Nogales M C, Serrano M C, Suárez E, Corpas R, Pérez L, Claro R, Jarana R, Romero-Gómez M, Martín-Mazuelos E
Servicio de Microbiología, Hospital Universitario de Valme, Sevilla, Spain.
Gastroenterol Hepatol. 2004 Nov;27(9):515-20. doi: 10.1016/s0210-5705(03)70518-4.
To determine hepatitis B virus (HBV) genotypes in southern Seville (Spain) and investigate the development of lamivudine-resistance mutations by using a hybridization technique with specific probes and by comparing the results with those of the direct sequencing technique. To evaluate the temporal relationship between variations in the level of HBV-DNA and detection of mutant variants. To analyze the influence of several genotypes on the pattern of mutations developed and on values of viral load and alanine aminotransferase (ALT) after their development.
In 37 patients with chronic HBV infection, HBV genotype was determined using the LiPA technique. In 10 of these patients undergoing lamivudine treatment for a mean of 19.2 months, the development of lamivudine-resistant mutations was investigated. In these 10 patients, the LiPA technique was compared with direct sequencing. During lamivudine treatment, we determined HBV-DNA by polymerase chain reaction (PCR) and ALT every 3-6 months.
The most frequent genotypes were D (45.9%) and A (18.9%); 2 patients were genotype B while 18.9% had mixed genotypes. Sequencing showed identical results except in one mixed genotype. Mutations were found in 60% of the cases. The results of sequencing were in agreement, except in the detection of mixed populations composed of mutants and wild-type (WT). Patients with genotype A showed the pattern M204I+WT in the first 12 months and those with genotype D showed the pattern L180M+M204V with or without WT at 18 months. In 5/6 cases, an increase of > 1 log10 in HBV-DNA was observed 3-8 months before the mutation was detected by LiPA. In patients with genotype B, levels of HBV-DNA and ALT after the development of mutations was lower than basal levels and was also lower than those in patients with genotypes A and D.
The LiPA technique for determination of HBV genotype and detection of lamivudine-resistance mutations shows excellent correlation with the most complex sequencing technique. Genotype D predominates in southern Seville. During lamivudine treatment, an increase in the level of HBV-DNA detected by PCR predicts the development of mutations before these are demonstrated by LiPA.
确定西班牙塞维利亚南部地区的乙型肝炎病毒（HBV）基因型，采用特异性探针杂交技术并与直接测序技术的结果进行比较，研究拉米夫定耐药突变的发生情况。评估HBV-DNA水平变化与突变体变异检测之间的时间关系。分析几种基因型对突变发生模式以及突变发生后病毒载量和丙氨酸氨基转移酶（ALT）值的影响。
对37例慢性HBV感染患者，采用线性探针分析（LiPA）技术确定HBV基因型。其中10例接受拉米夫定治疗平均19.2个月的患者，研究拉米夫定耐药突变的发生情况。对这10例患者，将LiPA技术与直接测序进行比较。在拉米夫定治疗期间，每3 - 6个月通过聚合酶链反应（PCR）测定HBV-DNA并检测ALT。
最常见的基因型是D型（45.9%）和A型（18.9%）；2例为B型，18.9%为混合基因型。除一个混合基因型外，测序结果一致。60%的病例发现有突变。除了检测由突变体和野生型（WT）组成的混合群体外，测序结果相符。A型基因型患者在最初12个月表现为M204I + WT模式，D型基因型患者在18个月时表现为L180M + M204V模式，有或无WT。在5/6的病例中，在LiPA检测到突变前3 - 8个月观察到HBV-DNA增加> 1 log10。B型基因型患者突变发生后HBV-DNA和ALT水平低于基础水平且也低于A型和D型基因型患者。
用于确定HBV基因型和检测拉米夫定耐药突变的LiPA技术与最复杂的测序技术显示出极好的相关性。D型基因型在塞维利亚南部占主导。在拉米夫定治疗期间，PCR检测到的HBV-DNA水平升高预示着在LiPA检测到突变之前突变的发生。
