Assay for hepatitis C virus in peripheral blood mononuclear cells enhances sensitivity of diagnosis and monitoring of HCV-associated hepatitis

Abstract

Hepatitis C virus (HCV) is a major etiological factor in chronic hepatitis affecting up to 24% of blood donors in Egypt. Since fluctuating levels of HCV RNA loads, including undetectable values, have been frequently observed in sera of chronic hepatitis patients, this study was designed to assess the sensitivity of PCR amplification for the plus- and minus-RNA strands in peripheral blood mononuclear cells (PBMC) compared to single serum PCR assay. Since the latter test detects viremia in only 79.5% of seropositive cases, the highest sensitivity for HCV diagnosis was achieved (93.20% when applying the combined triple test including PCR amplification of plus-strand in serum, together with plus-strand in PBMC and minus-strand in PBMC.The results of this study indicate that the triple test provides significant information on extrahepatic replication of HCV in a sizable sample of seropositive subjects (429 cases) and improves the assessment of HCV viremia. The cost/effectiveness and speed were upgraded by using capillary/air rapid thermal cycler. The use of the triple assay in HCV diagnosis and post-therapy monitoring is recommended.

Introduction

Hepatitis C virus (HCV) is a small RNA virus that is the major causative agent of post-transfusional non-A non-B hepatitis [1], [2], [3]. The genome consists of a single ORF (open reading frame) encoding a large polyprotein precursor that is cleaved to yield individual structural and non-structural viral proteins [2], [4]. HCV is taxonomically related to flaviviruses and pestiviruses. By analogy with these viruses, the HCV genome is presumed to replicate by using the negative-strand as a template [4], [5]. HCV infection is a major etiological cause of transfusion-associated hepatitis [6], [7], [8]. In Egypt, it represents the highest donor infection rates recorded so far, where 14–24% of blood donors are anti- HCV positive [9], [10], [11], [12], [13]. Monitoring the viremia pre- and post-antiviral therapy through the detection of viral RNA by use of qualitative reverse transcription-polymerase chain reaction (RT-PCR) and various quantitative methods has become the most frequently used and sensitive technique. Although these quantitative assays accurately determine the amount of virus in plasma or serum, HCV is also found in bone marrow and peripheral blood mononuclear cells (PBMCs) [14], [15], [16], [17], suggesting that the virus may have an extrahepatic localization and pathogenetic role. These cellular reservoirs of HCV are not detected by plasma RNA detection methods [18]. In addition, a major problem associated with assessment of viral loads in serum samples is that fluctuating levels of RNA are commonly observed including periods during which viremia is undetectable [19], [20], [21], [22], [23]. Consequently, routine serum or plasma measurements of HCV RNA may not provide an accurate determination of the total HCV load present in peripheral blood. The accurate determination of HCV RNA is important, since high serum levels of HCV RNA appear to correlate with a poor response to interferon therapy [24].

The aim of this work is to assess HCV viremia by use of simultaneous detection of plus and minus RNA strands in PBMC, together with serum RNA. The advantage of the triple assay of HCV RNA in serum and in PBMC (both plus and minus strands) over serum RNA and other possible combined assays in assessment of the status of HCV viremia in 429 infected patients was evaluated.