Association between thrombotic risk factors and extent of fibrosis in patients with non-alcoholic fatty liver diseases

AIM: To evaluate the prevalence of genetic and acquired prothrombotic risk factors and their association with the extent of fibrosis and fatty infiltration in patients with non-alcoholic fatty liver disease (NAFLD).METHODS: Forty-four patients with chronic hepatitis (28 men and 16 women, with mean age of 45±11 and 49±12 years, respectively) constituted the patient population of this study. The groups were divided as follows: 15 patients with fatty liver (FL); 15 with non-alcoholic steatohepatitis (NASH); 14 with chronic viral hepatitis (CH) diagnosed by histology and liver technetium scan or ultrasound; and 10 healthy individuals. Thrombophilic, coagulation factors and genetic mutations were diagnosed by standard hemostatic and molecular coagulation assays.RESULTS: Activated protein C (APC) resistance and protein S were the most prevalent thrombotic risk factors (6% and 10% in NAFLD vs 21% and 14% in CH; P＜0.01 and P＜0.05, respectively). One thrombotic risk factor was identified in 41% of patients (23% mild fibrosis, 18% severe fibrosis) and two thrombotic risk factors in 6% of patients with NAFLD and severe fibrosis. While no differences in APC ratio, lupus anticoagulant, fibrinogen, factor V Leiden,prothrombin, and MTHFR mutation were found. Protein S levels were significantly lower in NASH patients than in patients with FL alone (92±19 vs106±2, P＜0.01). Protein C levels were markedly higher in patients with NAFLD and mild or severe fibrosis as compared to the patients with CH, respectively (128±40 vs96±14, P＜0.001 or 129±36 vs 88±13, P＜0.01).CONCLUSION: Up to 46% of patients with NAFLD may have thrombotic risk factors, and the presence of thrombotic risk factors is correlated with the extent of hepatic fibrosis,suggesting a crucial role of the coagulation system in the pathogenesis of hepatic fibrosis.

Molecular epidemiology and putative origin of hepatitis C virus in random volunteers from Argentina

AIM:To study the subtype prevalence and the phylogenetic relatedness of hepatitis C virus(HCV)sequences obtained from the Argentine general population,a large cohort of individuals was analyzed.METHODS:Healthy Argentinian volunteers(n=6251)from 12 provinces representing all geographical regions of the country were studied.All parents or legal guardians of individuals younger than 18 years provided informed written consent for participation.The corresponding written permission from all municipal authorities was obtained from each city or town where subjects were to be included.HCV RNA reverse transcription-polymerase chain reaction products were sequenced and phylogenetically analyzed.The 5’untranslated region(5’UTR)was used for RNA detection and initial genotype classification.The NS5B polymerase region,encompassing nt 8262-8610,was used for subtyping.RESULTS:An unexpectedly low prevalence of HCV infection in the general population(0.32%)was observed.Our data contrasted with previous studies that reported rates ranging from 1.5%to 2.5%,mainly performed in selected populations of blood donors or vulnerable groups.The latter values are in keeping with the prevalence reported by the 2007 Argentinian HCV Consensus(approximately 2%).HCV subtypes weredistributed as follows:1a(25%),1b(25%),2c(25%),3a(5%),and 2j(5%).Two isolates ascribed either to genotype 1(5%)or to genotype 3(5%)by 5’UTR phylogenetic analysis could not be subtyped.Subtype 1a sequences comprised a highly homogeneous population and clustered with United States sequences.Genotype1b sequences represented a heterogeneous population,suggesting that this genotype might have been introduced from different sources.Most subtype 2c sequences clustered close to the 2c reported from Italy and Southern France.CONCLUSION:HCV has a low prevalence of 0.32%in the studied general population of Argentina.The pattern of HCV introduction and transmission in Argentina appears to be a consequence of multiple events and different for each subtype.

New real-time-PCR method to identify single point mutations in hepatitis C virus

AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80 K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. Light Cycler methods, based on real-time PCR with sequencespecific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10%(mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80 K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b in our setting. CONCLUSION A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.

ACE Score Identifies HBeAg-negative Inactive Carriers at a Single-point Evaluation,Regardless of HBV Genotype

Background and Aims:Hepatitis B virus(HBV)biomark-ers have been used for a better categorization of patients,even though the lack of simple algorithms and the impact of genotypes limit their application.Our aim was to assess the usefulness of noninvasive markers for the identification of HBV inactive carriers(ICs)in a single-point evaluation and to design a predictive model for their identification.Meth-ods:This retrospective-prospective study included 343 consecutive HBeAg-negative individuals.Clinical,analytical,and virological data were collected,and a liver biopsy was performed if needed.Subjects were classified at the end of follow-up as ICs,chronic hepatitis B and gray zone.A pre-dictive model was constructed,and validated by 1000-boot-strap samples.Results:After 39 months of follow-up,298 subjects were ICs,36 were chronic hepatitis B CHB,and nine were gray zone.Eighty-nine(25.9%)individuals re-quired a liver biopsy.Baseline HBV DNA hazard ratio(HR)6.0,p<0.001),HBV core-related antigen(HBcrAg)(HR 6.5,p<0.001),and elastography(HR 4.6,p<0.001)were inde-pendently associated with the IC stage.The ACE score(HBV DNA,HBcrAg,elastography),obtained by bootstrapping,yielded an area under the receiver operating characteris-tics(AUROC)of 0.925(95%CI:0.880-0.970,p<0.001)for identification of ICs.The AUROC for genotype D was 0.95,0.96 for A,0.90 for E,and 0.88 for H/F.An ACE score of<1 had a positive predictive value of 99.5%,and a score≤12 points had a diagnostic accuracy of 93.8%.Conclusions:Low baseline HBV DNA,HBcrAg,and liver stiffness were in-dependently associated with the IC phase.A score including those variables identified ICs at a single-point evaluation,and might be applied to implement less intensive follow-up strategies.