Assay for hepatitis C virus in peripheral blood mononuclear cells enhances sensitivity of diagnosis and monitoring of HCV-associated hepatitis
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.
Section snippets
Patients
The subjects were 429 HCV seropositive patients including 347 males and 82 females. Patients were referred to the medical service unit at the National research Center over a period of 1 year. No patients received antiviral therapy throughout the study. Three patients (one healthy carrier and two with chronic hepatitis) were referred weekly to assess fluctuation of viral loads and intracellular replication during a 9-week period.
Methods
Anti-HCV antibodies were assayed using IgG third generation
Fluctuation of HCV viremia
To examine the phenomenon of temporary disappearance of serum HCV RNA, three patients (two with chronic hepatitis and one healthy carrier) were subjected to a weekly assay of HCV RNA in serum as well as the detection of both genomic and antigenomic RNA strands in PBMC. These results (Table 1) demonstrated that the two patients with chronic hepatitis displayed intermittent negativity of serum HCV viremia. Patient 2 (chronic hepatitis) had no detectable viral RNA over the fourth and fifth weeks
Discussion
HCV is known to circulate in peripheral blood in a variety of forms [32], [33]. Consequently, quantitative measurements of plasma or serum do not provide a complete estimate of the circulating peripheral blood HCV burden [18]. 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]. In addition, a major problem is associated with assessment of viral loads in serum samples in that fluctuating
References (51)
- et al.
Serum antibodies against porphyric hepatocytes in patients with porphyria cutanea tarda and liver disease
Gastroenterology
(1983) - et al.
Hepatitis C virus infection in Egyptian volunteer blood donors in Riyadh
Lancet
(1991) - et al.
Detection of hepatitis C viral sequences in non-A, non-B hepatitis
Lancet
(1990) - et al.
Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
Anal Biochem
(1987) - et al.
Quantification of HCV replication using one step competitive RT-PCR and a solid phase colorimetric detection method
J Hepatol
(1994) - et al.
False detection of negative-strand hepatitis C virus RNA
Lancet
(1994) - et al.
Quantitative analysis in molecular diagnosis
Hum Pathol
(1994) - et al.
Infection of peripheral blood mononuclear cell by hepatitis C α virus
J Hepatol
(1992) - et al.
Molecular cloning of the human hepatitis C Virus genome from Japanese patients with non A, non B hepatitis
Proc Natl Acad Sci USA
(1990) - et al.
Genetic organization and diversity of the hepatitis C virus
Proc Natl Acad Sci USA
(1991)
Structure and organization of the hepatitis C virus genome isolated from human carriers
J Virol
Hepatitis C virus shares amino acid sequence similarity with pestiviruses as well as members of two plant virus supergroups
Proc Natl Acad Sci USA
Isolation and characterisation of an HCV-specific RNA dependent RNA polymerase activity from extracts of infected liver tissue
An assay for circulating antibodies to a major etiologic virus of human non A non B hepatitis
Science
Hepatitis virus: the major causative agent of viral non A, non B hepatitis
Br Med Bull
Hepatitis C virus infection in blood donors in Egypt
J Egyptian Public Health Assoc
Hepatitis C virus infection among Egyptian in Dammam
Ann Saudi Med
High HCV prevalence in Egyptian blood donors
Lancet
The epidemiology of antibody to hepatitis C in Egypt
J Infect Dis
Peripheral blood leucocytes may serve as a possible extrahepatic site for hepatitis C virus replication
J Gen Virol
Replication of hepatitis C in peripheral blood mononuclear cells
J Hepatol
Hepatitis C virus is detected in a monocyte/macrophage subpopulation of peripheral blood mononuclear cells of infected patients
J Infect Dis
Distribution of hepatitis C virus RNA in CD19 peripheral blood mononuclear cells of chronically infected patients
J Infect Dis
Distribution of hepatitis C virus HCV in whole blood and blood cell fractions: plasma HCV RNA analysis underestimates circulating virus load
J Infect Dis
Early events in hepatitis C virus infection of Chimpanzees
Proc Natl Acad Sci USA
Cited by (35)
Understanding molecular pathogenesis: The biological basis of human disease and implications for improved treatment of human
2018, Molecular Pathology: The Molecular Basis of Human DiseaseSpontaneous clearance of chronic hepatitis C infection is associated with an internal ribosomal entry site IIId stem loop structure variant
2015, Indian Journal of Medical MicrobiologyUnderstanding Molecular Pathogenesis: The Biological Basis of Human Disease and Implications for Improved Treatment of Human Disease
2009, Molecular Pathology: The Molecular Basis of Human DiseaseHepatitis C virus lymphotropism: lessons from a decade of studies
2007, Digestive and Liver DiseaseProtocol for Classification Single-Cell PBMC Types from Pathological Samples Using Supervised Machine Learning
2023, Methods in Molecular Biology