High HCV subtype heterogeneity in a chronically infected general population revealed by high-resolution hepatitis C virus subtyping.

OBJECTIVES: This study aimed to characterize the chronically infected general hepatitis C virus (HCV) population in Barcelona using a highly sensitive subtyping method that can identify the 67 recognized HCV subtypes and diagnose mixed infection by various genotypes/subtypes in a single individual. The resulting information has implications for selecting optimal direct-acting antiviral (DAA) treatment for each patient and establishing public healthcare policies in our setting. METHODS: Consecutive HCV patients (treatment-naïve or interferon-based failures) attending Vall d'Hebron Hospital outpatient clinics from February 2015 to May 2016 (N=1473) were included in the study. Patient samples were characterized using HCV subtyping by next-generation ultra-deep pyrosequencing. RESULTS: The following genotypes (G) were found: G1 (1126/1473 (76.4%)), G4 (145/1473 (9.8%)), G3 (135/1473 (9.2%)), G2 (51/1473 (3.5%)), and G5 (1/1473 (0.1%)). Twenty-two subtypes were seen: 1b (790/1473 (53.6%)), 1a (332/1473 (22.5%)), 3a (133/1473 (9.0%)), 4d (105/1473 (7.1%)), 4a (29/1473 (2.0%)), and 2c (25/1473 (1.7%)), with 16 low-prevalence subtypes accounting for the remaining 3.0% (44/1473). There was a worrisome 1.0% (15/1473) of mixed infections. G2 (51/1473 (3.5%)) showed a high level of heterogeneity. Analyses by age groups showed a predominance of G1b over G1a (428/506 (84.6%) vs. 24/506 (4.7%)) in patients born before 1950 (N=506/1473), and similar percentages of these subtypes in those born between 1951 and 1975 (N=834/1473) (315/834, 37.8% vs. 266/834, 31.9%) and after 1976 (N=133/1473) (47/133, 35.3% vs. 42/133, 31.6%). CONCLUSIONS: Subtype distribution showed a higher level of heterogeneity than was expected, particularly for G2. Prevalence of mixed infections was around 1%. HCV subtype distribution related to patient age group suggested that patients born from 1936 to 1975 in our setting should undergo screening for the infection. Next-generation sequencing enabled better classification of candidates for DAA-based treatment.

Removal from liver transplantation list of a hepatitis C virus-HIV co-infected patient after successful treatment with sofosbuvir and daclatasvir.

We present a human immunodeficiency virus-infected patient with severe decompensated hepatitis C virus-related cirrhosis awaiting liver transplantation (LT) who received a 24-week course of interferon/ribavirin-free antiviral treatment with sofosbuvir and daclatasvir on a compassionate basis. Rapid viral suppression was associated with progressive improvement of his liver function tests. The patient achieved a sustained virological response and concomitant clinical improvement, which prompted removal from the LT list 12 weeks after the end of treatment.

Early predictors of antiviral treatment response in liver transplant recipients with recurrent hepatitis C genotype 1.

The success of current antiviral treatment for hepatitis C virus (HCV) recurrence in liver transplant (LT) recipients remains limited. We aimed at evaluating the value of IL28B genotype and early viral kinetics to predict response to standard treatment in the transplant setting. We retrospectively evaluated 104 LT recipients treated for HCV genotype 1 recurrence between 2001 and 2010. Baseline variables, including IL28B genotype, and early viral kinetics were compared among patients who did or did not achieve a sustained virological response (SVR). Logistic regression analyses of candidate variables were conducted to generate a reliable predictive model based on the minimum set of variables. Twenty-nine (28%) achieved an SVR. On multivariate analysis, the magnitude of HCV RNA decline at 4 weeks (OR: 3.74, 95% CI: 1.64-9.39; P = 0.003) and treatment compliance (OR: 35.27, 95% CI: 3.35-365.54; P = 0.003) were the only independent predictors of SVR. Favourable recipient IL28B genotype significantly correlates with virological response at week 4 (OR 3.23; 95% CI, 1.12-9.15; P = 0.03). By logistic regression analysis, a model including donor age, recipient rs12979860 genotype and viral load at 4 weeks showed the best predictive value for SVR with an area under the receiver operating curve of 0.861. Favourable recipient IL28B genotype strongly correlates with the viral response at week 4 which is the strongest predictor of response. The combination of recipient IL28B genotype and donor age with the week 4 response reliably estimates the probability of SVR early on-treatment and may facilitate therapeutic strategies incorporating new antiviral agents.

Hepatitis C virus reinfection among prisoners with sustained virological response after treatment for chronic hepatitis C.

BACKGROUND & AIMS: We estimated HCV reinfection rate and its associated risk factors in inmates with chronic hepatitis C who had achieved sustained virological response (SVR) after completing combination therapy while in prison. METHODS: Individuals who had achieved an SVR after treatment provided from January 2003 to December 2009 at four prisons in Catalonia, had been tested annually for HCV RNA and were in prison during 2010, were invited to complete a questionnaire regarding risk factors for reinfection. Incidence rate was calculated as 100 person-years of follow-up. Risk factors potentially associated with reinfection were evaluated by bivariate log-rank test and multivariate Cox regression analysis. RESULTS: One hundred and nineteen subjects who had achieved an SVR agreed to participate. 98% were male, with a median age of 33.3 ± 6.3 years and 81% had a history of injection drug use (IDU). After a mean follow-up of 1.4 years, HCV reinfection was identified in nine former IDUs, seven with HCV genotype switch, for an overall reinfection rate of 5.27 cases per 100 person-years. Reinfection incidence was significantly higher among active drug users (HR=12.47; 95% CI: 2.90-53.71), HIV co-infected (HR=9.95; 95% CI: 1.73-57.34), and those engaging in more than one risk behaviors after treatment (HR=7.47; 95% CI: 1.19-46.89). CONCLUSIONS: HCV reinfection among inmates after successful treatment is high especially in those with ongoing IDU. Preventative interventions at diagnosis and during and after HCV treatment should be strongly reinforced.

IL28B genetic variation and hepatitis C virus-specific CD4(+) T-cell responses in anti-HCV-positive blood donors.

Epidemiological, viral and host factors are associated with the outcome of hepatitis C virus (HCV) infection, and strong host immune responses against HCV favour viral clearance. Recently, genome-wide association studies have shown a strong correlation between single-nucleotide polymorphisms (SNPs) near the interleukin-28B (IL28B) gene and spontaneous or treatment-induced HCV clearance. We have investigated whether protective IL28B genetic variants are associated with HCV-specific T-cell responses among Spanish blood donors. The rs12979860 IL28B haplotype was determined in 69 anti-HCV-positive blood donors (21 HCV RNA negative and 48 HCV RNA positive) and 30 seronegative donors. In all cases, HCV-specific CD4(+) T-cell responses to HCV recombinant proteins (core, NS3 and NS3 helicase) were assessed by ex vivo interferon-γ ELISpot assay. The rs12979860-CC genotype was highly overrepresented in donors with spontaneous HCV clearance when compared to those with chronic infection (76.2%vs 29.2%, P < 0.001; odds ratio, 7.77; 95% confidence interval, 2.4-25.3, P < 0.001). HCV-specific CD4(+) T-cell responses were detected in 16 (76.2%) spontaneous resolvers especially towards nonstructural proteins, but with no correlation with IL28B genotype. Chronic individuals had a significantly lower overall T-cell response again irrespective of IL28B genotype. When spontaneous resolvers and chronic individuals were stratified according to their IL28B genotype, significantly stronger T-cell responses were only observed among those with non-CC haplotypes. Although the protective rs12979860 IL28B CC genotype is associated with spontaneous HCV clearance, stronger CD4(+) T-cell responses towards NS3 were only evident among those with non-CC haplotypes.

Reversal of nonstructural protein 3-specific CD4(+) T cell dysfunction in patients with persistent hepatitis C virus infection.

Hepatitis C virus (HCV)-specific T cell responses are essential for HCV control, and chronic infection is characterized by functionally altered antigen-specific T cells. It has been proposed that the early inactivation of specific CD4(+) T cell responses may be involved in establishment of HCV persistence. We have investigated whether HCV-specific CD4(+) T cells dysfunction can be reversed in vitro. Nonstructural protein 3 (NS3) and core-specific CD4(+) T cells from eight chronically infected and eight spontaneously resolved HCV individuals were selected through transient CD154 (CD40 ligand) expression, and their functional profile (IFN-γ, IL-2, TNF-α, IL-10 and IL-4 production by enzyme-linked immunospot assay, cytometric bead array and intracellular cytokine staining, and proliferation by carboxy-fluorescein diacetate succinimidyl ester dilution assay) was determined both ex vivo and after in vitro expansion of sorted CD154-expressing cells in the absence of specific antigen in IL-7/IL-15-supplemented medium. Ex vivo bulk CD4(+) T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11%vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-γ. Antigen-free in vitro expansion of NS3-specific CD154(+) cells from chronic patients restored IFN-γ and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4(+) T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.

Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in healthy cattle, sheep and swine herds in Northern Spain.

Three-hundred and forty-five herds (17 swine, 122 dairy sheep, 124 beef and 82 dairy cattle) were investigated for prevalence of Shiga toxin-producing Escherichia coli (STEC). Rectal faecal samples were selectively enriched and then examined by immunodetection techniques (Immunomagnetic Separation with anti-E. coli O157 Dynabeads, ImmunoMagnetic cell Separation (IMS) and automated enzyme-linked fluorescent immunoassay using VIDAS) and polymerase chain reaction (PCR) (rfbE and fliC genes) to assess the prevalence of E. coli O157:H7. Prevalence of non-O157 STEC was estimated by PCR screening for stx genes of 10 lactose-positive colonies grown on MacConkey agar after enrichment. PCR was used on all STEC isolates to detect stx(1), stx(2), eaeA and E-hlyA genes. Both immunodetection methods showed a moderate-good level of agreement (kappa = 0.649) but IMS showed 87.5% complementary sensitivity. Prevalence of positive herds for E. coli O157:H7 was estimated at 8.7% for sheep and 3.8% for cattle, whereas all the porcine herds tested negative. Non-O157 STEC were also absent from swine, but were isolated more frequently from ovine (50.8%) than bovine herds (35.9%). Within-herd prevalences of excretion of E. coli O157:H7 established by individual testing of 279 sheep (six herds) and 30 beef cattle (one herd) were 7.3% and 6.7% respectively. PCR analysis of 49 E. coli O157:H7 and 209 non-O157 isolates showed a different distribution of virulence genes. All E. coli O157:H7 were stx(2) gene-positive, eaeA was detected in 95.9%, and the toxigenic profile stx(2)/eaeA/E-hlyA was present in 75.5% of the isolates. Among the non-O157 STEC, prevalence of eaeA was significantly lower (5.3%) and E-hlyA was present in 50.2% of the isolates but only sporadically associated with eaeA. stx(2) was predominant in non-O157 isolates from cattle, whereas in sheep the combination stx(1)/stx(2) was more prevalent. This study demonstrated the wide distribution of STEC in ruminant herds, which represent an important reservoir for strains that pose a potential risk for human infections.

Prevalence and strain diversity of thermophilic campylobacters in cattle, sheep and swine farms.

AIMS: To determine prevalence and strain diversity of thermophilic campylobacters in healthy ruminants and swine. METHODS AND RESULTS: Faecal samples collected from 343 herds (120 sheep, 124 beef cattle, 82 dairy cattle and 17 swine) in the Basque Country were screened in pools for thermophilic campylobacters. Two hundred and three herds were positive (67.1% dairy cattle, 58.9% beef cattle, 55.0% sheep and 52.9% pig), and species-specific PCR identified Campylobacter jejuni in 20.7% of the herds and Campylobacter coli in 6.4%. Campylobacter coli was isolated from the four production systems and was the most prevalent species in swine, where C. jejuni was not found. Other thermophilic campylobacters were found in all production systems. Four hundred and ninety-three animals from 11 positive herds were individually analysed, detecting significantly higher within-herd prevalences in dairy cattle (66.7%) and swine (57.8%) than in sheep (8.8%) or beef cattle (5.4%). flaA PCR-RFLP and pulsed-field gel electrophoresis analysis of a selection of isolates showed high genetic diversity. CONCLUSIONS: Healthy swine, cattle and sheep are important reservoirs of thermophilic campylobacters of different species and high genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient farm-based intervention measures are needed to reduce risk of infection. Non-C. jejuni/C. coli species should be monitored to investigate their significance for infection.

Long-term follow-up of the hepatitis C HENCORE cohort: response to therapy and occurrence of liver-related complications.

The aims of the study were to verify the long-term effect of time on viral clearance in hepatitis C virus (HCV) patients and to find out factors possibly associated with disease progression. A total of 1641 patients recruited from eight European centres in 1996-1997 were re-analysed 5-7 years after inclusion. The occurrence of decompensated cirrhosis, hepatocellular carcinoma (HCC) and liver transplantation was analysed in relation to different host and viral factors. Ninety-three per cent of the HCV patients who had cleared the virus (spontaneously or after antiviral therapy) remained HCV-RNA-negative during follow up and may be considered as 'cured'. Among patients who were sustained responders at inclusion, 2.3% developed liver complications during follow up, and 31% of non-responders did. Advanced age at infection and presence of the human leucocyte antigen (HLA) DRB1*1201-3 allele were possibly associated with a higher rate of progression to decompensated cirrhosis or HCC. Decompensated cirrhosis might be further associated with male gender, non-response to previous therapy, and lack of HLA DRB1*1301 allele, whereas HCC seems to be associated with the presence of the HLA DQ02 allele. Long-term follow up of HCV patients indicates that virological response persists over time and is associated with a very low incidence of liver complications. Advanced age at inclusion, advanced age at infection, viral genotype 1, non-response to previous therapy and possibly some specific HLA alleles are factors independently associated with a faster rate of progression towards liver complications. The large proportion of patients lost to follow up stresses the need for a strengthened and optimized management of HCV patients.

Peginterferon alpha-2b plus ribavirin vs interferon alpha-2b plus ribavirin for chronic hepatitis C in HIV-coinfected patients.

Treatment of chronic hepatitis C in human immunodeficiency virus (HIV)-infected patients is associated with low response rates and high incidence of side effects. One hundred twenty-one hepatitis C virus (HCV)-HIV-coinfected patients were randomized to receive interferon alpha-2b (3 MU thrice weekly; n = 61) or peginterferon alpha-2b (1.5 microg/kg/week; n = 60), plus ribavirin (800 mg daily), for 24 (genotype 2 or 3) or 48 weeks (genotype 1 or 4). We assessed early virological response at 4, 8 and 12 weeks to predict sustained virological response (SVR). Safety assessment included frequent blood lactate measurement and relative quantitation of mitochondrial DNA (mtDNA) content in peripheral blood mononuclear cells. In intention-to-treat analysis, the SVR rate was higher in the peginterferon group (55%vs 26%; P = 0.002). The difference for HCV genotypes 1 and 4 was 45%vs 14% (P = 0.009) and 50%vs 27% (P = 0.387), respectively, and for genotype 2 or 3, 71%vs 43% (P = 0.12) Viral response at 4, 8 and 12 weeks of treatment was highly predictive of SVR. Among genotype 3 patients, 17 of 20 (85%) whose HCV RNA was already undetectable at 4 weeks had an SVR after 24 weeks of treatment. Hyperlactataemia occurred in 22 patients and was clinically significant in six, two of whom died. mtDNA decreased significantly 4-12 weeks after the start of treatment in patients developing clinically significant hyperlactataemia. Peginterferon alpha-2b plus ribavirin was more effective than interferon alpha-2b plus ribavirin in HIV-coinfected patients. Frequent monitoring of virological response may be very helpful to optimize treatment compliance, to tailor treatment duration and to minimize side effects.

Hepatotoxicity of antiretroviral drugs in HIV HCV patients with congenital coagulopathies followed at an Haemophilia Unit during a decade.

The aim of the study was to assess the incidence and the cumulative probability of cytolytic and cholestatic hepatotoxicity during antiretroviral treatment in a group of HIV HCV haemophiliacs. We evaluated 47 patients that received 246 courses of antiretroviral treatment [98 courses of pre-highly active antiretroviral therapy (pre-HAART) and 148 HAART treatments]. Liver function tests were assessed at baseline of each treatment, after 1 month and at least every 4 months thereafter. Cytolytic and cholestatic hepatotoxicity was recorded. Of the 246 treatments, 28 (12.45%) were followed by cytolytic hepatotoxicity and 32 (13%) by cholestatic hepatotoxicity. Cytolytic hepatotoxicity was similar in HAART (16/148; 10.8%) and in pre-HAART treatment (12/98; 12.2%) and cholestatic hepatotoxicity was more frequent in HAART (29/148; 19.6%) than in pre-HAART treatment (3/98; 3.1%) (P < 0.001). The actuarial probability of developing cytolytic and cholestatic hepatotoxicity at 10 years of onset of antiretroviral treatments was 39% and 56%, respectively. Most enzyme elevations were asymptomatic, but in eight cases therapy was discontinued or changed and in one case a cirrhotic patient died of progressive liver failure. In HIV HCV haemophiliacs, the cumulative probability of developing hepatotoxicity during follow-up is high and although in the most cases the toxicity is mild, fatal cases can occur.

Serological evidence of Leptospira interrogans serovar Bratislava infection and its association with abortions in cattle in northern Spain.

A cross-sectional study was carried out in the Basque Country of Spain to determine the seroprevalence of 10 Leptospira serovars in a population of dairy cattle with poor fertility, and a case-control study was carried out in another northern area to investigate the role of Leptospira interrogans serovar Bratislava in abortions. L. Bratislava was the most prevalent serovar in the cross-sectional study, with 25.4 per cent of the cows testing positive in the microagglutination test when a cut-off of 1:10 or higher was applied, followed by Leptospira Hardjo (8.2 per cent), Leptospira Pomona (7.7 per cent), Leptospira Autumnalis (0.7 per cent) and Leptospira Copenhageni (0.1 per cent). In the case-control study the seroprevalence of L. Bratislava was significantly higher among the cows which had aborted when a titre of 1:300 or more was used as a cut-off (9.7 per cent v 3.4 per cent, P=0.008); 69 per cent of the L. Bratislava-infected cows that had aborted apparently aborted as a result of the infection.

Analysis of INF-gamma, TNF-alpha and dendritic cells to predict hepatitis C virus recurrence in liver transplant patients.

INTRODUCTION: Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease and the reason for more than 50% of liver transplantations (OLT). Recurrent HCV infection occurs in almost all transplant recipients and has an unfavorable course. Although immunosuppressive agents are necessary to avoid allograft rejection, these drugs may favor viral replication facilitating viral-mediated graft injury. METHODS: To predict the evolution of two HCV(+) patients who underwent OLT, we studied INF-gamma and TNF-alpha production and the maturation capacity of dendritic cells (DCs) at three time points: before transplantation (Pre-Tx) and at 2 (2M) and 6 (6M) months after transplantation. Cytometric bead assays were used to quantify INF-gamma and TNF-alpha production in the supernates of mixed leukocyte reactions (MLR) between spleen cells from the liver donor and CD4(+) cells from the recipients. Immature and mature DCs were generated in vitro from patient monocytes. RESULTS: The one patient who experienced recurrent HCV showed loss of CD4(+) responses to donor antigens and INF-gamma and TNF-alpha production after OLT. In contrast, the other patient maintained detectable levels of these cytokines after OLT. It was possible to generate mature DCs from monocytes with the aid of CD40L in both cases, but decreased expression of HLA-DR, CD80, and CD86 markers was observed upon posttransplantation analyses in the patient with recurrent HCV. CONCLUSION: Loss of the proliferative response as well as INF-gamma and TNF-alpha production, together with a decreased HLA-DR, CD80, and CD86 (markers of mature DCs), indicated an inadequate immune response to viral progression in the liver transplant recipient with relapsing HCV infection.

Subtype mutations in the envelope 2 region including phosphorylation homology domain of hepatitis C virus do not predict effectiveness of antiviral therapy.

The aim of this study was to determine whether specific sequences of the phosphorylation homology domain (PePHD) region could be correlated with differences in response to antiviral therapy in patients infected with hepatitis C virus subtypes 1b, 2c, 3a and 4c/d. We included 43 patients (22 sustained responders and 21 nonresponders or relapsers) in the study, who were classified according to early viral decline during the first weeks of antiviral treatment and response at end of follow up. Type of mutations, mutation frequency, genetic diversity and phylogenetic relationships were compared at the PePHD and flanking regions. Phylogenetic trees showed that each sequence clustered together with those of the same subtype. Sequences from subtypes 1b and 4c/d resembled more closely the phosphorylation sites of protein kinase R and eIF2 alpha than sequences from genotypes 2c and 3a, the latter with higher response rates to interferon-alpha (IFN alpha) treatment. However, within specific subtypes, no separate clusters of responders and nonresponders were observed either at the beginning or at the end of follow up. We were not able to find any particular sequence or mutation in the PePHD region or in any other subregion of the fragment studied that allowed prediction of treatment response.

Early detection of nonresponse to interferon plus ribavirin combination treatment of chronic hepatitis C.

We have investigated the value of early hepatitis C virus (HCV) RNA decline (DeltaHCV RNA) to predict response to combination therapy in 66 chronic hepatitis C patients treated with IFN-alpha2b (3 MU thrice weekly) and ribavirin (800 mg daily) for 12 months [25 sustained responders (SR) and 41 nonresponders or relapsers (NR)]. Serum HCV RNA was retrospectively measured in samples obtained at baseline and 4, 8 and 12 weeks after treatment onset, using a commercially available quantitative RT-PCR assay. At 4 weeks, serum HCV RNA had decreased a mean of 2.6 +/- 0.8 logs among SR as compared with only 0.5 +/- 0.8 logs in NR (P < 0.001), and was already undetectable (< 600 IU/mL) in 12 (48%) of the SR but in none of the NR. At 8 weeks, HCV RNA was undetectable in 21 SR and in 2 NR and mean DeltaHCV RNA were 4.2 +/- 1.3 and 0.8 +/- 1.0 logs, respectively (P < 0.001). At week 12 all SR had undetectable HCV RNA as compared with only five NR (P < 0.001). Stepwise logistic regression analysis identified DeltaHCV RNA at 12 weeks as the strongest predictor of sustained response. Receiver operating characteristic (ROC) curves of DeltaHCV RNA for sustained response prediction identified sensitivity peaks with 100% negative predictive value corresponding to DeltaHCV RNA > 1 log at 4 weeks, > 2 logs at 8 weeks and > 3 logs at 12 weeks. Our results show that early changes in the HCV RNA level may reliably identify patients having no chance of a sustained virological response during the first 3 months of combination therapy, thus providing an excellent tool for optimizing antiviral treatment of chronic hepatitis C.

Longitudinal evaluation of the structure of replicating and circulating hepatitis C virus quasispecies in nonprogressive chronic hepatitis C patients.

In previous cross-sectional studies, we demonstrated that, in most patients with chronic hepatitis C, the composition and complexity of the circulating hepatitis C virus (HCV) population do not coincide with those of the virus replicating in the liver. In the subgroup of patients with similar complexities in both compartments, the ratio of quasispecies complexity in the liver to that in serum (liver/serum complexity ratio) of paired samples correlated with disease stage. In the present study we investigated the dynamic behavior of viral population parameters in consecutive paired liver and serum samples, obtained 3 to 6 years apart, from four chronic hepatitis C patients with persistently normal transaminases and stable liver histology. We sequenced 359 clones of a genomic fragment encompassing the E2(p7)-NS2 junction, in two consecutive liver-serum sample pairs from the four patients and in four intermediate serum samples from one of the patients. The results show that the liver/serum complexity ratio is not stable but rather fluctuates widely over time. Hence, the liver/serum complexity ratio does not identify a particular group of patients but a particular state of the infecting quasispecies. Phylogenetic analysis and signature mutation patterns showed that virtually all circulating sequences originated from sequences present in the liver specimens. The overall behavior of the circulating viral quasispecies appears to originate from changes in the relative replication kinetics of the large mutant spectrum present in the infected liver.

Interferon and ribavirin combination therapy for chronic hepatitis C in human immunodeficiency virus-infected patients with congenital coagulation disorders.

We have conducted an open, prospective trial to assess the safety and efficacy of interferon alfa-2b and ribavirin in combination for the treatment of chronic hepatitis C in human immunodeficiency virus (HIV)-infected hemophiliacs. Twenty hemophiliacs coinfected with HIV and hepatitis C virus (HCV), 18 of them under highly active antiretroviral therapy (HAART), with a mean CD4(+) cell count of 490 +/- 176 cells/mm(3) and undetectable (n = 9) or low-level HIV RNA (<10,000 copies/mL; n = 11), were treated with interferon-alfa2b (3 MU thrice weekly) and ribavirin (800 mg/d) for 6 or 12 months according to virologic response. Patients were monitored for tolerance and response at 4, 8, 12, 24, 36, and 48 weeks during treatment and every other month thereafter. All 20 patients enrolled completed at least 6 months of treatment with no major side effect requiring treatment withdrawal, dose reduction, or modification of HAART. Overall, 8 patients (40%) achieved a sustained virologic response at the end of the 6-month post-treatment follow-up. Sustained responders had lower baseline HCV-RNA levels (5.7 +/- 0.8 vs. 6.3 +/- 0.4 log10 IU/mL, P =.041) but were otherwise similar to nonresponders. All sustained responders had a decrease in HCV-RNA level of at least 1 log per month during the first 2 months and undetectable levels at 6 months. In conclusion, our results provide evidence that combination therapy with interferon and ribavirin is safe in HIV-infected hemophiliacs with stable CD4 cell count and undetectable or low-level HIV replication, and leads to eradication of HCV in 40% of these patients.

Evaluation of hepatitis C virus RNA RT/PCR qualitative and quantitative second generation assays.

Hepatitis C virus (HCV) RNA qualitative and quantitative second generation assays (Amplicor HCV v2.0 and Amplicor HCV Monitor v2.0, respectively) were evaluated by testing serum samples from 132 blood donors anti-HCV positive HCV RNA negative by first generation qualitative assay and 326 viremic patients. An HCV RNA transcript was synthesized and ten-fold dilutions were used to assess sensitivity. Second generation assays were one log more sensitive than their respective first generation tests (10(2) copies per ml vs. 10(3) for the qualitative tests; 10(3) copies per ml vs. 10(4) for the quantitative tests). From the 132 anti-HCV positive RNA negative subjects, 6 (5%) were positive by Amplicor v2.0. Quantification figures by Monitor v2.0 were similar in genotypes 1, 2 and 3, whereas Monitor 1.0 values were higher in genotype 1 than in genotype 2 or 3. In 114 patients, branched-DNA v2.0 obtained higher values than Monitor v2.0 and Monitor v1.0 (6.6+/-0.6 log RNA copies per ml, 6.4+/-0.6, and 5.3+/-0.7, respectively, P<0.001). HCV RNA qualitative and quantitative second generation assays are more sensitive and genotype independent than first generation assays.