Circulating microRNAs associated with liver fibrosis in chronic hepatitis C patients.

A major challenge in hepatitis C research is the detection of early potential for progressive liver disease. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and can be biomarkers of pathological processes. In this study, we compared circulating miRNAs identified in hepatitis C virus (HCV)-infected patients presenting two extremes of liver disease: mild/moderate fibrosis and cirrhosis. The patients in the cirrhosis group subsequently developed hepatocellular carcinoma (HCC). We identified 163 mature miRNAs in the mild/moderate fibrosis group and 171 in the cirrhosis group, with 144 in common to both groups. Differential expression analysis revealed 5 upregulated miRNAs and 2 downregulated miRNAs in the cirrhosis group relative to the mild/moderate fibrosis group. Functional analyses of regulatory networks (target gene and miRNA) identified gene categories involved in cell cycle biological processes and metabolic pathways related to cell cycle, cancer, and apoptosis. These results suggest that the differentially expressed circulating miRNAs observed in this work (miR-215-5p, miR-483-5p, miR-193b-3p, miR-34a-5p, miR-885-5p, miR-26b-5p and miR -197-3p) may be candidates for biomarkers in the prognosis of liver disease.

Prevalence and predictive factors of moderate/severe liver steatosis in chronic hepatitis C (CHC) infected patients evaluated with controlled attenuation parameter (CAP).

A novel controlled attenuation parameter (CAP) using FibroScan® has been developed for assessment of liver steatosis. The aim was to evaluate the frequency and associated factors for moderate/severe steatosis evaluated by CAP in CHC patients submitted to transient elastography (TE) by FibroScan® . CHC patients underwent TE with CAP evaluation. The classification of steatosis was defined as: CAP < 222 dB/m  =  S0; CAP ≥ 222 dB/m and <233dB/m  =  S1; ≥233 dB/m < 290dB/m  =  S2 and >= 290 dB/m  =  S3. The prevalence of moderate/severe steatosis (CAP ≥ S2) and the related independent factors were identified by a logistic regression analysis. A significance level of 5% was adopted. 1104 CHC patients, 85% genotype-1 were included (mean age 55 ± 11 years; 46% male, mean BMI 25 ± 4 Kg/m2 ). Systemic arterial hypertension and type 2 diabetes mellitus prevalences were 39% and 17%, respectively. Liver stiffness measurement ≥ 9.5 kPa was observed in 39% of patients and steatosis was identified in 50% (S1 = 7%, S2 = 28% and S3 = 15%). The variables independently associated with moderate/severe steatosis were: male gender (OR=1.35; P = .037; 95% CI:1.01-1.81); systemic arterial hypertension (OR=1.57; P = .002; 95% CI:1.17-2.10) and BMI (OR=1.17; P < .01;95% CI:1.12-1.22). In conclusion, when CAP was adopted as a tool to detect steatosis, genotype 1 CHC patients presented a high prevalence of moderate/advanced steatosis. In these patients, liver steatosis was associated mostly to metabolic factors (arterial hypertension and high BMI).

Lower levels of dehydroepiandrosterone sulfate are associated with more advanced liver fibrosis in chronic hepatitis C.

Chronic infection with the hepatitis C virus induces liver fibrosis, but it is unknown why some patients progress to advanced fibrosis while others remain with mild disease. Recently, an inverse association between serum levels of dehydroepiandrosterone sulphate (DHEA-S) and liver fibrosis in patients with nonalcoholic fatty liver disease was described, and it was postulated that dehydroepiandrosterone (DHEA) has antifibrotic effects. Our aim was to compare serum DHEA-S levels with liver fibrosis in hepatitis C patients. We collected serum samples from hepatitis C patients at the same day they underwent a liver biopsy. S-DHEA was compared to different stages of fibrosis. Binary logistic regression models were applied to evaluate independent variables associated to fibrosis. We included 287 patients (43.9% male). According to fibrosis stages 0, 1, 2, 3 and 4, median serum DHEA-S levels were 103 (26-462), 73 (5-391), 46 (4-425), 35 (6-292) and 28 (2-115) µg/dL, respectively (P < .001). Median serum DHEA-S levels were 74 (5-462) vs 36 (2-425) µg/dL for mild (F0-1) vs significant (F2-4) fibrosis, respectively (P < .001). Median serum DHEA-S levels were 64 (4-462) vs 31 (2-292) µg/dL for non advanced (F0-2) vs advanced fibrosis (F3-4), respectively (P < .001). The same association was found when the subgroup of HCV patients with and without steatosis or steatohepatitis was analysed. The association between lower DHEA-S levels and advanced fibrosis was independent of age, gender, diabetes mellitus, obesity and steatosis. Lower circulating DHEA-S levels are associated with more advanced stages of liver fibrosis in hepatitis C patients.

Comparison of oral fluid collection methods for the molecular detection of hepatitis B virus.

BACKGROUND: This study aims to compare the efficiency of four oral fluid collection methods (Salivette, FTA Card, spitting and DNA-Sal) to detect HBV DNA by qualitative PCR. MATERIALS AND METHODS: Seventy-four individuals (32 HBV reactive and 42 with no HBV markers) donated serum and oral fluid. In-house qualitative PCR to detect HBV was used for both samples and commercial quantitative PCR for serum. RESULTS: HBV DNA was detected in all serum samples from HBV-infected individuals, and it was not detected in control group. HBV DNA from HBV group was detected in 17 samples collected with Salivette device, 16 samples collected by FTA Card device, 16 samples collected from spitting and 13 samples collected by DNA-Sal device. Samples that corresponded to a higher viral load in their paired serum sample could be detected using all oral fluid collection methods, but Salivette collection device yielded the largest numbers of positive samples and had a wide range of viral load that was detected. CONCLUSION: It was possible to detect HBV DNA using all devices tested, but higher number of positive samples was observed when samples were collected using Salivette device, which shows high concordance to viral load observed in the paired serum samples.

Simultaneous detection of hepatitis C virus antigen and antibodies in dried blood spots.

BACKGROUND: Enzyme immunoassays (EIA) designed to detect hepatitis C virus (HCV) core antigen and anti-HCV antibodies (HCV AgAb) simultaneously can improve the early detection of HCV infection when molecular diagnostic methods are not widely available. OBJECTIVES: To evaluate the suitability of dried blood spot (DBS) samples for detecting HCV AgAb using commercial EIAs. STUDY DESIGN: Paired serum and DBS samples were assayed using two commercial EIAs for HCV AgAb (Monolisa™ HCV AgAb ULTRA and Murex HCV AgAb). Manufacturer's recommendations were followed for sera while sample volume, incubation time and cut-off (CO) determination were evaluated for the DBS samples. The values of sensitivity, specificity, inter-rater agreement, detection limit, assay precision and stability of DBS samples at different conditions (22-26°C, 2-8°C and -20°C) were determined. RESULTS: It was necessary to increase the DBS sample volume fourfold compared to the sera samples to approximate the DBS Optical Density (OD) values to the sera OD values. Using ROC curve to recalculate CO values for the DBS samples, sensitivity was 97.5% for both EIAs, while the specificity was 99.71% for Monolisa™ HCV AgAb ULTRA and 95.95% for Murex HCV AgAb. Accurate testing results were obtained with DBS samples for 60 days at all conditions evaluated; storage at -20°C resulted in low OD variation. Both EIAs demonstrated the same limit of detection among DBS samples [estimated viral load of 3.1 International Units per millilitre (IU/mL)] and low OD value variability in repetitivity and reproducibility studies. CONCLUSION: DBS samples can be used for the detection of HCV AgAb by EIA as they present comparable performance characteristics and excellent stability among various storage conditions.

Dried blood spot samples: optimization of commercial EIAs for hepatitis C antibody detection and stability under different storage conditions.

This study was undertaken to optimize and compare the efficiency of two commercial EIAs for anti-HCV detection (HCV Ab Radim, Pomezzia, Italy and ETI-AB-HCVK-4 DiaSorin, Vercelli, Italy), in dried blood spot (DBS) samples. The long-term stability of anti-HCV on DBS samples stored at three environmental conditions was also evaluated at: 2-8 °C, 20-25 °C, and -20 °C. Paired DBS and serum samples were obtained from individuals with or without anti-HCV. The type of elution buffer, sample and conjugate volume, sample incubation time and cut-off values were evaluated. For both EIAs, a larger sample volume was used, and the cut-off value determined by the manufacturer was employed for Radim EIA; however, ROC curve analysis was used for the DiaSorin EIA. The sensitivity and specificity of Radim EIA on DBS were 97.5% and 99.5%, respectively, and of DiaSorin EIA were 88.9% and 98.9%, respectively. Accurate results were obtained for a period of 117 days using DBS samples stored at all storage conditions, but storage at -20 °C resulted in the lowest variation among the absorbance values. Both EIAs demonstrated the same limit of detection (until dilution of 1:10(4) with estimated viral load of 3.1 × 10(-1) UI/ml), but the Radim EIA was associated with the best performance because a low coefficient of variation was observed in the repetition and reproducibility studies. In conclusion, commercial EIAs can be optimized for anti-HCV detection in DBS samples that are extremely stable at different conditions for more than 100 days.

An evaluation of different saliva collection methods for detection of antibodies against hepatitis C virus (anti-HCV).

BACKGROUND: Saliva samples can be used as an alternative fluid for against hepatitis C virus (anti-HCV) detection owing to the ease of collection and excellent acceptability. This study was conducted to optimize a commercial enzyme immunoassay (EIA) to detect anti-HCV in saliva samples. METHODS: Ninety-six individuals donated paired serum and saliva samples that were obtained, using a commercial device (Salivette) and spitting into a sterile container. Initially, elution buffer for the Salivette samples, sample volume, incubation time and temperature, and two different anti-HCV EIAs were evaluated. Using the optimized assay, three methods for cut-off calculation were also evaluated. RESULTS: A 20-fold increase in the sample volume for both collection methods was needed. Moreover, the Radim assay was the most appropriate assay for anti-HCV detection in saliva samples, and the quality parameters were increased when a ROC curve was used to determine the cut-off value. Using this optimized assay, the sensitivities, specificities, accuracies, positive and negative predictive values were above 90% for saliva obtained using both the Salivette and spitting methods. Using this assay, discordant false-negative results were obtained for only two Salivette samples and five spitting samples. The concordance kappa was 93% for the Salivette method and 86.1% for the spitting method, demonstrating excellent performance. CONCLUSIONS: Saliva samples obtained for both methods can be employed for anti-HCV detection among HCV-infected or HCV-suspected cases, but several modifications must be performed on commercial EIAs to obtain good results. Moreover, samples obtained with commercial devices are more appropriate for anti-HCV detection in saliva samples.

The incidence of GB virus C / hepatitis G virus infection in Brazilian patients who received blood transfusion during cardiac surgery.

The incidence of GBV-C/hepatitis G virus (GBV-C/HGV) infection after blood transfusion is unknown in Brazil. Many studies have so far addressed its relationship with blood transfusion, but its association with liver disease was not confirmed. A prospective study was carried out between 1996 and 1999 in Rio de Janeiro. Ninety three patients who received blood transfusion during cardiac surgery were followed for six months and blood samples were drawn before and after surgery to determine antibodies to GBV-C/hepatitis G virus (anti-HGenv) using a step sandwich immunoassay and GBV-C/HGV-RNA using reverse transcriptase polymerase chain reaction. The alanine aminotransferase (ALT) levels were serially determined as well as clinical data compiled related to hepatitis. Prior to surgery, anti-HGenv was present in 35.5% (33/93) of patients and 4.3%(4/93) were found to be viremic. Seroconversion following transfusion was observed in 9 patients and 4 additional individuals became viremic for a total incidence of 23% (13/56). Six months after blood transfusion, only 4 of those nine patients previously antibody positive still had anti-HGenv detectable in serum. No patients had clinical or laboratory evidence of acute hepatitis and no correlation was found with GBV-C/HGV infection and number of blood units transfused (p = 0.37). This study highlights the importance of using both HGV-RNA PCR and anti-HGenv to accurately estimate the magnitude of GBV-C/HGV infection. The observed high prevalence and incidence rates show that this infection is common in Brazil; however, no clinical or biochemical evidence of liver disease was demonstrated in the period of study and longer longitudinal observation is needed to define any pathogenic effect.