Monitoring Drug Resistance in Chronic Hepatitis B Virus (HBV)-Infected Patients during Lamivudine Therapy: Evaluation of Performance of INNO-LiPA HBV DR Assay

Sensitive and early detection of emerging hepatitis B virus (HBV) drug resistance may not only help monitor the viral dynamics associated with lamivudine treatment but could also improve therapeutic decision making. This is especially important when new antivirals effective against lamivudine-resistant HBV become available. A total of 159 serum samples from 33 chronic HBV patients receiving lamivudine treatment were analyzed at four centers for the presence of lamivudine-resistant mutations at codons 528 [180] (proposed revised nomenclature according to Stuyver et al. [Hepatology 33:751-757, 2001] shown in brackets), 552 [204], and 555 [207] of the HBV polymerase. Sequencing data were compared with results generated by the INNO-LiPA HBV DR line probe assay (LiPA), an assay based on reverse hybridization of amplified HBV DNA fragments with specific nucleotide probes immobilized on nitrocellulose strips. LiPA provided at least the same information as sequencing for 97.5% of all codons analyzed for codon 528 [180], 95% for codon 552 [204], and 100% for codon 555 [207]. The most common reason for discrepant or indeterminate results (0.4% and 1.5%, respectively) in a small percentage of the population tested could be attributed to polymorphisms not yet covered by LiPA probes. In at least five patients, a mutant could be detected earlier by LiPA than by sequencing. In 15 patients, LiPA detected mixed wild-type and mutant virus populations before viral breakthrough. These results demonstrate that INNO-LiPA HBV DR is a highly sensitive and easily applicable assay for the detection and monitoring of lamivudine-resistant mutations in chronic hepatitis B patients and that the assay is more sensitive than sequencing in detecting mixed mutant and wild-type sequences.

早期灵敏地检出新发乙型肝炎病毒（HBV）耐药性，不仅有助于监测拉米夫定治疗相关的病毒动力学变化，还可优化治疗决策。当针对拉米夫定耐药HBV的新型抗病毒药物问世时，该检测的临床价值尤为突出。本研究从4家研究中心纳入33名接受拉米夫定治疗的慢性HBV感染者的159份血清样本，针对HBV聚合酶的528[180]（依据Stuyver等[Hepatology 33:751-757, 2001]提出的修订命名规则，方括号内为修订后编号）、552[204]及555[207]位密码子的拉米夫定耐药突变进行了分析。将测序所得数据与INNO-LiPA HBV DR线探针检测试剂盒（INNO-LiPA HBV DR line probe assay，简称LiPA）的检测结果进行比对；该检测基于扩增的HBV DNA片段与固定于硝酸纤维素膜条上的特异性核苷酸探针的反向杂交原理。在所有分析的密码子位点中，LiPA与测序结果的一致性表现为：528[180]位密码子达97.5%，552[204]位达95%，555[207]位达100%，其提供的检测信息至少与测序相当。在少量受检样本中，结果出现不一致或不确定的比例分别为0.4%和1.5%，最常见的诱因是LiPA探针尚未覆盖的多态性位点。至少有5名患者的样本中，LiPA可比测序更早检出突变株。在15名患者中，LiPA可在病毒学突破前检出野生型与突变型病毒的混合种群。本研究结果证实，INNO-LiPA HBV DR检测试剂盒可实现灵敏且便捷的拉米夫定耐药突变检测与监测，适用于慢性乙型肝炎患者的相关临床管理；且在检出混合野生型与突变型病毒序列方面，其灵敏度优于传统测序技术。