Juan Ignacio Esteban

Transmission of Hepatitis C Virus by a Cardiac Surgeon

BACKGROUND In the course of a study conducted in 1992 through 1994 of the efficacy of screening blood donors for antibodies to hepatitis C virus (HCV), we found that two patients had acquired hepatitis C after cardiac surgery, with the transmission apparently unrelated to blood transfusions. Because their surgeon had chronic hepatitis C, we sought to determine whether he was transmitting the virus to his patients. METHODS Of 222 of the surgeons patients who participated in studies of post-transfusion hepatitis between 1988 and 1994, 6 contracted postoperative hepatitis C, despite the use of only seronegative blood for transfusions. All six patients had undergone valve-replacement surgery. Analyses were performed to compare nucleotide sequences encompassing the hypervariable region at the junction between the coding regions for envelope glycoproteins E1 and E2 in the surgeon, the patients, and 10 controls infected with the same HCV genotype. RESULTS The surgeon and five of the six patients with hepatitis C unrelated to transfusion were infected with HCV genotype 3; the sixth patient had genotype 1 and was considered to have been infected from another source. Thirteen other patients of the surgeon had transfusion-associated hepatitis C and were also infected with genotype 1. The average net genetic distance between the sequences from the five patients with HCV genotype 3 and those from the surgeon was 2.1 percent (range, 1.1 to 2.5 percent; P < 0.001), as compared with an average distance of 7.6 percent (range, 6.1 to 8.3 percent) between the sequences from the patients and those from the controls. The results of phylogenetic-tree analysis indicated a common epidemiologic origin of the viruses from the surgeon and the five patients. CONCLUSIONS Our findings provide evidence that a cardiac surgeon with chronic hepatitis C may have transmitted HCV to five of his patients during open-heart surgery.

Evaluation of Antibodies to Hepatitis C Virus in a Study of Transfusion-Associated Hepatitis

BACKGROUND The hepatitis C virus (HCV) is now known to be the chief cause of transfusion-associated non-A, non-B hepatitis, but the prevalence of HCV among blood donors and the frequency of transmission by blood transfusion are unknown. METHODS To assess the sensitivity and specificity of a test for antibody to HCV, we tested serum samples from participants in a large study of transfusion-associated hepatitis. Samples were obtained prospectively from consecutive adults undergoing open-heart surgery in Spain, but were tested retrospectively, after the antibody enzyme immunoassay for anti-HCV became available. RESULTS Of 280 transfusion recipients given a total of 1109 units of blood, 27 (9.6 percent) had transfusion-associated non-A, non-B hepatitis (mean follow-up, 52 weeks) and 24 of the 27 seroconverted to anti-HCV-positive, whereas only 2 (0.8 percent) of the remaining transfusion recipients seroconverted. Among the 1044 donor specimens available for testing, 16 (1.5 percent) had anti-HCV antibody. Only 1 additional seropositive donor was found when 44 implicated donors who had been seronegative were retested 9 to 12 months later. Of the 16 recipients of anti-HCV-positive blood, 14 (88 percent) had transfusion-associated hepatitis and seroconverted to anti-HCV-positive. The remaining two recipients had neither hepatitis nor anti-HCV antibody. Among 25 patients with non-A, non-B hepatitis for whom all transfused blood was tested, 14 had received blood positive for anti-HCV. CONCLUSIONS About 90 percent of blood donors with antibody to HCV have infectious virus in their blood. The screening of blood donors for anti-HCV antibody should prevent about half the cases of transfusion-associated hepatitis, but the donors with infectious virus who are anti-HCV-negative may remain seronegative for prolonged periods.

High rate of infectivity and liver disease in blood donors with antibodies to hepatitis C virus.

OBJECTIVE To determine the epidemiologic, clinical, serologic, and histologic importance of antibodies to hepatitis C virus (anti-HCV) in blood donors. DESIGN Cross-sectional identification and prospective evaluation of seropositive donors; retrospective assessment of infectivity; and nested case-control study for risk factors. SETTING Liver unit of a referral-based university hospital. SUBJECTS Of 30,231 consecutive donors, 368 (1.2%) were found to be anti-HCV-reactive by enzyme-linked immunosorbent assay (ELISA). Two hundred and fifty-four of these 368 donors were evaluated for risk factors by comparison with 284 age- and sex-matched controls. Eighty-six spouses of seropositive donors were also evaluated. MEASUREMENTS AND MAIN RESULTS Twenty-four percent of the seropositive donors had a history of percutaneous exposure to blood. This rate increased to 45% when only those donors confirmed to be anti-HCV positive by a second-generation recombinant immunoblot assay (RIBA-2) were considered. A family history of liver disease (odds ratio, 2.8; 95% Cl, 1.6 to 4.8), previous blood transfusion (odds ratio, 6.1; 95% Cl, 3 to 12.5), and a history of tattooing or intravenous drug abuse (odds ratio, 8.4; 95% Cl, 2.3 to 31) were associated with anti-HCV seropositivity. An elevated alanine aminotransferase (ALT) level was found in 58% of the seropositive donors. Of the 150 donors tested, 104 (69%; Cl, 62% to 77%) were confirmed by RIBA-2 to be anti-HCV positive. Of the 105 donors who had a biopsy, 16% had normal histologic findings, 11% had minimal changes, 21% had chronic persistent hepatitis, 45% had chronic active hepatitis, and 7% had active cirrhosis. All 77 donors with RIBA-2-confirmed seropositivity had histologic abnormalities. Of 43 donors evaluated in an infectivity study, 82% were implicated in previous HCV transmission. Only 2.3% of the spouses were anti-HCV positive. The ELISA, RIBA-2, and ALT results correlated with infectivity and abnormal histologic findings. CONCLUSIONS In our geographic area, almost 70% of donors who are anti-HCV positive by ELISA are confirmed to be positive by RIBA-2; most of these donors appear to be chronic carriers of HCV and have substantial liver disease.

Quality of life and cognitive function in hepatitis C at different stages of liver disease

BACKGROUND/AIMS Hepatitis C has been associated with a decrease in quality of life and with neurological abnormalities. The aim of our study was to investigate the relationship between quality of life and cognitive function. METHODS Quality of life, clinical variables and neuropsychological function were evaluated in 120 patients with hepatitis C (mild chronic hepatitis, compensated cirrhosis and decompensated cirrhosis) and in healthy controls (n=40, in each group). RESULTS Patients with chronic hepatitis or compensated cirrhosis showed a decrease in quality of life, in spite of unimpaired neuropsychological tests. Patients with decompensated cirrhosis exhibited a further decrease in quality of life and neuropsychological abnormalities. The decrease in quality of life was associated with the severity of liver failure, neuropsychological abnormalities and treatment with beta-blockers or diuretics. However, in the multivariable analysis, only treatment with beta-blockers or diuretics (which was limited to decompensated cirrhosis) was independently associated with quality of life. CONCLUSIONS Hepatitis C causes a decrease in quality of life even in the absence of major cognitive impairment. The mechanisms that worsen quality of life are unknown. However, in cirrhotic outpatients with prior decompensations, treatment with beta-blockers or diuretics appears to have an important effect on quality of life.

Nucleotide and Amino Acid Complexity of Hepatitis C Virus Quasispecies in Serum and Liver

ABSTRACT The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral replication. Several studies have tried to unravel, through the parameters that characterize HCV circulating quasispecies, prognostic markers of the disease. In a previous work we demonstrated that the parameters of circulating viral quasispecies do not always reflect those of the intrahepatic virus. Here, we have analyzed paired serum and liver quasispecies from 39 genotype 1b-infected patients with different degrees of liver damage, ranging from minimal changes to cirrhosis. Viral level was quantified by real-time reverse transcription-PCR, and viral heterogeneity was characterized through the cloning and sequencing of 540 HCV variants of a genomic fragment encompassing the E2-NS2 junction. Although in 95% of patients, serum and liver consensus HCV amino acid sequences were identical, quasispecies complexity varied considerably between the viruses isolated from each compartment. Patients with HCV quasispecies in serum more complex (26%) than, less complex (28%) than, or similarly complex (41%) to those in liver were found. Among the last, a significant correlation between fibrosis and all the parameters that measure the viral amino acid complexity was found. Correlation between fibrosis and serum viral load was found as well (R = 0.7). With regard to the origin of the differences in quasispecies complexity between serum and liver populations, sequence analysis argued against extrahepatic replication as a quantitatively important contributing factor and supported the idea of a differential effect or different selective forces on the virus depending on whether it is circulating in serum or replicating in the liver.

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

Summary.  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 μg/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.

High-Resolution Hepatitis C Virus Subtyping Using NS5B Deep Sequencing and Phylogeny, an Alternative to Current Methods

ABSTRACT Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.

Ultra-Deep Pyrosequencing (UDPS) Data Treatment to Study Amplicon HCV Minor Variants

We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.

Ultra-Deep Pyrosequencing Detects Conserved Genomic Sites and Quantifies Linkage of Drug-Resistant Amino Acid Changes in the Hepatitis B Virus Genome

Background Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region. Methodology/Principal Findings Serum samples obtained from chronic HBV-infected patients at pre-treatment and during sequential NA treatment with lamivudine, adefovir, and entecavir were analyzed by ultra-deep pyrosequencing (UDPS) using the GS-FLX platform (454 Life Sciences-Roche). The pre-treatment HBV quasispecies was not enriched with NA-resistant substitutions. The frequencies of this type of substitutions at pre-treatment did not predict the frequencies observed during lamivudine treatment. On linkage analysis of the RT region studied, NA-resistant HBV variants (except for rtA181T) were present in combinations of amino acid substitutions that increased in complexity after viral breakthrough to entecavir, at which time the combined variant rtL180M-S202G-M204V-V207I predominated. In the overlapping surface region, NA-resistant substitutions caused selection of stop codons in a significant percentage of sequences both at pre-treatment and during sequential treatment; the rtA181T substitution, related to sW172stop, predominated during treatment with lamivudine and adefovir. A highly conserved RT residue (rtL155), even more conserved than the essential residues in the RT catalytic motif YMDD, was identified in all samples. Conclusions UDPS methodology enabled quantification of HBV quasispecies variants, even those harboring complex combinations of amino acid changes. The high percentage of potentially defective genomes, especially in the surface region, suggests effective trans-complementation of these variants.

Response of hepatitis C virus to long-term passage in the presence of alpha interferon: multiple mutations and a common phenotype.

ABSTRACT Cell culture-produced hepatitis C virus (HCV) has been subjected to up to 100 serial passages in human hepatoma cells in the absence or presence of different doses of alpha interferon (IFN-α). Virus survival, genetic changes, fitness levels, and phenotypic traits have been examined. While high initial IFN-α doses (increasing from 1 to 4 IU/ml) did not allow HCV survival beyond passage 40, a gradual exposure (from 0.25 to 10 IU/ml) allowed the virus to survive for at least 100 passages. The virus passaged in the presence of IFN-α acquired IFN-α resistance as evidenced by enhanced progeny production and viral protein expression in an IFN-α environment. A partial IFN-α resistance was also noted in populations passaged in the absence of IFN-α. All lineages acquired adaptative mutations, and multiple, nonsynonymous mutations scattered throughout the genome were present in IFN-α-selected populations. Comparison of consensus sequences indicates a dominance of synonymous versus nonsynonymous substitutions. IFN-α-resistant populations displayed decreased sensitivity to a combination of IFN-α and ribavirin. A phenotypic trait common to all assayed viral populations is the ability to increase shutoff host cell protein synthesis, accentuated in infections with IFN-α-selected populations carried out in the presence of IFN-α. The trait was associated with enhanced phosphorylation of protein kinase R (PKR) and eIF2α, although other contributing factors are likely. The results suggest that multiple, independent mutational pathways can confer IFN-α resistance to HCV and might explain why no unified picture has been obtained regarding IFN-α resistance in vivo.