Agnello V, Abel G, Knight G B, Muchmore E
Department of Laboratory Medicine, Lahey Clinic Medical Center, Burlington, MA 01805, USA.
Hepatology. 1998 Aug;28(2):573-84. doi: 10.1002/hep.510280240.
The controversial question of the extent of hepatocyte infection in chronic hepatitis C was re-examined in both chimpanzees and humans using a newly modified in situ hybridization (ISH) method for detecting hepatitis C virus (HCV) RNA. The specificity of the methodology for distinguishing positive- and negative-strand synthetic HCV RNA was at least six magnitudes greater than the reverse-transcription polymerase chain reaction (RT-PCR) assay for HCV. The sensitivity of the methodology as determined by cell culture assay was 14 +/- 2 genomic equivalents (gE) of HCV positive strand per cell, which was three magnitudes less sensitive than RT-PCR quantitation of HCV. In contrast to previous studies in both humans and chimpanzees with chronic hepatitis C, a high percentage of hepatocytes positive for both positive- and negative-strand HCV RNA was found in most specimens studied. In humans, the extent of hepatocyte infection varied with histological activity index (HAI). In the two chimpanzees studied, the liver biopsies showed minimal histological disease activity, but high percentages of hepatocytes were HCV-positive by ISH that correlated with hepatocyte ultrastructural changes associated with HCV infection. Hepatocyte infection was confirmed by RNA extraction and RT-PCR techniques for detecting HCV RNA that minimize the false detection of negative strands. In both human and chimpanzee liver biopsies showing minimal HAI, the hepatocyte concentration of HCV was estimated to be very low. These findings suggested the hypothesis that persistent infection in the liver may be caused in part by low-level HCV replication. The theoretical and clinical implications of these findings are discussed.
利用一种新改良的原位杂交（ISH）方法检测丙型肝炎病毒（HCV）RNA，在黑猩猩和人类中重新审视了慢性丙型肝炎中肝细胞感染程度这一有争议的问题。该方法区分正链和负链合成HCV RNA的特异性比HCV的逆转录聚合酶链反应（RT-PCR）检测至少高六个数量级。通过细胞培养检测确定的该方法的灵敏度为每个细胞14±2个HCV正链基因组当量（gE），比HCV的RT-PCR定量灵敏度低三个数量级。与之前在人类和慢性丙型肝炎黑猩猩中的研究不同，在大多数研究标本中发现，同时对正链和负链HCV RNA呈阳性的肝细胞比例很高。在人类中，肝细胞感染程度随组织学活动指数（HAI）而变化。在研究的两只黑猩猩中，肝活检显示组织学疾病活动最小，但ISH检测显示高比例的肝细胞HCV呈阳性，这与HCV感染相关的肝细胞超微结构变化相关。通过RNA提取和用于检测HCV RNA的RT-PCR技术证实了肝细胞感染，该技术将负链的假检测降至最低。在显示HAI最小的人类和黑猩猩肝活检中，HCV的肝细胞浓度估计非常低。这些发现提出了一个假说，即肝脏中的持续感染可能部分是由低水平的HCV复制引起的。讨论了这些发现的理论和临床意义。
