We enrolled patients with HCV-related chronic liver disease attending the Infectious Diseases Unit of the A.O.U. Federico II of Naples, from January 2017 to March 2018, treated with DAAs. Fibrosis was evaluated by the mean of Fibroscan ®. The severity of liver disease was graduated according to the degree of liver fibrosis by Metavir stage or clinical signs. The Metavir score was estimated with a FibroScan© exam performed within 6 months before the beginning of the antiviral treatment. Moreover, clinical cirrhosis was identified according to the presence of clinical, biochemical and ultrasound signs including a blood platelet count lower than 100,000/mm3, hypertrophy of the caudate lobe, nodularity of the liver surfaces, altered straightness of hepatic veins, ascites, porto-systemic encephalopathy, esophageal varices and ultrasound evidence characterizing liver cirrhosis. Decompensated cirrhosis was defined as a cirrhosis in Child-Pugh stage of at least B7, while advanced liver fibrosis was defined as the presence of Metavir score ≥ F4 or clinical cirrhosis [21].
For each patient, a fecal sample was collected within 1–2 weeks before starting therapy and by SVR12 time. Subjects with the following inclusion and exclusion criteria were enrolled:
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Inclusion criteria: positivity for HCV-RNA; HBsAg negativity; anti-HIV negativity; age > 18 years; intention to participate in the study and signature of the informed consent.
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Exclusion criteria: age < 18 years; coinfection with HBV or HIV; inability to understand or sign informed consent; any other condition that in the opinion of the experienced person could make the patient unsuitable for enrollment or that could interfere with the patient's participation in the study and its completion; use of any antibiotic, antifungal or probiotic in the past 90 days; BMI (Body Mass Index) > 30.
The enrolled subjects were equipped with a special kit for the collection of faecal samples, together with the instructions for sampling. The storage of these samples was carried out at -80 ° C until the time of analysis. The collection, transport and storage were carried out following the Standard Operating Procedures (SOP) defined by the International Human Microbiome Standard Consortium (IHMC SOP 04).
Microbial Dna Extraction, 16s Rrna Gene Sequencing And Data Analysis
Fecal microbial DNA was extracted using the procedures outlined by the IHMC SOP 07 at the Department of Agricultural Sciences of the University of Naples Federico II. Faecal microbiota was analysed by sequencing of the V3-V4 regions of the 16S ribosomal RNA gene, with a procedure and primers previously described [22, 23]. Amplicons were purified, indexed and sequenced on a MiSeq Illumina platform according to the Illumina 16S metagenomic sequencing library preparation protocol, yielding to 2x250-bp, paired-end reads.
Demultiplexed, forward, and reverse reads were joined by using FLASH [24]. Joined reads were quality trimmed (Phred score > 20) and short reads (< 250 bp) were discarded by using Prinseq [25]. High-quality reads were then imported into QIIME 1.9 [26]. Operational taxonomic units (OTU) were picked at 97% of similarity using a de novo approach and the uclust method, and taxonomic assignment was obtained by using the RDP classifier and the Greengenes database [23], following a pipeline previously reported [23]. In order to avoid biases due to the different sequencing depth, OTU tables were rarefied to the lowest number of sequences per sample. The α-diversity indices were calculated by QIIME.
The taxonomy tables were imported into the R software (http://www.r-project.org/) for statistica analyses and visualization. Non-parametric tests (Wilcoxon-Mann-Whitney test, Kruskal-Wallis non-parametric ANOVA) were used to evaluate differences in diversity indices or in specific taxa abundance in subjects grouped according to available categorical variables. All significance values were adjusted to take into account the effect of multiple comparisons, using the Benjamini-Hochberg correction and a False Discovery Rate value < 0.05 was considered statistically significant.
The study was conducted in compliance with the Declaration of Helsinki and the principles of good clinical practice. For this study informed consent was obtained from all subjects and/or their legal guardian(s). The study was approved by the "Federico II" Ethics Committee.