Phylogenetic analysis in the clinical risk management of an outbreak of hepatitis C virus infection among transfused thalassaemia patients in Italy.

BACKGROUND: Occurrence of hepatitis C virus (HCV) infection is reduced by effective risk management procedures, but patient-to-patient transmission continues to be reported in healthcare settings. AIM: To report the use of phylogenetic analysis in the clinical risk management of an HCV outbreak among 128 thalassaemia outpatients followed at a thalassaemia centre of an Italian hospital. METHODS: Epidemiological investigation and root-cause analysis were performed. All patients with acute hepatitis and known chronic infection were tested for HCV RNA, HCV genotyping, and NS3, NS5A, and NS5B HCV genomic region sequencing. To identify transmission clusters, phylogenetic trees were built for each gene employing Bayesian methods. FINDINGS: All patients with acute hepatitis were infected with HCV genotype 1b. Root-cause analysis, including a lookback procedure, excluded blood donors as the source of HCV transmission. The phylogenetic analysis, conducted on seven patients with acute infection and eight patients with chronic infection, highlighted four transmission clusters including at least one patient with chronic and one patient with acute HCV infection. All patients in the same cluster received a blood transfusion during the same day. Two patients with acute hepatitis spontaneously cleared HCV within four weeks and nine patients received ledipasvir plus sofosbuvir for six weeks, all achieving a sustained virological response. CONCLUSION: Combined use of root-cause analysis and molecular epidemiology was effective in ascertaining the origin of the HCV outbreak. Antiviral therapy avoided the chronic progression of the infection and further spread in care units and in the family environment.

HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels.

OBJECTIVES: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen. METHODS: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays. RESULTS: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11). CONCLUSIONS: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

A recent epidemiological cluster of acute hepatitis B genotype F1b infection in a restricted geographical area of Italy.

In this study, by phylogenetic analysis, we identified an epidemiological cluster involving eight individuals diagnosed with acute hepatitis B virus (HBV) infection related to unprotected sexual intercourse in a restricted area of central Italy (time period: 2011-2014). Notably, these patients (six of eight Italians) were infected by subgenotype F1b, which is not commonly found in western countries. Ultra-deep pyrosequencing confirmed a superimposable composition of HBV quasi-species in these patients. Despite the availability of effective vaccination, this study highlights the importance of not underestimating the risk of HBV infection, of continuing to set up surveillance programmes for HBV infection, and of investigating the pathogenetic potential of these atypical genotypes.