HCV in serum, peripheral blood mononuclear cells and lymphocyte subpopulations in C-hepatitis patients

Abstract

To define the best marker for the follow-up and evaluation of HCV infections we determined anti-HCV antibodies, serum transaminases and HCV RNA in patients diagnosed with chronic hepatitis for C virus and treated with α-interferon. The presence/absence of HCV RNA was determined in serum, peripheral blood mononuclear cells (PBMC) and lymphocyte subpopulations. Samples were submitted to RT-PCR and subsequent nested PCR. Treatment with α-interferon induced a fall in the number of HCV RNA positive patients from an initial 88 to 25% at the end of the treatment. The withdrawal of treatment was associated with a significant increase in the number of HCV RNA positive patients (43% at the 12-month follow-up). In 61% of the patients the PCR analysis of the PBMC population detected the presence of HCV RNA. In 87% of cases the cell fraction identified as CD19 resulted positive in the PCR test and the viral genome was undetectable in PBMC subpopulations in only 13% of cases. In one third of the patients whose serum was negative for PCR the analysis demonstrated the presence of HCV RNA in PBMC. Conclusions: The disappearance of the viral genome in serum, a criterion of treatment response, is not necessarily followed by its disappearance in PBMC. The joint determination of HCV by PCR technique in serum and blood cells should be used as a particular instrument with each patient.

Introduction

The diagnosis of chronic hepatic damage due to hepatitis C virus (HCV) is based on clinical and biochemical criteria, with increased transaminases initiating suspicion of this infection. The anti-HCV determination and the histological study confirm the diagnosis. However, the presence of anti-HCV indicates either a past cured infection or a chronic infection. This fact, together with the late appearance of anti-HCV in the course of HCV-induced acute hepatitis, has led to the development of diagnostic tests to provide accurate and early information on HCV infection and its replicative state [1], even when the concentration of HCV in the infected serum and tissues is very low.

The polymerase chain reaction (PCR) technique can recognize and amplify specific portions of the HCV genome [2]. The variability of the nucleotide sequence in some regions of the genome, which could affect the detection of the viral RNA, has been solved by the amplification of genome segments belonging to the 5′ non-coding region, whose nucleotide sequence is the most constant and conserved with a lower mutation rate in all the viruses isolated [3]. The use of localized primers in this region has significantly increased the detection rate of HCV-RNA (to 90%) in patients with HCV-related chronic hepatitis [4]. Moreover, the adoption of nested type PCR techniques has reduced contamination and increased sensitivity and specificity, markedly reducing the possibility of false positives [5].

It seems to be proven that HCV does not have an exclusive tropism for the liver [6], [7], [8].The infective particle circulates freely in the serum, colonizing not only the liver but also other sites such as peripheral blood mononuclear cells (PBMC) and various lymphocyte subpopulations [9], [10]. Although infected lymphocytes have not been demonstrated to release viral particles into the peripheral blood, it has been postulated that the localization of the virus in the cells may allow it to escape the immune system, thus contributing to the chronicity of the viral infection [11] and to recurrences after treatment with α-interferon. The colonization of PBMC and subpopulations could result from phagocytosis, but there is the disquieting possibility that the cells may be truly affected as part of the infectious process of the disease and could form a temporary reservoir for the virus.

We designed a clinical-experimental study on a series of patients with HCV-related chronic hepatitis treated with recombinant interferon α-2b. Our objectives were: (1) to up-date and standardize the technique of HCV determination in blood samples by PCR after inverse transcription (RT-PCR); (2) to follow-up and evaluate the response to α-interferon treatment of patients with hepatitis C, using clinical and analytical parameters and concomitant RT-PCR determination of HCV in serum; and (3) to study the presence of the viral genome in PBMC and lymphocyte subpopulations both before and after α-interferon treatment.