Viral dynamic modelling of Hepatitis C and resistance-associated variants in haemophiliacs.

AIM: Chronic hepatitis C virus (HCV) infection is an important source of morbidity and mortality among haemophiliacs. Limited data are available regarding treatment intervention using direct-acting antivirals (DAAs) and theoretical concerns regarding accumulation of drug-associated resistance variants (RAVs) remain. We conducted a pilot study of treatment with telaprevir/pegylated interferon-alfa/ribavirin to evaluate treatment response and the role of lead-in DAA therapy on mutational selection of resistance variants. METHODS: Ultra-deep sequence analysis was performed at baseline, 48 hours and 168 hours after treatment initiation. RESULTS: No dominant RAVs were identified at baseline, but low-level RAVs were noted at baseline in all subjects. Viral dynamic models were used to assess treatment responses. The efficacy parameter (Ɛ) for lead-in ranged from 0 to 0.9745 (mean = 0.514). Subsequent addition of telaprevir resulted in a mean efficacy of more than 0.999. This was comparable to subjects who started all three medications simultaneously. A total of 80% achieved SVR. While rapid shifts in the RAV population following DAA initiation were observed, treatment failure associated with A156V was observed in only one patient. Adverse event profiles were similar to that observed in non-haemophilia cohorts. There was no evidence of factor inhibitor formation. There was no evidence that lead-in provided benefit in terms of response efficacy. CONCLUSION: These data support DAA-based therapy in those with inherited bleeding disorders.

Hepatitis C virus NS3 mutations in haemophiliacs.

Haemophiliacs have high hepatitis C virus (HCV) exposure risk from blood products that did not undergo heat inactivation or disease-specific screening prior to 1987. Repeated exposure to infected factor concentrates predisposes haemophiliacs to higher likelihood of HCV from multiple sources. HIV coinfection could result in impaired clearance of less fit variants resulting in enrichment of quasispecies carrying resistance mutations. We postulated that haemophiliacs demonstrate increased prevalence of baseline signature mutations in the HCV NS3/4 serine protease coding domain. We examined the prevalence of putative HCV protease inhibitor mutations, mutations, subclassified into dominant mutations if changes conferred resistance, and minor variants not associated with drug resistance, in patients with haemophilia A or B, infected with HCV or HCV/HIV, prior to HCV PI exposure. A total of 151 subjects were evaluated, including 22 haemophiliacs and 129 non-haemophilic controls. Of the 58 mutations detected, 55 (95%) were resistance mutations and three (5%) were minor variants. Dominant mutations were detected in 10 (45.5%) haemophiliacs and in 43 (33.3%) controls (OR 1.67, 95% CI 0.67-4.16). There was no statistical difference in proportion of dominant mutations (P = 0.27) or minor variants (P = 0.47) between groups, despite adjustment for HIV status (P = 0.44). No significant differences in dominant or minor resistance mutations between haemophiliacs and non-haemophiliacs were observed. HIV presence or prior HAART exposure did not affect baseline distribution. We conclude that haemophiliacs are not at higher risk for pre-existing HCV PI mutations, and prospective studies of response to PI-based regimens with HCV activity are indicated.

Hepatitis E infection in HIV-infected liver and kidney transplant candidates.

Hepatitis E virus (HEV) has been reported to cause acute and chronic hepatitis in those with HIV infection and among solid organ transplant recipients in Europe. Limited data indicate that HEV is endemic in the United States, but the prevalence and significance of HEV infection among those with HIV and awaiting solid organ transplantation is unknown. We evaluated anti-HEV IgM and IgG antibodies and HEV RNA in 166 HIV-infected solid organ transplant candidates enrolled in the NIH HIV-Transplant Cohort. Overall prevalence of anti-HEV IgG approached 20% in both liver and renal transplant candidates. Evidence of recent infection was present in approximately 2% of liver transplant candidates and none of the kidney transplant candidates. HEV RNA was not detected in any patient. We conclude that markers of HEV infection are frequent among candidates for transplantation, but active, ongoing viremia is not seen. Evidence of recent infection (acute on chronic) liver disease was present in liver but not kidney recipients.

Mutations associated with occult hepatitis B virus infection result in decreased surface antigen expression in vitro.

Occult hepatitis B virus (HBV) infection is characterized by the absence of detectable hepatitis B surface antigen (HBsAg) in the serum, despite detectable HBV DNA. Investigations of the mechanisms underlying the development of occult HBV infection are lacking in the current literature, although viral mutations in the surface region, resulting in decreased HBsAg expression or secretion, represent one potential mechanism. Wild-type HBsAg expression vectors were constructed from genotype-matched chronic HBV sequences. Site-directed mutagenesis was then utilized to introduce three genotype A mutations - M103I, K122R and G145A - associated with occult HBV infection in vivo, alone and in combination, into the wild-type HBsAg vectors. Transfection of Huh7 and HepG2 cell lines was performed, and cell culture supernatants and cell lysates were collected over 7 days to assess the effects of these mutations on extracellular and intracellular HBsAg levels. The G145A mutation resulted in significantly decreased extracellular and intracellular HBsAg expression in vitro. The most pronounced reduction in HBsAg expression was observed when all three mutations were present. The mutations evaluated in vitro in the current study resulted in decreased HBsAg expression and potentially increased hepatic retention and/or decreased hepatic secretion of synthesized HBsAg, which could explain the lack of HBsAg detection that is characteristic of occult HBV infection in vivo.

Genomic variability associated with the presence of occult hepatitis B virus in HIV co-infected individuals.

Occult hepatitis B virus (O-HBV) infection is characterized by the presence of HBV DNA without detectable hepatitis B surface antigen (HBV DNA+/HBsAg-) in the serum. Although O-HBV is more prevalent during HBV/HIV co-infection, analysis of HBV mutations in co-infected patients is limited. In this preliminary study, HBV PreSurface (PreS) and surface (S) regions were amplified from 33 HIV-positive patient serum samples - 27 chronic HBV (C-HBV) and six O-HBV infections. HBV genotype was determined by phylogenetic analysis, while quasispecies diversity was quantified for the PreS, S and overlapping polymerase regions. C-HBV infections harboured genotypes A, D and G, compared to A, E, G and one mixed A/G infection for O-HBV. Interestingly, nonsynonymous-synonymous mutation values indicated positive immune selection in three regions for O-HBV vs one for C-HBV. Sequence analysis further identified new O-HBV mutations, in addition to several previously reported mutations within the HBsAg antigenic determinant. Several of these O-HBV mutations likely contribute to the lack of detectable HBsAg in O-HBV infection by interfering with detection in serologic assays, altering antigen secretion and/or decreasing replicative fitness.

Prevalence and genotypic variability of TTV in HIV-infected patients.

TT virus is a small, circular DNA virus, that has been associated with transfusion hepatitis. We sought to determine the prevalence of TT virus (TTV) in patients with human immunodeficiency virus (HIV) infection and to characterize the virus in terms of genotypic variability and in the relationship to CD4+, HIV viral loads, HCV/HIV coinfection, and ALT abnormalities. A cross-sectional analysis of HIV-infected patients in the United States, including 86 HIV-positive subjects and 118 HIV-negative controls was performed. TTV was detected using a seminested PCR technique. Samples underwent cloning and sequence analysis and/or RFLP to determine genotype. Thirty-eight percent of HIV-positive patients had TTV infection versus 14.4% of patients within the matching cohort (P = 0.0009). The highest rate of TTV infection was in patients with concurrent HCV/HIV infection (54% vs 30%, P = 0.038). HIV-infected subjects with TTV had lower ALT levels than those without TTV (P = 0.036). Intravenous drug use was the leading factor associated with TTV positivity among HIV-positive subjects. Mixed genotypes were more common in those with HIV. Therefore, TTV prevalence, ALT levels, and genomic heterogeneity of TTV all seem to be altered in patients with HIV.

Mechanism of indinavir-induced hyperbilirubinemia.

Indinavir is a viral protease inhibitor used for the treatment of HIV infection. Unconjugated hyperbilirubinemia develops in up to 25% of patients receiving indinavir, prompting drug discontinuation and further clinical evaluation in some instances. We postulated that this side-effect is due to indinavir-mediated impairment of bilirubin UDP-glucuronosyltransferase (UGT) activity and would be most pronounced in individuals with reduced hepatic enzyme levels, as occurs in approximately 10% of the population manifesting Gilbert's syndrome. This hypothesis was tested in vitro, in the Gunn rat model of UGT deficiency, and in HIV-infected patients with and without the Gilbert's polymorphism. Indinavir was found to competitively inhibit UGT enzymatic activity (K(I) = 183 microM) while concomitantly inducing hepatic bilirubin UGT mRNA and protein expression. Although oral indinavir increased plasma bilirubin levels in wild-type and heterozygous Gunn rats, the mean rise was significantly greater in the latter group of animals. Similarly, serum bilirubin increased by a mean of 0.34 mg/dl in indinavir-treated HIV patients lacking the Gilbert's polymorphism versus 1.45 mg/dl in those who were either heterozygous or homozygous for the mutant allele. Whereas saquinavir also competitively inhibits UGT activity, this drug has not been associated with hyperbilirubinemia, most likely because of the higher K(I) (360 microM) and substantially lower therapeutic levels as compared with indinavir. Taken together, these findings indicate that elevations in serum-unconjugated bilirubin associated with indinavir treatment result from direct inhibition of bilirubin-conjugating activity.

Hepatitis cRNA quasispecies complexity in patients with alcoholic liver disease.

Alcohol abuse is described as a major cofactor in the development of hepatitis C (HCV) associated liver disease and may play a role in the outcome of interferon-based treatment interventions. The association between HCV viral heterogeneity and alcohol has not been previously described. This study was designed to evaluate the quasispecies nature of the HCV population in patients with compensated and decompensated alcoholic liver disease, to test the hypothesis that alcoholics have greater complexity than matched nonalcoholic controls. A nonisotopic heteroduplex complexity assay (HCA) was first validated by comparison with results of quasispecies complexity determined by subcloning and sequencing of amplicon products from the E2/NS1 hypervariable coding region (HVR). Subsequently, this methodology was applied to comparison of 20 compensated (Child's-Pugh A) and decompensated (Child's-Pugh B/C) alcoholic and 20 nonalcoholic controls. The complexity of the HVR, as well as the 5' Untranslated (5'UT) and the NS5b coding domains were evaluated. The HCA methodology provided a reasonable semiquantitative measure of HCV RNA quasispecies variability compared with subclone sequence homology comparison. Overall, alcoholic patients had greater quasispecies complexity (2.65 bands) than nonalcoholic controls (1.6 bands; P =.01). Subset analysis revealed that compensated alcoholic patients had a mean of 3.1 homo/heteroduplex bands per sample whereas Child's-Pugh B/C alcoholics showed intermediate complexity. A similar quasispecies complexity difference was seen in the 5'UTR, but not in the NS5b coding domain. Quasispecies complexity was not associated with viral titer, complementary DNA concentration, or genotype. The differences in quasispecies complexity may help explain reports of poor interferon responsiveness in alcoholic patients.

Host response to mycobacterial infection in the alcoholic rat: male and female dimorphism.

Increased susceptibility to tuberculosis occurs in the alcoholic. One explanation for the altered susceptibility is a change in T-lymphocyte modulation. To evaluate this, 24 male and 24 female Sprague-Dawley rats were treated with either a Lieber-type liquid ethanol diet (LED) or an isocaloric control (LCD). After 2 weeks, half the subjects were infected with BCG (10(8) colony-forming units) and sacrificed after 42 days. Splenic helper (CD4) and suppressor/cytoxic (CD8) cells were quantitated by flow cytometry. By three-way analysis of variance, splenic cellularity was significantly increased by infection (p < 0.0001) but suppressed by LED (p = 0.0002). There was a marginal sexual difference (p = 0.065) with females exhibiting a 35% lower response while on alcohol. Examining lymphocyte subsets, the most significant changes were observed after infection (BCG) and alcohol treatment (LED). CD4 levels were diminished by LED (p = 0.0002) but markedly increased by infection (p < 0.0001), producing a highly significant interaction that affected both absolute number (p < 0.0001) and relative percent present (p = 0.0078). CD8 was influenced only by infection (p < 0.0001). This resulted in a infection-related increase in the CD4/CD8 ratio which was lower with LED (p = 0.0032). Splenic T-lymphocytes, predominately CD4, are involved in the host response to BCG hepatitis and are adversely influenced by LED, which may contribute to increased susceptibility.

Hepatitis C serotypes in nonalcoholic and alcoholic patients.

Genotyping of the hepatitis C virus (HCV) RNA can be performed by a variety of methods following polymerase chain reaction amplification of a stable RNA portion of the genome. The gold standard is amplification of the RNA from the NS5 region, followed by direct sequencing and homology comparison. This method is extremely labor intensive. In this study, we compared an immunoblot serotyping technique (HCV SIA) to a reverse-hybridization line-probe assay (LiPA) for genotype classification among non-alcoholic HCV infected patients. We then compared and contrasted the response in this cohort to a population of alcoholic patients with HCV infection. To validate the serotype assay, sera from 110 patients with chronic HCV infection was utilized. Serotyping (Chiron SIA) and genotyping by the LiPA (Line Probe Assay, Innogenetics) reverse-hybridization technique was performed. Additionally, both methods were compared to sequence-derived genotyping in 26 patients based on PCR amplification of the NS5 region. After the validation phase, sera from 105 alcoholic patients was genotypically classified by the serologic method. The nonalcoholic and alcoholic groups were then compared with regard to serotype, demographics, and frequency of untypable test results. Among typable pairs, the overall concordance rate between serotyping and LiPA-based genotyping was 93.75%. Patients with genotype 1 by reverse hybridization demonstrated a 95.8% concordance with serotype. Untypable samples were present for both techniques, but since they occurred in different patients, the techniques were complementary. Alcoholic patients were significantly more likely to be infected with untypable serotypes than those without a pattern of alcohol abuse. These patients were also more likely to be HCV RNA negative than sera from typable patients. Serotype 1 was associated with high HCV RNA titer and poor interferon treatment response among both nonalcoholic and alcoholic patients. An immunoblot method for the evaluation of genotype classification was rapid and easily performed compared to sequence-based genotyping. There was a high degree of concordance compared to reverse-hybridization and sequence-based genotype characterization methods. Failure to detect HCV RNA in the serum is associated with a higher likelihood of classification failure. This problem was particularly prevalent in the alcoholic population. HCV RNA titers and treatment outcomes were strongly associated with serotype classification results, demonstrating clinical utility of this assay technique.

Biphasic in vivo immune function after low- versus high-dose alcohol consumption.

A series of experiments was performed to assess the alterations in immune status in vivo that are associated with differences in the amount and duration of ethanol intake. Using a nonspecific delayed cutaneous hypersensitivity-like response to the intradermal injection of phytohemagglutinin, the area of induration (skin test response) was significantly enhanced (p = 0.008) after low-dose ethanol (0.5 g/kg) administered daily by gastric gavage for 5 days. High-dose ethanol (6.0 g/kg) significantly diminished this response (p = 0.03). Using an experimental model of Mycobacterium bovis hepatitis, the host immune response was also altered in a biphasic manner after chronic, 28-day ethanol consumption. With this model 0.43 +/- 0.03 g/kg/day (mean +/- SEM) of ethanol (low dose) was associated with a 40% improvement in the removal of the organisms from liver tissue (p = 0.002). High dose (12.1 +/- 0.5 g/kg/day) impaired removal, resulting in a 55% increase in the number of viable organisms (p = 0.001). The levels of three cytokines, MIF, TNF-alpha, and IL-2, known to be involved in the modulation of the host response to mycobacterial infections, were measured in sera after the infection. The serum levels of these cytokines in response to infection did not correlate with this biphasic response to different alcohol dose levels.

Human hepatocyte growth factor in alcoholic liver disease: a comparison with change in alpha-fetoprotein. Department of Veterans Affairs Cooperative Study Group 275.

To evaluate the hepatic regenerative response in patients with alcoholic liver disease, sera from 263 patients with severe alcoholic hepatitis and/or cirrhosis were analyzed for hepatocyte growth factor (HGF) and alpha-fetoprotein (AFP). HGF concentration was elevated above healthy controls in 95% of the patients (median level = 2.4 ng/ml), whereas AFP tended to be depressed below controls (median level = 4.1 ng/ml). Correlations with parameters of liver injury (i.e., ascites, encephalopathy, AST bilirubin, and protime) all showed a more significant correlation with HGF concentrations than those of AFP. Patients with HGF levels below the mean (4 ng/ml) exhibited significantly better survival (median survival = 35 months vs. 8.5 months for those with HGF > or = 4 ng/ml; p = 0.007). Serum HGF levels were associated with various specific histologic features of alcoholic hepatitis that included, but were not exclusively related to, necrosis.

The impact of age on alcohol toxicity in the rat.

This study was performed to determine whether the severity of chronic alcohol toxicity is altered by age and duration of drinking. Alcohol as 35% of calorie intake (ED treatment) was administered to Sprague-Dawley rats at predetermined ages beginning at 1, 6, 12, 18, 24 and 27 months for a duration of treatment varying from 1 to 3 months. The degree of injury was compared to controls (CD treatment) of comparable age and duration of treatment. ED was associated with significantly higher serum levels of AST, total bilirubin and alkaline phosphatase (P < 0.0001 for each test) without detectable differences due to age and duration of treatment. Liver triglycerides (as a measure of alcoholic fatty steatosis) were significantly increased by ED (P < 0.0001) and influenced by both age and duration of treatment. The greatest toxicity was observed in young animals. ED treatment beginning at 1 month of age was associated with an AST level 69% above CD and liver triglycerides 463% above CD; beginning at 18 months of age, ED produced an increase of 24% in AST and 175% in liver triglycerides. The hepatic regenerative capacity, as measured by 3H-thymidine uptake into nuclear DNA, was similarly affected by both ED and age. Regeneration was significantly higher in youth. ED produced a 62% increase above CD at 1 month compared to an 11% increase beginning at 18 months of age. These observations suggest that juveniles develop more severe injury from alcohol but that a greater regenerative capacity exists in youth. This may explain the observed clinical relationship between age and prognosis seen in patients with severe alcoholic liver injury.

Impact of chronic alcoholism on the aging rat: changes in nutrition, liver composition, and mortality.

The adverse effects of chronic alcohol consumption (mean 6.68 g/kg/d) were assessed in 150 male Sprague-Dawley rats over their life span (25 months). Evaluations were performed at 2, 3, 8, 13, 19, and 25 months of age for changes in nutrition status, biochemical tests for liver injury, compositional changes in liver, and hepatic regenerative capacity. In spite of nearly identical caloric intake, alcohol treatment was associated with nutritional levels 10-30% lower than controls. Maximal changes were observed at the two extremes of ages (2-3 months and 19-25 months). Hence, a nutritional contribution to other adverse changes could not be excluded. Fatty compositional increases (triglycerides) occurred early (5-fold increases after 1 month of treatment) then declined to levels only slightly above controls. Biochemical tests on sera for liver injury (AST and total bilirubin) were consistently higher with alcohol treatment. Regenerative capacity measured by [3H]thymidine uptake after partial hepatectomy was initially elevated in the alcoholic then rapidly declined beyond 7 months of age. In control animals, an age-related decline was also observed but occurred later beyond 12 months of age. Consistent with these adverse effects, ethanol diet survival was poorer than the pair-fed control groups by 15% (median survival for alcoholics, 17 months vs. 20 months in controls.

Biochemical and clinical effects of aspartame in patients with chronic, stable alcoholic liver disease.

Aspartame is an artificial sweetener completely metabolized in the gut and absorbed as aspartate, phenylalanine, and methanol. Phenylalanine is thought to mediate or exacerbate hepatic encephalopathy, and an impaired liver may not be able to cope with the ammoniagenic properties of the amino acid constituents, or adequately metabolize methanol. Thus, we compared the clinical and biochemical effects of a single ingestion of aspartame (15 mg/kg) to skim milk (phenylalanine content equimolar to aspartame) and placebo in patients with chronic, alcoholic liver disease in a randomized, crossover study. Aspartame produced an elevation of plasma phenylalanine significantly greater than milk and placebo (Cmax 14.55 +/- 7.38, 10.95 +/- 4.95, 8.84 +/- 4.55 mumol/dl, respectively; p < 0.01). However, quantified encephalopathic changes were observed only with milk (p < 0.05). Plasma aspartate, methanol, formate, and ammonia levels remained unchanged after all treatments. The lack of clinical derangements in encephalopathic indices, methanol accumulation, or biochemical changes in liver status suggests that a single large dose of aspartame (representing 5 times the average daily intake of adults) may be used safely by patients with chronic, stable liver disease.

Antibodies to hepatitis B virus and hepatitis C virus in alcoholic hepatitis and cirrhosis: their prevalence and clinical relevance. The VA Cooperative Study Group (No. 119)

Patients with overt alcoholic liver disease who had participated in a multicenter therapeutic trial and subgroups of controls (i.e., alcoholic patients without liver disease and patients with neither alcoholism nor liver disease) were tested for hepatitis B virus and hepatitis C virus antibodies to determine the prevalence of these antibodies to determine the prevalence of these antibodies and any clinical association in the progression and outcome of alcoholic liver disease. Antibodies to hepatitis B (anti-HBs and/or anti-HBc) were found in 29.2% of patients with alcoholic liver disease, in 26.1% of hospitalized alcoholic patients without liver disease and in 24.2% of hospitalized nonalcoholic patients without liver disease; frequencies were not significantly different from one another. HBsAg was not evaluated because HBsAg+ patients had been excluded from the original trial. The presence of these antibody markers correlated with ethnic origin of and immunoglobulin levels in the patients. In contrast, antibody to hepatitis C, as detected by enzyme immunoassay, was positive in 27.1%, 4.8% and 3.0% of the three groups, respectively, the first differing significantly from the other two. Antibody to hepatitis C virus positivity correlated significantly with clinical severity of the disease and with the presence of histological features that imply chronic viral infection (periportal inflammation, cirrhosis), despite the fact that the supplementary assay for antibody to hepatitis C virus, using recombinant immunoblot assay, reduced the positive rate by 79%.(ABSTRACT TRUNCATED AT 250 WORDS)

Host response to mycobacterial infection in the alcoholic rat.

Animals, chronically treated with alcohol, were inoculated with mycobacteria (bacillus Calmette-Guérin, 10.2 x 10(6) organisms) into the spleen to produce a granulomatous hepatitis. Before infection, chronic alcohol ingestion was associated with a depressed skin test response to phytohemagglutinin, 71.7% of baseline (P = 0.009). Mycobacterial (bacillus Calmette-Guérin) infection stimulated phytohemagglutinin skin test response to 417% of baseline in controls and 299% in alcoholics (P less than 0.001). The hepatic granuloma response was altered with smaller but more numerous granulomas (mean +/- SEM, 81.2 +/- 1.5 microns2 of area with a frequency of 1.8 granulomas per field in alcoholics vs. 129.8 +/- 5.71 microns2 and 1.2 granulomas per field in controls; P less than 0.001). These changes were associated with a 10-fold increase in colony-forming units per gram of liver (54.5 +/- 18.2 in alcoholics vs. 5.6 +/- 1.83 in controls; P = 0.0006). This model offers precise parameters for host response to infection and indicates that alcohol significantly impairs the clearing capacity for mycobacteria from the liver.