Umeoka Fumi, Iwasaki Yoshiaki, Matsumura Masayuki, Takaki Akinobu, Kobashi Haruhiko, Tatsukawa Masashi, Shiraha Hidenori, Fujioka Shin-ichi, Sakaguchi Kohsaku, Shiratori Yasushi
Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan.
J Gastroenterol. 2006 Jul;41(7):693-701. doi: 10.1007/s00535-006-1834-x.
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.
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.
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.
The FBH assay is an efficient method for detecting and quantifying HBV mutants, as early as 3 months after lamivudine administration.
长期使用拉米夫定治疗会导致拉米夫定耐药的乙型肝炎病毒（HBV）出现。本研究的目的是开发一种荧光双探针杂交（FBH）检测方法，用于检测和定量拉米夫定治疗患者临床过程中的HBV突变体，并评估其临床实用性。
我们开发了一种FBH检测方法来检测HBV DNA聚合酶基因中的突变。使用野生型/突变体质粒DNA的稀释系列来确定该检测方法的检测灵敏度。对27例接受拉米夫定治疗的患者采集的血样进行分析。
检测到低至总HBV DNA 10%的突变体DNA水平（灵敏度 = 100%，特异性 = 80%）。在拉米夫定给药后的平均20个月随访期间，27例患者中有5例检测到HBV突变体。在这5例患者中，有1例在拉米夫定治疗开始时检测到YIDD突变体，而其余4例患者在开始拉米夫定给药后3个月内被鉴定为具有YIDD突变体。在5例有HBV突变体的患者中，4例在检测到HBV突变体后10多个月出现突破型肝炎，随后HBV DNA再次出现或再次升高，其特征是以突变体为主。即使血清HBV DNA滴度低于市售定量聚合酶链反应的检测限，仍在1例患者中检测到YIDD突变体。
FBH检测方法是一种在拉米夫定给药后3个月即可有效检测和定量HBV突变体的方法。
