Hepatitis C Late Relapse in Patients With Directly Acting Antiviral-related Sustained Virological Response at Week 12

Mariantonietta Pisaturo; Carmine Minichini; Mario Starace; Mara Caroprese; Margherita Macera; Giuseppina Brancaccio; Stefania De Pascalis; Antonella Santonicola; Alfonso Galeota Lanza; Rosa Zampino; Gaetano Cotticelli; Evangelista Sagnelli; Giovanni Battista Gaeta; Nicola Coppola


Liver International. 2019;39(5):844-853. 

In This Article

Patients and Methods


All 129 consecutive HCV patients with a virological failure to an IFN-free regimen observed at the laboratory of infectious diseases of the University of Campania Luigi Vanvitelli, Naples, from October 2015 to October 2017 were enrolled.

All patients had been treated with a DAA regimen according to the HCV genotype, international guidelines and local availability in 19 clinical centres in Campania, southern Italy. These centres had cooperated in several clinical investigations using the same clinical approach.[13,14] In the study period, these 19 centres treated by DAA regimen 3259 anti-HIV negative subjects, with virological failure in 129. At the time of failure, the 129 failed patients were referred to the laboratory of infectious diseases of the University of Campania Luigi Vanvitelli for a genotype resistance test for a resistance-associated substitution (RAS) in the NS3, NS5A and NS5B HCV regions. At the time of enrolment (at failure to DAA regimen), the subjects filled in a questionnaire on their demographic data, risk factors for parenteral acquisition of infection and clinical and therapeutic history.

Before the start of the DAA regimen, the patients underwent a complete physical examination, full liver function tests, α-foetoprotein, viral markers (HBV, HCV, hepatitis delta virus-HDV and human immunodeficiency virus-HIV) and liver ultrasound scan. The risk factors for the acquisition of parenteral infections were evaluated by the anamnesis. The indication for antiviral therapy and the choice of the IFN-free regimen were made by the physician according to the international guidelines[10] and local availability. The dose of the different DAAs and the duration of treatment were chosen according to the international guidelines.[10] Sofosbuvir plus ribavirin for the patients with genotype 1 and 3, dasabuvir+paritaprevir-ritonavir+ombitasvir for those with genotype 2 or 3, and simeprevir plus daclatasvir were defined as suboptimal or not effective. Sofosbuvir plus daclatasvir or ledipasvir for all genotypes and dasabuvir+paritaprevir-ritonavir+ombitasvir for those with genotype 1 were defined as optimal.

All the patients were followed up as outpatients at week 4 and at the end of DAA treatment, and then at week 4, 12, 24, 48, 72 and 96 after stopping the treatment. The patients who became HCV-RNA-negative at month 3 after stopping treatment were considered as having an SVR. The patients with initial HCV-RNA negativization and a subsequent recurrence of HCV-RNA during DAA treatment were defined patients with a viral breakthrough. The patients who did not clear HCV-RNA during treatment were defined non-responders. Patients were defined relapsers when, after HCV-RNA negativization during treatment, they showed a virological reactivation by week 12 once treatment was discontinued. Finally, the patients were defined as having a late relapse when, after HCV-RNA negativization during treatment and at week 12 after stopping DAA treatment, they showed a virological reactivation after week 12. Risk factors for HCV re-infection were defined as the presence of active intravenous drug use, homosexuality, history of recent surgery (from 12 weeks before the start of the DAA regimen to the last observation) or imprisonment.

The patients with initial cirrhosis were diagnosed on the basis of a liver biopsy showing a fibrosis score of F4 according to the METAVIR score, or F5 or F6 according to Ishak, or, if not performed, a Fibroscan score of more than 12.4 kPa or on the basis of the presence of unequivocal clinical, biochemical and ultrasound signs including a blood platelet count lower than 100 000/mm3 associated with one or more of the following: ascites, portosystemic encephalopathy, oesophageal varices and ultrasound evidence characterizing cirrhosis.

Samples of serum were obtained from each patient at the time of enrolment, that is after the virological failure, and stored at −80°C until used for this investigation.

All patients provided written informed consent for the collection and storage of biological samples and for the anonymous use of their data in clinical research, in accordance with the international guidelines and with the Helsinki Declaration of 1975 and revised in 1983. The Ethics Committee of the Azienda Ospedaliera Universitaria of the Second University of Naples, now University of Campania, approved the study (1538/2015).


The anti-HCV antibody was sought using a 3rd generation commercial immunoenzymatic assay (Ortho Diagnostic Systems, Neckargemund, Germany). Liver biochemistry and routine analyses were performed by routine methods in a Cobas Modular 6000 automated analyzer using c501 biochemistry modules (Roche Diagnostics Ltd, Rotkreuz, Switzerland).

Viral RNA was extracted from 140 μL of plasma samples using a microspin column (QIAamp RNA viral kit, Qiagen GmbH, Hilden, Germany). HCV-RNA was quantified by performing a real-time polymerase chain reaction (PCR) in a Light cycler 1.5 (Roche Diagnostics, Branchburg, NJ, USA), as reported in a previous paper;[16] by this method, the detection limit in plasma samples is estimated at around 40 IU/mL. HCV genotypes were determined by HCV genotype Lipa assay (Bayer, France), following the manufacturer's instructions.

NS3, NS5A and NS5B Sequencing Methods

On the serum collected at virological failure, HCV genotype was re-evaluated by Sanger sequencing of NS5B, and RASs in NS3, NS5A and NS5B were sought.

Sanger sequencing of NS3, NS5A and NS5B was performed applying home-made protocols for all patients enrolled, as previously reported.[17] Briefly, the NS3 (181aa), NS5A (140aa) and NS5B (565aa) regions were amplified and sequenced from stored serum/plasma samples using ABI 3500 Genetic Analyzer (Applied Biosystems Inc, Foster City, CA, USA). The sequences were submitted to biotechnology analysis by comparison with reference sequences recognized in the literature and taken from Los Alamos database and other databases (GenBank accession numbers: HCV-1a:H77 NC_004102, HCV-1b: D90208). The phylogenetic trees were made using the Mega 6 program, and by comparison with the reference sequence, it was possible to establish the genotypes and possible contaminations of the samples. The mutations and quasispecies were identified by SeqScape program software (Applied Biosystems) with a >20% tolerance for improper sequencing. Finally, we interpreted the resistance mutations based on the reference sources, as previously reported.[17]

In the patients with a late relapse for whom also a serum sample collected before the DAA-failed regimen was available, NS3, NS5A and NS5B phylogenetic trees, which also included HCV-1b reference sequences, were performed. Bootstrap analysis with 1000 replicates was performed to assess the significance of the nodes; values greater than 70% were considered significant. A genotype 1a sequence (1a_NC004102_US) was used as the outgroup.

Statistical Analysis

We used the Kolmogorov-Smirnov test to check for a Gaussian distribution of quantitative variables. In cases of a Gaussian distribution, we used for comparisons the Student t test for unpaired variables. In cases of a non-Gaussian distribution, we used for comparisons the Mann-Whitney U test. The chi-square test (or Fisher's exact test) was used for categorical variables. A P-value < 0.05 was considered to be statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences version 18.0 (SPSS, Chicago, IL, USA).