM: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN-α), which results in an efficient reduction of the viral load only in 20-40% of treated patients. Mutations at HBV precore prevail in different clinical status of HBV infection. The roles of precore mutation in the progression of chronic hepatitis and interferon sensitivity are still unknown. The aim of this study was to explore if there was any relationship between HBV precore mutation and sensitivity to interferon in vitro. METHODS: HBV replication-competent recombinant constructs with different patterns of precore mutations were developed. Then the recombinants were transiently transfected into hepatoma cell line (Huh7) by calcium phosphate transfection method. With or without IFN, viral products in culture medium were collected and quantified 3 d after transfection. RESULTS: We obtained 4 recombinant constructs by orientation-cloning 1.2-fold-overlength HBV genome into pUC18 vector via the EcoRI and Hind lll and PCR mediated site-directed mutagenesis method. All the recombinants contained mutations within precore region. Huh7 cells transfected with recombinants secreted HBsAg and HBV particles into the cell culture medium, indicating that all the recombinants were replication-competent. By comparing the amount of HBV DNA in the medium, we found that HBV DNA in medium reflecting HBV replication efficiency was different in different recombinants. Recombinants containing precore mutation had fewer HBV DNAs in culture medium than wild type. This result: showed that recombinants containing precore mutation had lower replication efficiency than wild type. HBV DNA was decreased in pUC18-HBV1.2-WT recombinants after IFN was added while others with precore mutations were not, indicating that HBV harboring precore mutation was less sensitive to IFN in cell culture system. CONCLUSION: These data indicate that HBV harboring precore mutation may be resistant to IFN in vitro.

Molecular diagnosis and treatment of drug-resistant hepatitis B virus

Oral antiviral agents have been developed in the last two decades for the treatment of chronic hepatitis B(CHB).However,antiviral resistance remains an important challenge for long-term CHB therapy.All of the clinically available oral antiviral agents are nucleoside or nucleotide analogues that target the activity of viral reverse transcriptase(RT),and all are reported to have resistant mutations.Since the hepatitis B virus(HBV)RT,like other viral polymerases,lacks proofreading activity,the emergence of drug-resistance occurs readily under selective pressure from the administration of antiviral agents.The molecular diagnosis of drug-resistant HBV is based on sequence variations,and current diagnostic methods include sequencing,restriction fragment polymorphism analysis,and hybridization.Here,we will discuss the currently available molecular diagnosis tools,in vitro phenotypic assays for validation of drug-resistant HBV,and treatment options for drug-resistant HBV.

Clonal evolution of hepatitis B virus polymerase gene mutations during lamivudine-adefovir combination treatment

AIM:To identify hepatitis B virus polymerase gene mutations during antiviral therapy using lamivudineadefovir sequential monotherapy followed by lamivudine-adefovir combination therapy.METHODS:The patient cohort included four adult chronic hepatitis B patients who had undergone sequential monotherapy,first with lamivudine(LMV) and then,after developing viral breakthrough,with adefovir(ADV) therapy.All of the patients had non-response or viral breakthrough after LMV-ADV sequential monotherapy,which resulted in the switching of their antiviral regimen to LMV-ADV combination therapy.Eleven serum samples from the four patients who showed non-response to rescue LMV-ADV combination therapy were collected sequentially at a time before the antiviral treatment and then during the LMV monotherapy,ADV monotherapy,and LMV-ADV combination therapy.For the genotypic analysis,the whole 1310-bp polymerase gene region was amplified,cloned and sequenced.RESULTS:All patients had been previously treated with 100 mg of LMV once daily for a 15-to 26-mo period.The emergence of resistance mutations to LMV,such as rtM204V/I and/or rtL180M,were found in all patients.Their antiviral regimens were switched to ADV monotherapy as the second line treatment.All patients had viral breakthrough or non-response after the LMV-ADV sequential monotherapy.ADV-resistant mutations were detected after 13 to 19 mo of LMV-ADV sequential monotherapy.The rtA181V/T mutations were predominantly identified during the ADV treatment in the LMV-resistant patients.Twenty-seven of 38 clones were combined with an amino acid change at rt181;three clones had mutations in rt236 and one clone had a combined mutation.The rtA181V/T mutations were not suppressed by the LMV-ADV combination therapy.Thirty-nine of 64 clones showed an rtA181V/T mutation and six clones showed combined mutations in rt181 and rt236.Mutations in rt204 re-emerged during the combination treatment.The rt181 and rt204 mutations did not co-exist in one clone.CONCLUSION:Add-on lamivudine therapy with adefovir for adefovir resistance may not suppress the pre-existing adefovir-resistant mutation that develops during lamivudine-adefovir sequential monotherapy.

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.

The relationship between HBV lamivudine resistance and HBV genotypes or basic core promoter mutations

OBJECTIVE: To investigate the relationship between HBV Iamivudine resistance and HBV genotypes or basic core promoter (BCP) mutations. METHODS: The common coated probes were synthesized according to the conserved regions of the preC gene of hepatitis B virus (HBV). Different colorized probes were chosen from the sequences of different genotypes of HBV (A to F), BCP and YMDD wild types and mutants, respectively. HBV DNA levels, HBV genotypes, BCP and YMDD resistants were analyzed by PCR microplate hybridization ELISA at the zero and 6th month after the patients were treated with lamivudine. RESULTS: HBV genotyping results showed that HBV types B, C, D accounted for about 30%, 36% and 23% patients respectively. Thirteen BCP mutations (type B in 1 patient, type C in 8 and type D in 4) were found before treatment with lamivudine. HBV DNA levels were lower than 100 pg/ml in 2 patients anti higher than 100 pg/ml in 11. 9.4% of the HBV patients (5/43; type C in 3 and type D in 2) showed YMDD resistants and 4 BCP mutations at the same time. CONCLUSION: Oral treatment of lamivudine decreases the level of serum HBV DNA. The appearance of HBV YMDD resistants is related to certain HBV genotypes, and most of them are BCP mutations.

Clinical relevance of hepatitis B virus variants

The hepatitis B virus(HBV) is a global public health problem with more than 240 million people chronically infected worldwide, who are at risk for end-stage liver disease and hepatocellular carcinoma. There are an estimated 600000 deaths annually from complications of HBV-related liver disease. Antiviral therapy with nucleos/tide analogs(NA) targeting the HBV polymerase(P) can inhibit disease progression by long-term suppression of HBV replication. However, treatment may fail with first generation NA therapy due to the emergence of drugresistant mutants, as well as incomplete medication adherence. The HBV replicates via an error-prone reverse transcriptase leading to quasispecies. Due to overlapping open reading frames mutations within the HBV P can cause concomitant changes in the HBV surface gene(S) and vice versa. HBV quasispecies diversity is associated with response to antiviral therapy, disease severity and long-term clinical outcomes. Specific mutants have been associated with antiviral drug resistance, immune escape, liver fibrosis development and tumorgenesis. An understanding of HBV variants and their clinical relevance may be important for monitoring chronic hepatitis B disease progression and treatment response. In this review, we will discuss HBV molecular virology, mechanism of variant development, and their potential clinical impact.

Application of hepatitis B virus replication mouse model

AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents,the interferon inducer polyinosinic-polytidylin acid(polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1,and the inhibiting effect of these agents on HBV DNA replication was evaluated.To identify the model's value in a replication ability study of HBV drug-resistant mutants and a HBx-minus mutant,telbivudine resistance mutants(rtM204I,ayw subtype),adefovir resistance mutants(rtA181V + rtN236T,ayw subtype) and HBxminus mutants were injected respectively,and their corresponding HBV DNA replication intermediates in mouse liver were assessed.RESULTS:Compared with the wild type HBV replication mouse model without antiviral agent treatment,the HBV DNA replication intermediates of the polyICtreated group were decreased 1-fold;while in the entecavir-and adefovir-treated groups,the levels of HBV DNA replication intermediates were inhibited 13.6-fold and 1.4-fold,respectively.For the mouse models injected with telbivudine resistance mutant,adefovir resistance mutant and HBx-minus mutant,HBV DNA replication intermediates could still be detected,but the levels of HBV DNA replication intermediates of these mutants decreased 4.5-fold,5.6-fold and 2.9-fold respectively,compared with the mouse model with wild type HBV plasmid.CONCLUSION:The HBV replication mouse model we established was a useful and convenient tool to detect the efficacy of antiviral agents and to study the replication ability of HBV mutants in vivo.

Diagnostic and therapeutic progress of multi-drug resistance with anti-HBV nucleos(t)ide analogues

Nucleos(t)ide analogues(NA) are a breakthrough in the treatment and management of chronic hepatitis B.NA could suppress the replication of hepatitis B virus(HBV) and control the progression of the disease.However,drug resistance caused by their long-term use becomes a practical problem,which influences the long-term outcomes in patients.Liver transplantation is the only choice for patients with HBV-related end-stage liver disease.But,the recurrence of HBV after transplantation often caused by the development of drug resistance leads to unfavorable outcomes for the recipients.Recently,the multi-drug resistance(MDR) has become a common issue raised due to the development and clinical application of a variety of NA.This may complicate the antiviral therapy and bring poorly prognostic outcomes.Although clinical evidence has suggested that combination therapy with different NA could effectively reduce the viral load in patients with MDR,the advent of new antiviral agents with high potency and high genetic barrier to resistance brings hope to antiviral therapy.The future of HBV researches relies on how toprevent the MDR occurrence and develop reasonable and effective treatment strategies.This review focuses on the diagnostic and therapeutic progress in MDR caused by the anti-HBV NA and describes some new research progress in this field.

南京地区268例慢性HBV感染者天然耐药突变调查及影响因素分析

目的调查分析南京地区268例慢性乙型肝炎病毒(HBV)感染者天然耐药突变情况,并分析影响天然耐药突变发生的相关因素。方法回顾性分析2019年10月—2022年10月南京地区268例慢性HBV感染者的资料,统计天然耐药发生情况及耐药突变类型,比较发生突变和未发生突变患者临床特征,采用logistic分析影响天然耐药突变发生的因素。结果268例慢性HBV感染患者经基因测序检测,共有9例天然耐药突变,检出率为3.36%(9/268);9例天然耐药突变患者耐药位点分别为rt180M、rt204V、rt204I、rt250V、rt180M、rt204V+rt180M、rt213T、rt236T和rt204I+rt180M,发生突变患者年龄、HBV DNA水平、病程均高于未发生突变患者(P<0.05);Logistic多因素回归分析显示,年龄、HBV DNA水平、病程均是影响慢性HBV感染者发生天然耐药突变的独立危险因素(OR=4.162,4.411,5.766,P<0.05)。结论该地区慢性HBV感染者存在天然耐药突变,年龄、HBV DNA水平、病程均是影响慢性HBV感染者发生天然耐药突变的独立危险因素,应加强防范。

Molecular epidemiology of hepatitis B virus in Asia

Although safe and effective vaccines against hepatitis B virus(HBV) have been available for three decades, HBV infection remains the leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma(HCC) worldwide, especially in Asian countries. HBV has been classified into at least 9 genotypes according to the molecular evolutionary analysis of the genomic DNA sequence and shown to have a distinct geographical distribution. Novel HBV genotypes/subgenotypes have been reported, especially from Southeast Asian countries. The clinical characteristics and therapeutic effectiveness of interferon(IFN) and nucleos(t)ide analogues vary among different HBV genotypes. Mutations at T1653 C in subgenotype C2 from Japan and South Korea, C/A1753 T and C1858 T in subgenotype C1 from Vietnam, and C1638 T and T1753 V in subgenotype B3 from Indonesia were reported to be associated with advanced liver diseases including HCC. Genotype distribution in Japan has been changed by an increasing ratio of subgenotype A2 in chronic hepatitis B. While a large number of epidemiological and clinical studies have been reported from Asian countries, most of the studies were conducted in developed countries such as Taiwan, China, South Korea and Japan. In this review, the most recent publications on the geographical distribution of genetic variants of HBV and related issues such as disease progression and therapy in Asia are updated and summarized.

云南地区结核病患者与HBV、HCV、HIV共感染免疫功能及耐药性研究

目的探讨云南地区结核病患者合并感染HBV、HCV、HIV中合并感染率、免疫功能及耐药性状况。方法收集2020年4月至2023年3月昆明市第三人民医院收治的2271例云南地区结核病患者进行HBV、HCV、HIV血清学检测,同时进行淋巴细胞检测及结核菌的耐药基因检测,分析结核病分别感染HBV、HCV、HIV的感染率、免疫功能及耐药性。结果2271例结核病患者中,结核病合并HBV感染499例,感染率21.97%;合并HCV感染196例,感染率8.63%;合并HIV感染166例,感染率7.31%;单纯结核患者1410例,占比62.09%。结核病合并HBV、HCV、HIV感染率在不同年龄组比较,差异具有统计学意义(P<0.01)。结核病合并HBV、HCV患者中CD3^(+)、CD4^(+)和CD8^(+)T细胞的绝对数量表达较高,且两者的差异均具有统计学意义(P<0.05);结核病合并HIV患者CD3^(+)、CD4^(+)T细胞的绝对数量表达较低,CD8^(+)T细胞的绝对数量表达上升,两者的差异具有统计学意义(P<0.01)。CD3^(+)、CD4^(+)、CD8^(+)T细胞绝对数量在结核合并HIV的女性患者中表达较高,且差异均具有统计学意义(P<0.05);各年龄段中,结核合并HIV患者CD3^(+)、CD8^(+)T细胞绝对数量在46~65岁组最低,差异均有统计学意义(P<0.05)。2271例结核病患者中,耐任意1种一线药(单耐药)有391例,总体耐药率17.22%;其中,合并HBV耐药52例,耐药率10.42%;合并HCV耐药10例,耐药率5.10%;合并HIV耐药40例,耐药率24.10%;单纯结核患者耐药289例,耐药率20.50%。其中最多的是单耐药耐利福平33例,占19.88%。结论云南地区结核及合并HBV感染率最高,结核合并HIV患者的免疫功能最差,结核合并HIV的耐药率最高,单耐药以耐利福平最多;结核属于慢性消耗性疾病,免疫功能较一般人群差,易合并HBV感染,结核患者感染HIV有加重免疫功能减低及耐药性产生的趋势。

Nucleos(t)ide analogues causes HBV S gene mutations and carcinogenesis

BACKGROUND： The long-term use of nudeos（t）ide analogues causes drug resistance and mutations in the HBV reverse tran- scriptase （RT） region of the polymerase gene. The RT region overlaps the HBV surface gene （S gene） and therefore, the mutations in the RT region simultaneously modify S gene sequence. Certain mutations in the RT region bring about truncated S proteins because the corresponding changed S gene encodes a stop codon which results in the loss of a large portion of the C-terminal hydrophobic region of HBV surface protein. The rtA181T/sW172＊, rtM204I/sW196＊ and rtV191I/sW182＊ are the most frequently reported drug-resistant mutations with C-terminal truncation, these mutations have oncogenic potential. DATA SOURCES： PubMed and Web of Science were searched using terms： ＂hepatitis B virus＂, ＂HBV drug resistance mutation＂ ＂HBV surface protein＂ ＂HBV truncation＂, ＂hepatocellular carcinoma＂, ＂rtA181T/sW172＊＂, ＂rtM204I/sW196＊＂, ＂rtV191I/sW182＊＂, and relevant articles published in English in the past decades were reviewed. RESULTS： The rtA181T/sW172＊ and rtV191I/sW182＊ mutants occurred more frequently than the rtM204I/sW196＊ mutant both in chronic hepatitis B patients and the HBV-related hepatocellular carcinoma tissues. Although these mutations occur naturally, nudeos（t）ide analogues therapy is the main driving force. These mutations may exist alone or coexist with other HBV mutations. All these three mutants impair the virion secretion and result in HBV surface protein retention and serum HBV DNA level reduction. These mutations possess potential carcinogenic properties. The three mutations are resistant to more than one nucleos（t）ide analogue and therefore, it is difficult to treat the patients with the truncated mutations.CONCLUSIONS： Nucleos（t）ide analogues induce drug resistance and HBV S gene truncated mutations. These mutations have potential carcinogenesis.

DNA-guided hepatitis B treatment,viral load is essential,but not sufficient

Hepatitis B virus （HBV） infection is a global public health problem that concerns 350 million people worldwide. Individuals with chronic hepatitis B （CriB） are at increased risk of developing liver cirrhosis, hepatic de-compensation and hepatocellular carcinoma. To maintain undetectable viral load reduces chronic infection complications. There is no treatment that eradicates HBV infection. Current drugs are expensive, are associated with adverse events, and are of limited efficacy. Current guidelines try to standardize the clinical practice. Nevertheless, controversy remains about management of asymptomatic patients with CriB who are hepatitis B e antigen （HBeAg）-positive with normal alanine aminotransferase, and what is the cut-off value of viral load to distinguish HBeAg- negative CriB patients and inactive carriers. We discuss in detail why DNA level alone is not sufficient to begin treatment of CriB.

Long-term effects of lamivudine treatment in Japanese chronic hepatitis B patients

AIM： To analyze the association between the emergence of tyrosine-methionine-asparatate-asparatate （YMDD） mutants （reverse transcription; rtM204I/V） and deterioration of liver function during long-term lamivudine treatment of Japanese patients with chronic hepatitis B virus （HBV） infection. METHODS： The data of 61 consecutive Japanese pa- tients with chronic hepatitis B who underwent continu- ous lamivudine treatment for more than 24 mo and had a virological response were analyzed. Analysis of YMDD mutants was done by real-time polymerase chain reaction with LightCycler probe hybridization assay for up to 90 mo （mean, 50.8 too; range, 24-90 too）.RESULTS： A mixed mutant-type （YMDD ＋ tyrosine-iso- leucine-asparatate-asparatate： YIDD or tyrosine-valineasparatate-asparatate： YVDD） or a mutant-type （YIDD or YVDD） were found in 57.4% of 61 patients at i year, 78.7% of 61 patients at 2 years, 79.6% of 49 patients at 3 years, 70.5% of 34 patients at 4 years, 68.4% of 19 patients at 5 years, 57.1% of 14 patients at 6 years, and 33.3% of 6 patients at 7 years. Of the 61 patients, 56 （92%） had mixed mutant- or a mutant-type. Only 5 （8%） had no mutants at each observation point. Vi- rological breakthrough was found in 26 （46.4%） of 56 patients with YMDD mutants, 20 of whom had a hepa- titis flare-up： the remaining 30 （53.6%） had neither a virological breakthrough nor a flare-up. All 20 patients who developed a hepatitis flare-up had a biochemical and virological response after adefovir was added to the lamivudine treatment. CONCLUSION： Our results suggest that it is possible to continue lamivudine treatment, even after the emergence of YMDD mutants, up to the time that the patients develop a hepatitis flare-up.

Viral hepatitis and human immunodeficiency virus coinfections in Asia

Hepatitis B virus(HBV), hepatitis C virus(HCV),and human immunodeficiency virus(HIV) affect many people in Asian countries, although there are geographic differences. Both HBV and HIV(HBV/HIV) and HCV/HIV co-infections are highly prevalent in Asia. Hetero- and homosexual, injection drug use, and geographic area are strong predictors of HBV, HCV, and HIV serostatus. In HBV endemic regions, the prevalence and genotype distribution of HBV/HIV coinfection is almost comparable with that in the general population. In Japan, where HBV has low endemicity, the prevalence of HBV/HIV co-infection is approximately 10-fold higher than that in the general population, and HBV Ae is the most common subgenotype among HIV infected individuals. Highly active antiretroviral therapy(HAART) is an effective treatment for HIV/Acquired Immune Deficiency Syndrome. Lamivudine, a component of HAART, is an effective treatment for HBV, HIV, and HBV/HIV co-infection; however, cost, emerging drug resistance, antiretroviral-associated liver toxicity and liver-related morbidity due to HCV progression are particular concerns. HCV/HIV co-infection may accelerate the clinical progression of both HCV and HIV. The high prevalence of HBV/HIV and HCV/HIV co-infections in Asia underscores the need to improve prevention and control measures, as fewer evidencebased prevention strategies are available(compared with Western countries). In this review, the most recent publications on the prevalence of HBV/HIV and HCV/HIV co-infections and related issues, such as therapy and problems in Asia, are updated and summarized.

安徽淮北市CHB患者HBV基因型、耐药类别分布与耐药突变位点的分析

目的了解安徽淮北市慢性乙型肝炎(CHB)患者乙型肝炎病毒(HBV)基因型、耐药类别分布特征及耐药位点。方法选取2018年1月—2020年12月淮北矿工总医院及其下属医院收治的HBV-DNA阳性CHB患者141例,收集血清。采用荧光定量PCR反应进行HBV-DNA定量检测。采用PCR-RFLP法检测HBV基因分型。采用Sanger法测序分析P区基因序列核苷类似物耐药相关突变位点和耐药情况。结果141例CHB患者中有109例成功分型,其中HBV基因型以C型为主(71.56%),B型(17.43%)次之,B+C型最少(11.01%)。CHB患者耐药突率为25.69%,且C型(28.21%)最高。常用核苷类似物的耐药性,以拉米夫定及阿德福韦最高(60.71%),替比夫定次之(25.00%)、恩替卡韦最低(3.57%)。P区检出16种耐药突变类型和3种突变模式,均以C型为主。其中阿德福韦以rtN236T和rtA181T/S常见;拉米夫定以rtL180M+M204I/V为主,rtA181T及V207M次之;恩替卡韦以rtA181S+T184N为主;替比夫定以rtL180M+M204I/V常见。结论安徽淮北市HBV基因型以C型为主,C型耐药突变率最高。核苷类似物耐药均以C型为主,且突变模式较复杂。不同类型CHB患者耐药突变类别及位点存在差异性,因此有必要对HBV基因型及耐药相关突变进行检测,以提高核苷类似物抗病毒治疗的疗效。

High Frequency of Antiviral Resistance Mutations in HBV Genotypes A2 and H: Multidrug Resistance Strains in Mexico

Background and Aims:Lamivudine(3TC),telbivudine(LdT),entecavir(ETV),adefovir(ADF),and tenofovir(TFV)are drugs used to treat hepatitis B virus(HBV)infection,but specific mutations allow some viruses to become resistant to antiviral drugs or to acquire immune escape capacities.These mutations have not been thoroughly investigated in Mexico.This study aimed to estimate the prevalence of HBV antiviral resistance and escape mutations.Methods:This cross-sectional study analyzed 158 samples.HBV DNA was extracted,amplified,and sequenced in serum samples using the spin column method,PCR assay,and Sanger’s sequencing,respectively.HBV genotypes were determined,and HBV mutations were tested using the Geno2pheno tool.Results:Overall,68.4%(108/158)of HBV patients were infected with genotype H,followed by G(11.4%,18/158),A2(10.8%,17/158),F1b(6.9.0%,11/158),D(1.9%,3/158),and E(0.6%,1/158),and 5.1%(8/158)had evidence of recombination.The prevalence of resistance mutations was 8.2%(13/158)and the most common combined mutation was rt180M+rt204V.Notably,we found the combinations rt180M+rt204V+rt173L(n=2)and rt180M+rt204V+rt202G(n=1)that confer multidrug resistance to 3TC,LdT,and ETV.Resistance mutations were found in genotypes A2(11.8%,2/17),and H(10.2%,11/108),and escape mutations were detected in HBV genotypes A2(11.8%,2/17),H(10.2%,11/108),F1b(9.1%,1/11)and G(5.6%,1/18).Conclusions:The highest prevalence of antiviral resistance mutations or escape mutations was detected in HBV genotypes A2 and H.The earliest cases of HBV multidrug resistance were detected in Mexico.

甘肃地区乙型肝炎病毒分型和耐药突变分析

目的 探讨甘肃地区乙型肝炎病毒(HBV)基因型分布特征及耐药情况,为乙型肝炎患者抗病毒用药提供指导依据。方法 选择2018年1月至2019年8月于该院就诊的131例乙型肝炎患者,通过Sanger测序法分析患者HBV基因型和耐药突变位点分布情况。结果 131例乙型肝炎患者共检出B、C、D 3种基因型。其中B型2例(1.53%),C型126例(96.18%),D型3例(2.29%)。检出51例核苷酸类似物耐药,总耐药率为38.93%,其中B型1例,C型49例,D型1例。耐药突变位点最多见于M204V/I,多位点突变以M204V/I+M204V联合突变多见,常在此基础上发生3位点、4位点甚至5位点突变。与阿德福韦酯相关的A181V/T、N236T、A181V/T+N236T耐单药突变最常见,共15例(29.41%)。耐多药突变36例(70.59%),主要以拉米夫定和替比夫定联合耐药为主,在此基础上多有恩替卡韦联合耐药发生。结论 甘肃地区HBV患者基因型以C型为主,耐药形势严峻,突变组合模式复杂多样,应及时检测患者HBV基因型及耐药突变位点,以评价乙型肝炎患者临床疗效,并指导临床抗病毒治疗合理用药。