Pawlotsky J M, Bastie A, Lonjon I, Rémiré J, Darthuy F, Soussy C J, Dhumeaux D
Department of Bacteriology and Virology, Hôpital Henri Mondor, Université Paris XII, Créteil, France.
J Virol Methods. 1997 May;65(2):245-53. doi: 10.1016/s0166-0934(97)02196-4.
Detection of hepatitis B virus (HBV) DNA in serum allows monitoring of HBV replication and assessing responses to antiviral treatment. HBV DNA quantification measures virus replication and can be used as a prognosis indicator of liver disease and an index of response to antiviral drugs. The aim of this study was to compare the performances of three HBV DNA detection and/or quantification techniques for assessing HBV replication. Three hundred unselected sera with a request for HBV DNA detection and quantification were tested with a molecular hybridisation technique without amplification (Digene Hybrid-Capture, Murex Diagnostics Ltd), a signal amplification assay based on branched DNA technology (Quantiplex HBV DNA, Chiron diagnostics), and an 'in-house' qualitative, non quantitative target amplification assay based on the polymerase chain reaction (PCR) with primers located in the S gene of the HBV genome. Hybrid-capture and branched DNA gave concordant results in 278 cases (93%). In the 128 samples positive by both assays, DNA titres in pg/ml were related significantly (r = 0.70, P < 0.0001). but branched DNA titres increased more rapidly than hybrid-capture titres when the amount of HBV DNA in the sample increased. Twenty-two sera (7%) were negative by hybrid-capture, but positive in branched DNA (detection rate gain: 15%). In these 22 patients, DNA titres were low, HBsAg was present in all instances and alanine aminotransferase activity was elevated in 18 patients (82%); HBeAg was present in seven patients (32%) and anti-HBe antibodies in 18 patients (82%); liver biopsy, undertaken in 18 patients, revealed chronic active hepatitis in all instances, associated with cirrhosis in eight cases. Qualitative, non-quantitative HBV DNA PCR was positive in 75 (50%) of the 150 hybrid-capture-negative, branched DNA-negative samples, including a significant proportion of patients without evidence of ongoing HBV-related liver disease. The results show that in general, the branched DNA assay detects HBV DNA in more patients than hybrid-capture and that this improved detection rate is relevant clinically and genome equivalents/ml are preferred to pg/ml to quantify HBV DNA in clinical specimens and finally qualitative, non-quantitative polymerase chain reaction can detect HBV DNA in patients without evidence of active HBV-related liver disease. This study emphasizes the need for more sensitive, university standardised quantitative HBV DNA assays and for the definition of clinically relevant cutoffs with these assays.
检测血清中的乙型肝炎病毒（HBV）DNA有助于监测HBV复制并评估抗病毒治疗的反应。HBV DNA定量可测量病毒复制情况，并可作为肝病的预后指标及对抗病毒药物反应的指标。本研究的目的是比较三种HBV DNA检测和/或定量技术评估HBV复制的性能。对300份要求进行HBV DNA检测和定量的未经选择的血清，采用无扩增的分子杂交技术（Digene Hybrid-Capture，Murex诊断有限公司）、基于分支DNA技术的信号放大检测法（Quantiplex HBV DNA，Chiron诊断公司）以及一种基于聚合酶链反应（PCR）的“内部”定性、非定量靶标扩增检测法进行检测，后者的引物位于HBV基因组的S基因中。杂交捕获法和分支DNA法在278例（93%）病例中结果一致。在两种检测均为阳性的128份样本中，pg/ml水平的DNA滴度显著相关（r = 0.70，P < 0.0001），但当样本中HBV DNA量增加时，分支DNA滴度比杂交捕获滴度升高得更快。22份血清（7%）杂交捕获法检测为阴性，但分支DNA法检测为阳性（检测率提高：15%）。在这22例患者中，DNA滴度较低，所有患者均存在HBsAg，18例患者（82%）丙氨酸转氨酶活性升高；7例患者（32%）存在HBeAg，18例患者（82%）存在抗-HBe抗体；18例患者进行了肝活检，所有病例均显示为慢性活动性肝炎，其中8例伴有肝硬化。在150份杂交捕获法和分支DNA法均为阴性的样本中，定性、非定量的HBV DNA PCR有75份（50%）为阳性，其中包括相当比例无持续HBV相关肝病证据的患者。结果表明，总体而言，分支DNA检测法比杂交捕获法能检测出更多患者的HBV DNA，且这种提高的检测率具有临床相关性，临床标本中定量HBV DNA时每毫升基因组当量比pg/ml更可取，最后，定性、非定量的聚合酶链反应可在无活动性HBV相关肝病证据的患者中检测出HBV DNA。本研究强调需要更敏感、统一标准化的定量HBV DNA检测法以及用这些检测法确定临床相关的临界值。
