The study included patients treated at the liver disease outpatient clinics of the Santa Casa de Misericórdia do Pará Foundation and the João de Barros Barreto University Hospital. Consecutive patients with chronic HCV were included. The HCV group consisted of 101 patients with chronic hepatitis C, characterized by clinical changes, abnormal liver tests and HCV RNA positivity.
The inclusion criteria adopted for the individuals were as follows: age 18 or older, both sexes, and positivity for HBsAg for more than 6 months or positivity for HCV RNA. Individuals coinfected with hepatitis B virus (HBV), hepatitis delta virus, or human immunodeficiency virus (HIV) were excluded from the study.
A control group was formed to compare the genotypic and allelic frequencies of the investigated polymorphisms, which included 300 blood samples from volunteer donors from the Foundation Center for Hemotherapy and Hematology of Pará (Fundação Centro de Hemoterapia e Hematologia do Pará). These volunteers were matched with the patient group for age and sex; were seronegative for HCV, HBV, HIV-1, human T-lymphotropic virus 1/2; and patients who used or were using specific antiviral therapy against HCV.
DNA was extracted from peripheral-blood leukocytes using the Puregene kit (Gentra Systems, Minneapolis, Minnesota, USA) according to the manufacturer's protocol. The procedure included the steps of cell lysis, protein precipitation, DNA precipitation, and DNA hydration.
Polymorphisms were genotyped by quantitative real-time polymerase chain reaction in the StepOne PLUS Sequence Detector (Applied Biosystems, Foster City, CA, USA). The assay used for each polymorphism contained a pair of primers and a pair of VIC- and FAM-labeled probes for the respective alleles. For both polymorphisms, predesigned and customized TaqMan® SNP Genotyping Assays were used: C_7514879_10 for TNFA -308G>A and C_1747360_10 for IL10 -1082A>G (Thermo Fisher, Carlsbad, California, USA). For each reaction, 2X TaqMan® Universal PCR Master Mix, 1X TaqMan® Assay (diluted from 20X), and 20 ng of DNA was used in a final reaction volume of 10 µL. The following temperature cycle was used for the amplification: 60 °C for 30 seconds, 95 °C for 10 minutes, and 50 cycles of 92 °C for 30 seconds and 60 °C for 1 minute and 30 seconds.
All selected patients were clinically evaluated and subjected to a complimentary investigation consisting of hematological, biochemical, serological, virological, and ultrasound examinations. These data were transcribed from the medical records into a form developed specifically for this study.
Liver biopsy specimens were obtained only from patients with medical indications for the investigation of liver parenchyma changes, in compliance with the clinical care protocol. The liver biopsies were performed by a medical professional from one of the study hospitals using a Tru-Cut needle under ultrasound guidance. The sample was sent to the Department of Pathological Anatomy of Federal University of Pará, where they were examined following the department’s routines, which included hematoxylin–eosin (HE), chromotrope aniline blue (CAB), Gomori’s reticulin, and Shikata’s orcein staining.
The histopathological diagnosis followed the METAVIR classification , which classifies the activity of the portal and periportal inflammatory infiltrate from 0 to 3 (A0-A3), A0-A1 indicating absent to mild inflammation and A2-A3 indicating moderate to severe inflammation. The structural changes in the liver parenchyma (degree of fibrosis) were classified from 0 to 4 (F0-F4), F0-F1 indicating absent to mild liver fibrosis, F2 indicating moderate liver fibrosis, and F3-F4 indicating liver fibrosis that has progressed to cirrhosis. All data regarding the histopathological profile were obtained from the patients’ medical records.
Hardy-Weinberg equilibrium analysis was performed in all samples through the chi-squared test. Comparative analyses of the allelic and genotypic frequencies were done using the G-test and chi-squared test. Comparisons of viral load levels (HCV RNA) were done using the Kruskal-Wallis test and the Mann-Whitney test. Statistical analyses were done with BioEstat software version 5.3 , adopting a significance level of p< 0.05.