title: Quantification of hepatitis C virus-infected peripheral blood mononuclear cells by in situ reverse transcriptase-polymerase chain reaction.
abstract: Hepatitis C virus (HCV) is known to infect peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C, but the proportion of HCV-infected circulating cells is not detectable by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and the pathogenic significance of HCV lymphotropism is still unclear. Therefore, we have devised an in situ RT-PCR technique using fluorescein-labeled HCV-specific primers revealed by flow cytometry. PBMC were isolated from 28 patients with chronic HCV-related liver disease; of these, 6 had previously received an orthotopic liver transplantation (OLT) and were on immuno-suppressive treatment. Fourteen patients (50%) were found positive for HCV genome within PBMC by in situ RT-PCR, the proportion of HCV-infected cells ranging from 0.2% to 8.1%. All 6 OLT patients tested positive. The fluorescent signal, corresponding to the HCV-specific 340-bp amplicon, was confined to part of the cytoplasmic compartment of scattered PBMC. Of these 14 patients, 12 had also negativestrand HCV RNA within PBMC detected by "tagged" RT-PCR. We conclude that HCV may infect a significant proportion of PBMC in chronic hepatitis C patients, especially immunosuppressed OLT cases, and that viral replication within PBMC is a common occurrence. Over time, the persistence of HCV-infected immune system cells might interfere with normal immunologic mechanisms and play a role in the pathogenic processes leading to extrahepatic disorders such as mixed cryoglobulinemia and B-cell malignant lymphoma.
authors.affiliation: Cattedra di Medicina Interna II, Università di Bologna, Policlinico S. Orsola, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Muratori
authors.forename: D
authors.initials: D
authors.lastname: Gibellini
authors.forename: M
authors.initials: M
authors.lastname: Lenzi
authors.forename: M
authors.initials: M
authors.lastname: Cataleta
authors.forename: P
authors.initials: P
authors.lastname: Muratori
authors.forename: M C
authors.initials: MC
authors.lastname: Morelli
authors.forename: F B
authors.initials: FB
authors.lastname: Bianchi
mesh_terms: D000328:Adult; D000368:Aged; D003593:Cytoplasm / Q000821:virology; D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016679:Genome, Viral; D016174:Hepacivirus / Q000302:isolation & purification*; D006526:Hepatitis C / Q000601:surgery / Q000821:virology*; D006801:Humans; D007165:Immunosuppression Therapy; D007958:Leukocyte Count / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000821:virology*; D016031:Liver Transplantation; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D016037:Single-Blind Method; D014766:Viremia / Q000601:surgery / Q000821:virology*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral