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Clinical and Therapeutic Implications of Hepatitis C Virus Compartmentalization

2006; Elsevier BV; Volume: 131; Issue: 1 Linguagem: Inglês

10.1053/j.gastro.2006.04.016

1528-0012

Gaétana Di Liberto, Anne–Marie Roque–Afonso, Rachid Kara, Delphine Ducoulombier, Guillaume Fallot, Didier Samuel, Cyrille Féray,

Monoclonal and Polyclonal Antibodies Research

Background & Aims: Blood mononuclear cells (BMCs) frequently are infected by hepatitis C virus (HCV) variants that are not found in plasma. The influence of this compartmentalization on the natural and therapeutic outcome of hepatitis C is unknown. Methods: We studied 119 patients with previously untreated chronic HCV infection. Sixty-five of these patients started first-line treatment with pegylated interferon-alfa and ribavirin after enrollment in the study. The internal ribosomal entry site (IRES) of HCV RNA was amplified and compared between plasma and BMCs by means of single-strand conformational polymorphism (SSCP) analysis, line-probe assay, and cloning sequencing. Results: The IRES SSCP patterns differed between plasma and BMCs in 54 (48%) of 113 assessable patients. Twenty-seven (24%) of these patients were co-infected by 2 HCV types or subtypes, only 1 of which was detectable in BMCs (n = 25) or in plasma (n = 2). SSCP-defined compartmentalization was more frequent in former drug users than in others (35/56 [60%] vs 19/56 [34%]; P < .01), and less frequent in patients with genotype 1 HCV in plasma (26/73 [24%] vs 28/40 [65%]; P < .01). The only variables that were independently predictive of a sustained virologic response were SSCP-defined compartmentalization (25/31 vs 10/32; P = .0001) and genotype 2 or 3 infection of BMCs (22/31 vs 8/34; P = .002). Conclusions: A significant proportion of patients with hepatitis C are co-infected by 2 or more HCV variants with distinct IRES sequences and distinct cellular tropism. This compartmentalization is a strong independent predictor of treatment efficacy. Background & Aims: Blood mononuclear cells (BMCs) frequently are infected by hepatitis C virus (HCV) variants that are not found in plasma. The influence of this compartmentalization on the natural and therapeutic outcome of hepatitis C is unknown. Methods: We studied 119 patients with previously untreated chronic HCV infection. Sixty-five of these patients started first-line treatment with pegylated interferon-alfa and ribavirin after enrollment in the study. The internal ribosomal entry site (IRES) of HCV RNA was amplified and compared between plasma and BMCs by means of single-strand conformational polymorphism (SSCP) analysis, line-probe assay, and cloning sequencing. Results: The IRES SSCP patterns differed between plasma and BMCs in 54 (48%) of 113 assessable patients. Twenty-seven (24%) of these patients were co-infected by 2 HCV types or subtypes, only 1 of which was detectable in BMCs (n = 25) or in plasma (n = 2). SSCP-defined compartmentalization was more frequent in former drug users than in others (35/56 [60%] vs 19/56 [34%]; P < .01), and less frequent in patients with genotype 1 HCV in plasma (26/73 [24%] vs 28/40 [65%]; P < .01). The only variables that were independently predictive of a sustained virologic response were SSCP-defined compartmentalization (25/31 vs 10/32; P = .0001) and genotype 2 or 3 infection of BMCs (22/31 vs 8/34; P = .002). Conclusions: A significant proportion of patients with hepatitis C are co-infected by 2 or more HCV variants with distinct IRES sequences and distinct cellular tropism. This compartmentalization is a strong independent predictor of treatment efficacy. Hepatitis C virus (HCV) is a positively stranded RNA virus that infects more than 1% of the world’s population and is a major cause of cirrhosis and hepatocellular carcinoma. Phylogenetic analysis of HCV strains collected worldwide has identified at least 6 types and multiple subtypes.1Simmonds P. Alberti A. Alter H.J. Bonino F. Bradley D.W. Brechot C. et al.A proposed system for the nomenclature of hepatitis C viral genotypes.Hepatology. 1994; 19: 1321-1324Crossref PubMed Scopus (1049) Google Scholar Combination therapy with pegylated interferon-alfa and ribavirin yields a sustained virologic response (SVR) after 24 weeks in more than 80% of patients with genotype 2 or 3 HCV infection (based on plasma sampling), but in less than 50% of patients with genotype 1 or 4 infection treated for 48 weeks. Many studies have shown the presence of positively and negatively stranded HCV RNA in blood mononuclear cells (BMCs) of chronically infected individuals.2Zignego A.L. Macchia D. Monti M. Thiers V. Mazzetti M. Foschi M. et al.Infection of peripheral mononuclear blood cells by hepatitis C virus.J Hepatol. 1992; 15: 382-386Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 3Muller H.M. Pfaff E. Goeser T. Kallinowski B. Solbach C. Theilmann L. Peripheral blood leukocytes serve as a possible extrahepatic site for hepatitis C virus replication.J Gen Virol. 1993; 74: 669-676Crossref PubMed Scopus (228) Google Scholar, 4Lerat H. Berby F. Trabaud M.A. Vidalin O. Major M. Trepo C. et al.Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.J Clin Invest. 1996; 97: 845-851Crossref PubMed Scopus (259) Google Scholar, 5Laskus T. Radkowski M. Wang L.F. Vargas H. Rakela J. The presence of active hepatitis C virus replication in lymphoid tissue in patients coinfected with human immunodeficiency virus type 1.J Infect Dis. 1998; 178: 1189-1192Crossref PubMed Scopus (66) Google Scholar, 6Laskus T. Radkowski M. Piasek A. Nowicki M. Horban A. Cianciara J. et al.Hepatitis C virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1 evidence of active replication in monocytes/macrophages and lymphocytes.J Infect Dis. 2000; 181: 442-448Crossref PubMed Scopus (202) Google Scholar, 7Radkowski M. Wilkinson J. Nowicki M. Adair D. Vargas H. Ingui C. et al.Search for hepatitis C virus negative-strand RNA sequences and analysis of viral sequences in the central nervous system evidence of replication.J Virol. 2002; 76: 600-608Crossref PubMed Scopus (245) Google Scholar Positive and negative HCV-RNA strands also were detected in BMCs harvested from long-term responders to interferon–ribavirin combination therapy8Pham T.N. MacParland S.A. Mulrooney P.M. Cooksley H. Naoumov N.V. Michalak T.I. Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C.J Virol. 2004; 78: 5867-5874Crossref PubMed Scopus (283) Google Scholar, 9Radkowski M. Gallegos-Orozco J.F. Jablonska J. Colby T.V. Walewska-Zielecka B. Kubicka J. et al.Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C.Hepatology. 2005; 41: 106-114Crossref PubMed Scopus (273) Google Scholar and from treatment-naive HCV-seropositive patients with undetectable viremia.10Radkowski M. Horban A. Gallegos-Orozco J.F. Pawelczyk A. Jablonska J. Wilkinson J. et al.Evidence for viral persistence in patients who test positive for anti-hepatitis C virus antibodies and have normal alanine aminotransferase levels.J Infect Dis. 2005; 191: 1730-1733Crossref PubMed Scopus (44) Google Scholar Finally, a recent report11Pal S. Sullivan D. Kim S. Lai K.K. Kae J. Cotler S. et al.Productive replication of hepatitis C virus in perihepatic lymph nodes in vivo implications of HCV lymphotropism.Gastroenterology. 2005; 30: 1107-1116Google Scholar showed the presence of HCV proteins and positive and negative HCV-RNA strands in lymph nodes. Infection of immune cells could be a mechanism by which HCV evades the host response, but no relation has been reported between BMC infection and the natural or therapeutic outcome of HCV infection. HCV-RNA polymerase lacks proofreading ability, and this leads to strong genomic variability. In infected patients, HCV circulates as a population of closely related variants that are referred to collectively as a quasispecies. This quasispecies distribution may be involved in viral persistence in the early stages of infection.12Farci P. Shimoda A. Coiana A. Diaz G. Peddis G. Melpolder J. et al.The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies.Science. 2000; 288: 339-344Crossref PubMed Scopus (770) Google Scholar HCV compartmentalization is a concept in which viral variants are distributed nonrandomly among the different sites of replication.13Roque-Afonso A.M. Jiang J. Penin F. Tareau C. Samuel D. Petit M.A. et al.Nonrandom distribution of hepatitis C virus quasispecies in plasma and peripheral blood mononuclear cell subsets.J Virol. 1999; 73: 9213-9221PubMed Google Scholar Studies of the hypervariable region of the HCV envelope E2 show that specific variants are frequent particularly in B lymphocytes and monocytes.14Ducoulombier D. Roque-Afonso A.M. Di Liberto G. Penin F. Kara R. Richard Y. et al.Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes.Hepatology. 2004; 39: 817-825Crossref PubMed Scopus (133) Google Scholar HCV compartmentalization also can be detected by analyzing the 5′ untranslated part of HCV RNA (the most strongly conserved region), corresponding to the internal ribosomal entry site (IRES) that regulates viral polyprotein translation. Some patients have been found to have different HCV genotypes in their brain and plasma.15Laskus T. Radkowski M. Bednarska A. Wilkinson J. Adair D. Nowicki M. et al.Detection and analysis of hepatitis C virus sequences in cerebrospinal fluid.J Virol. 2002; 76: 10064-10068Crossref PubMed Scopus (137) Google Scholar, 16Forton D.M. Karayiannis P. Mahmud N. Taylor-Robinson S.D. Thomas H.C. Identification of unique hepatitis C virus quasispecies in the central nervous system and comparative analysis of internal translational efficiency of brain, liver, and serum variants.J Virol. 2004; 78: 5170-5183Crossref PubMed Scopus (205) Google Scholar In a recent study of 65 immunocompetent patients and 44 liver transplant recipients,17Roque-Afonso A.M. Ducoulombier D. Di Liberto G. Kara R. Gigou M. Dussaix E. et al.Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells.J Virol. 2005; 79: 6349-6357Crossref PubMed Scopus (131) Google Scholar we found that the plasma and BMC IRES sequences differed in 39% of patients, and that the BMCs of 9% of patients harbored a genotype that could not be detected in their plasma. Infection through drug use or liver transplantation was associated with the presence of divergent BMC variants. The impact of such mixed infections on the natural and therapeutic outcome of HCV infection is not known. Here we examined possible relations between IRES-defined HCV compartmentalization and the main clinical, histologic, and virologic characteristics of HCV infection, focusing on the SVR status more than 1 year after the end of interferon–ribavirin combination therapy. A total of 119 patients managed by 2 of the authors (C.F. and D.S.) at Paul Brousse Hospital, France, were enrolled in this study with their written consent. The inclusion criteria were anti-HCV and plasma HCV-RNA positivity, hepatitis B surface antigen and anti–human immunodeficiency virus negativity, no previous treatment with interferon or ribavirin, no anticancer chemotherapy or therapeutic immunosuppression, no intranasal or intravenous illicit drug use in the past 3 years, current alcohol intake less than 20 g/day, and normal liver function (prothrombin time >70%). When transfusion or intravenous/intranasal drug use was clearly the route of infection, 2 distinct durations of HCV infection and 2 distinct ages at infection were estimated, from the years of first and last transfusion (before 1991) and the years of first and last drug use. Among patients transfused before 1991, those who also used drugs formed a separate subgroup. Patients with no history of drug use or transfusion were classified as having an unknown route of infection, even in cases of previous tattooing, piercing, invasive medical investigations, or surgery. A liver biopsy specimen obtained the day of blood sampling was available for 111 of 119 patients. Hepatic fibrosis and inflammatory activity were graded with the METAVIR system.18Bedossa P. Poynard T. The METAVIR Cooperative Study GroupAn algorithm for the grading of activity in chronic hepatitis C.Hepatology. 1996; 24: 289-293Crossref PubMed Google Scholar Forty of the 119 patients were included in our previous study of IRES-defined BMC compartmentalization.17Roque-Afonso A.M. Ducoulombier D. Di Liberto G. Kara R. Gigou M. Dussaix E. et al.Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells.J Virol. 2005; 79: 6349-6357Crossref PubMed Scopus (131) Google Scholar Finally, anti–HBc was positive in 16 of 55 patients who were former drug users and in 8 of 64 other patients (P = .04). Sixty-five patients (52%) started treatment between January 2001 and January 2003, based on weight-dosed pegylated interferon-alfa 2b (Virapeg; Schering-Plough, Raritan, NJ) 1–1.5 μg/kg/wk (depending on predicted tolerability) and ribavirin 10–15 mg/kg (according to renal function and the red cell count). Therapy was stopped if viremia persisted at the 12th week. Otherwise, it was continued for another 12 weeks in patients with genotype 2 or 3 infection and for 36 weeks in other patients. Virologic responses and SVRs, respectively, were defined by undetectable HCV viremia (50 copies/mL) in the Amplicor Cobas assay (Roche Diagnostic, Meylan, France) at the 12th week after treatment initiation and the 12th month after treatment completion. The main characteristics of the treated and untreated patients are shown in Table 1.Table 1Characteristics of Treated and Untreated PatientsVariablesNot treated (n = 54)Treated (n = 65)P valueMen29 (51%)32 (52%)NSAge at inclusion46 ± 1243 ± 11NSAge at infection26 ± 924 ± 7NSDuration of infection18 ± 917 ± 7NSHCV RNA (log copies/mL)5.7 ± 5.15.9 ± 6.2NSRoute of infectionIVDU25 (45%)30 (46%)NSTransfusion15 (28%)15 (23%)NSIVDU and transfusion2 (4%)3 (5%)NSUnknown12 (22%)17 (26%)NSPlasma HCV genotypes<.001 140 (74%)38 (58%) 21 (2%)12 (18%) 33 (6%)13 (20%) 410 (19%)2 (3%)Biopsy examination48 (89%)63 (97%)NSFibrosis scoreNS 07 (15%)5 (8%) 123 (48%)28 (44%) 24 (8%)15 (24%) 31 (2%)8 (13%) 4 (cirrhosis)13 (28%)7 (11%)NS, not significant; IVDU, intravenous drug use. Open table in a new tab NS, not significant; IVDU, intravenous drug use. Blood was collected on the day of liver biopsy examination. BMCs were isolated from 10 mL of blood with the Vacutainer CPT (Becton Dickinson, Le Pont-De-Claix, France). RNA was extracted from 140 μL of plasma with the QIAmp Viral RNA kit (Qiagen GmBH, Germany), and from BMCs with the RNeasy minikit (Qiagen, Courtaboeuf, France). To detect the HCV genome, one fifth of the plasma extract or 1 μg of cellular RNA extract was subjected to reverse-transcription polymerase chain reaction with Ready-To-Go reverse-transcription polymerase chain reaction beads (Pharmacia Biotech, Uppsala, Sweden). Three microliters of the first polymerase chain reaction (PCR) product was subjected to a second round of PCR using Ready-To-Go PCR beads, according to the manufacturer’s instructions. A 250–base pair (bp) fragment (nucleotides 100–350) of the 5′untranslated region (5′UTR) was amplified with a nested reverse-transcription polymerase chain reaction protocol using previously published primers.17Roque-Afonso A.M. Ducoulombier D. Di Liberto G. Kara R. Gigou M. Dussaix E. et al.Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells.J Virol. 2005; 79: 6349-6357Crossref PubMed Scopus (131) Google Scholar A 1500-bp fragment (nucleotides 100–1629) spanning the 5′UTR, core, E1, and HVR1 regions was amplified in some cases with Expand Taq polymerase (Roche) and previously published primers.14Ducoulombier D. Roque-Afonso A.M. Di Liberto G. Penin F. Kara R. Richard Y. et al.Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes.Hepatology. 2004; 39: 817-825Crossref PubMed Scopus (133) Google Scholar HCV RNA was quantified in plasma and BMCs by using real-time PCR as described elsewhere.19Takeuchi T. Katsume A. Tanaka T. Abe A. Inoue K. Tsukiyama-Kohara K. et al.Real-time detection system for quantification of hepatitis C virus genome.Gastroenterology. 1999; 116: 636-642Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar Glyceraldehyde-3-phosphate dehydrogenase RNA was quantified (Roche) to normalize BMC HCV-RNA values. Serial 10-fold dilutions of titered HCV and cellular (BMC) RNA were run in each experiment and used to construct standard curves for HCV RNA and glyceraldehyde-3-phosphate dehydrogenase RNA. The HCV 5′UTR quasispecies were compared first by using the single-strand conformation polymorphism (SSCP) assay. This widely used mutation-scanning technique is based on the relation between the electrophoretic mobility of single-strand DNA and its nucleotide sequence.20Orita M. Kanazawa H. Hayashi H. Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms.Proc Natl Acad Sci U S A. 1989; 86: 2766-2770Crossref PubMed Scopus (3365) Google Scholar SSCP analysis was performed as previously described.17Roque-Afonso A.M. Ducoulombier D. Di Liberto G. Kara R. Gigou M. Dussaix E. et al.Compartmentalization of hepatitis C virus genotypes between plasma and peripheral blood mononuclear cells.J Virol. 2005; 79: 6349-6357Crossref PubMed Scopus (131) Google Scholar Briefly, the target sequences were amplified by PCR, then the reaction product was mixed with gel-loading buffer, held at 95°C for 10 minutes, and applied to polyacrylamide gels (GeneGel Excel 12.5/24; Amersham-Pharmacia Biotech) on a thermostated device (Multiphor II system; Amersham-Pharmacia) at 5°C, 600 V, 25 mA, and 15 W. The performance of our 5′UTR SSCP assay was assessed by testing 160 cloned and sequenced variants derived from 250-bp 5′UTR amplicons. We found that 1, 2, and 3 mutations yielded different SSCP patterns in 10%, 44%, and 69% of cases, respectively. SSCP patterns always were different when 4 or more mutations were present, and never were different when the sequences were identical. Plasma and BMC SSCP patterns were considered distinct if they differed by at least 1 band in each compartment. SSCP-defined compartmentalization was assessed blindly to clinical status. PCR products were cloned with the pGEM-T Easy Vector system (Promega Corporation, Madison, WI) and transformed in Escherichia coli JM109 competent cells (Promega). After overnight incubation at 37°C, insertion was checked on white colonies by PCR with the 5′UTR inner primer pair. Sequences were read bidirectionally with inner primers on an ABI377 sequencer using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit with Amplitaq DNA polymerase (Perkin Elmer/Applied Biosystems Division, Foster City, CA). Ten to 20 clones from plasma and BMCs were sequenced for each patient. Nucleotide sequences of cloned products were aligned by using the CLUSTAL W program version 1.5. Pairwise nucleotide distances were calculated by using the Kimura 2-parameter method with a transition-to-transversion ratio of 2. Phylogenetic trees were constructed with the neighbor-joining algorithm, a cluster-analysis method suited to sequences with high similarity scores. Statistical analysis of the topology thus obtained was performed with 1000 replications of bootstrap sampling. The 5′UTR genotyping was performed with the INNO-LiPA HCV II kit (Innogenetics, Ghent, Belgium), according to the manufacturer’s instructions. Briefly, the 5′UTR is amplified with biotinylated primers, and biotin-labeled PCR products then are reverse-hybridized to specific probes attached to nitrocellulose strips. The HCV type is deduced from the patterns of hybridizing bands by using the LiPA interpretation chart.21Stuyver L. Wyseur A. van Arnhem W. Hernandez F. Maertens G. Second generation line-probe assay for hepatitis C virus genotyping.J Clin Microbiol. 1996; 34: 2259-2266PubMed Google Scholar The following variables were analyzed: sex, route of infection (single transfusion, >2 transfusions before 1991, intranasal or intravenous drug use, or undetermined), birth in areas with higher HCV prevalence rates than in France (sub-Saharan Africa or the Middle East), age at enrollment, minimal and maximal durations of infection and minimal and maximal ages of infection (see previously), the plasma genotype (1–4), plasma and BMC viral loads, histologic activity and fibrosis scores in patients with liver biopsy examination, alcohol consumption (±10 g/day), SSCP-defined compartmentalization (yes/no), genotype 1 or 4 detection in BMCs (yes/no), and genotype 2 or 3 detection in BMCs (yes/no). Univariate and multivariate analyses were performed with StatView software (Abacus Concept, Berkeley, CA). The Fisher exact test was used to compare 2 nominal variables, and a t test was used for nominal and continuous variables. Logistic regression and multiple regression were used for multivariate analysis of dependent variables. The 5′UTR HCV RNA was detected in both BMCs and plasma of 113 of 119 patients (94%). The mean viral load was 5.8 ± 5.0 log copies/mL in plasma and 4.2 ± 3.8 log copies/μg of BMC RNA. SSCP profiles differed between plasma and BMCs in 54 (48%) of these 113 patients. Among the 65 patients who started treatment after enrollment, blood samples were obtained from 10 patients with SVR and 4 patients without SVR more than 1 year after treatment completion. HCV RNA was detected in BMCs from 4 of 4 patients without SVR and from 2 of 10 patients with SVR. In 15 patients, IRES sequences were amplified and cloned from plasma and BMC HCV RNA, and at least 10 clones were isolated and amplified from each compartment (number of clones = 385). Among these 15 patients, 5 had similar plasma and BMC SSCP profiles and, in each case, cloning showed fewer than 5 nucleotide changes (on a 250-bp amplicon; 2%) between plasma and BMC variants. We cloned the 5′UTR to hypervariable region (HVR) (nt 100–1619) of HCV RNA in BMCs and plasma from 2 of these patients and confirmed that all variants found in BMCs (or plasma) belonged to the same type or subtype (Figure 1). However, analysis of the HVR showed BMC-specific variants in both patients (data not shown). Among the other 10 patients whose IRES-SSCP profiles differed between plasma and BMCs, cloning of the IRES showed the presence of specific variants in BMCs. Genetic (IRES) distances between plasma and BMCs were higher than .1 in 8 of 10 patients (Figure 2). Sequence analysis revealed that a type or subtype was present in BMCs but not in plasma in these 8 patients. In 2 patients (patients 18 and 25) in whom genotype 1 was detected in both plasma and BMCs, we cloned the 5′UTR to HVR region and confirmed the InoLipa subtyping results. Analysis of the HVR in these 2 patients also showed the presence of BMC-specific variants (data not shown).Figure 2Phylogenetic analysis of plasma and BMC variants in 4 patients with different genotypes in plasma and BMCs. The genetic distances between plasma and BMC strains were long. ◊, Plasma; •, BMCs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Among the 54 patients who had SSCP-defined HCV compartmentalization, paired BMCs and plasma HCV RNA were analyzed by line-probe assay, and different types or subtypes were found in plasma vs BMCs in 27 patients (Table 2). In 19 patients, 1 genotype was detected in both compartments and another type or subtype was detected only in BMCs; in 6 patients the plasma and BMC genotypes differed; and in 2 patients 2 different genotypes were detected in plasma but only 1 of them was detected in BMCs. Among the 27 patients with genotypic compartmentalization, 10 of 11 of those with non-1 genotype in plasma had genotype 1 in BMCs and 15 of 16 of those with genotype 1 in plasma also had genotype 1 in BMCs (Table 2). Cloning of UTR or of UTR-to-HVR (n = 2) sequences were performed in 10 of these patients and confirmed the line probe assays results. We also genotyped plasma and BMC HCV RNA from 59 patients who had similar plasma and BMC-SSCP profiles and confirmed that the same genotype always was present in the 2 compartments.Table 2Characteristics of the 27 Patients With Different Types or Subtypes in Plasma and BMCsNo.BitherapyRoute of infectionAgeSexDuration of infectionActivityFibrosisPlasma HCV RNA (log copies/mL)Plasma genotypeBMC HCV RNA (log copies/μg)BMC genotypeCloningSVR3NIVDU44M22116.013.01 + 20NA5NUnknown42MNA144.81a3.11a+2+30NA1NIVDU45M10NANA5.51a2.41a+30NA14NTransfusion66F25144.81a3.11a+30NA18NIVDU37M5NANA5.81a3.01b1NA6NIVDU and transfusion58M40046.11b4.21b+20NA16NUnknown42FNA225.21b2.61b+20NA9NIVDU32F13215.21+24.21a0NA27NIVDU27F3005.433.82+31NA10NUnknown40FNANANA4.643.11+2+30NA11PIVDU35M17125.01a3.71a+30N15PIVDU42M19236.01b4.01b+20N21PTransfusion51F20245.324.21b+21N19PIVDU27M10005.51b+33.51b1N12PIVDU40M20224.632.61+30N25PIVDU42M15225.21b4.21a1P23PIVDU and transfusion33F5315.71a3.21a+1b1P26PIVDU46F25214.01a3.21a+21P13PIVDU30M11214.71a3.01a+30P4PUnknown42MNA246.11b4.030P7PIVDU20F1NANA4.931.51+2+30P17PIVDU47F14214.333.01+30P20PIVDU34M13014.032.81+31P22PIVDU32M1512NA31.81a1P8PIVDU35F18215.331.91b+30P24PIVDU39F15314.644.21+41P2NIVDU38M16006.343.51a+20PIVDU, intravenous drug use; NA, not available; N, negative; P, positive. Open table in a new tab IVDU, intravenous drug use; NA, not available; N, negative; P, positive. Different plasma and BMC-SSCP profiles (ie, compartmentalization) were less frequent in patients infected by transfusion (7/29 [24%]) than in nontransfused former drug users (31/51 [61%]; P < .001) SSCP-defined compartmentalization also was less frequent in patients with plasma genotype 1 infection (26/73 [36%] vs 28/40 [70%]; P = .001) (Table 3).Table 3SSCP-Defined and Genotype-Defined Compartmentalization: Relation to Clinical, Virologic, and Histologic ParametersSSCP unequal between plasma and BMCs (n = 54)SSCP equal between plasma and BMCs (n = 59)P valueGenotype 2 or 3 in BMCs (n = 40)No genotype 2 or 3 in BMCs (n = 71)P valueMen28/54 (52%)30/59 (52%)NS2138NSAge at inclusion43 ± 1246 ± 12NS46 ± 1244 ± 12NSMaximal duration of infectionaAmong patients infected by transfusion or former drug usage.17 ± 818 ± 7NS17 ± 817 ± 8NSMinimal duration of infectionaAmong patients infected by transfusion or former drug usage.11 ± 514 ± 7NS10 ± 612 ± 9NSAge at infectionaAmong patients infected by transfusion or former drug usage.25 ± 726 ± 10NS27 ± 925 ± 8NSHCV RNA (log copies/mL)5.6 ± 5.76.0 ± 6.2NS5.2 ± 4.95.6 ± 5.0NSRoute of infectionn = 113.007n = 113NSIVDU31 (57%)20 (34%)21 (52%)30 (58%)Transfusion7 (12%)22 (37%)6 (15%)24 (33%)IVDU and transfusion4 (7%)1 (7%)2 (5%)3 (4%)Unknown12 (22%)16 (27%)11 (28%)16 (22%)Plasma genotypen = 113.001n = 113<.001 126 (48%)47 (80%)12 (30%)65 (89%) 27 (13%)6 (10%)13 (32%)0 (0%) 313 (24%)2 (3%)14 (35%)1 (1%) 48 (15%)4 (7%)1 (2%)7 (10%)Fibrosisn = 106NSn = 106NS 06 (12%)6 (10%)2 (6%)9 (13%) 120 (42%)29 (50%)16 (44%)32 (46%) 210 (21%)8 (14%)7 (19%)12 (17%) 35 (10%)3 (5%)4 (11%)4 (6%) 47 (15%)12 (21%)7 (19%)13 (19%)NS, not significant; IVDU, intravenous drug use.a Among patients infected by transfusion or former drug usage. Open table in a new tab NS, not significant; IVDU, intravenous drug use. Logistic regression analysis showed that SSCP-defined compartmentalization was less frequent in patients with plasma genotype 1 infection and in patients infected by transfusion, independently of other factors. Neither SSCP-defined compartmentalization nor the presence of genotype 2 or 3 in BMCs was related to the severity of liver disease (Metavir activity or fibrosis scores), anti–hepatitis B core antigen positivity, sex, age, age at infection, the duration of infection (minimal or maximum), or plasma or BMC viral load. Among the 65 patients who started antiviral treatment, 39 (60%) became plasma HCV-RNA negative at the end of the third month (Table 4). Eight of these 39 patients subsequently relapsed and 31 (47%) had an SVR.Table 4Variables Related to the SVR Status in 65 Treated PatientsSVR (n = 31)No SVR (n = 34)P valueMen13 (41%)19 (54%)NSAge at inclusion44 ± 1344 ± 10NSMaximal duration of infectionaAmong patients infected by transfusion or IVDU.16 ± 719 ± 6NSMinimal duration of infectionaAmong patients infected by transfusion or IVDU.11 ± 614 ± 10NSAge at infectionaAmong patients infected by transfusion or IVDU.25 ± 724 ± 8NSHCV RNA (log copies/mL)6.0 ± 6.15.5 ± 5.9NSRoute of infectionNSIVDU15 (48%)15 (44%)Transfusion5 (16%)10 (29%)IVDU and transfusion2 (6%)1 (3%)Unknown9 (29%)8 (24%)Plasma genotype.0006 110 (32%)28 (82%) 29 (29%)3 (9%) 310 (32%)3 (9%) 42 (7%)0Fibrosis (n = 63)NS 01 (3%)4 (12%) 117 (56%)11 (33%) 26 (20%)9 (27%) 32 (7%)6 (18%) 44 (13%)3 (9%)Compartmentalization Genotype 1 or 4 in BMCs (n = 65)17/31 (55%)30/34 (88%)NS Genotype 2 or 3 in BMCs (n = 65)22/31 (71%)8/34 (24%).0002 SSCP ≠ (n = 63)25/31 (80%)10/32 (31%).0001NS, not significant; IVDU, intravenous drug use.a Among patients infected by transfusion or IVDU. Open table in a new tab NS, not significant; IVDU, intravenous drug use. SVR was less frequent in patients infected by genotype 1 (10/38 [26%]) than in patients infected by genotype 2 (9/12 [75%]; P < .001) or genotype 3 (10/13 [77%]; P < .001). SVR was more frequent in patients with SSCP-defined compartmentalization (25/31 [80%]) than in other patients (10/32 [31%]; P = .0001). It also was more frequent in patients who had genotype 2 or 3 infection of BMCs (22/31 [71%]) than in other patients (8/34 [24%]; P = .0002). SVR was not related to the presence of anti–hepatitis B core antigen or to plasma or BMC viral loads.