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Abstract

Background

This real-world clinical setting study characterized the virological patterns in genotype-1 patients failing interferon (IFN)-free regimens and evaluated the efficacy of re-treatment.

Methods

A total of 73 consecutive patients failing IFN-free regimens were enrolled (17 genotype-1a and 56 −1b). At failure Sanger sequencing of NS3, NS5A and NS5B regions was performed by home-made protocols.

Results

In patients having failed an NS3 inhibitor, the prevalence of NS3-RASs was higher in the 10 with genotype-1a than in the 24 with genotype-1b (80% versus 41.6%). In patients treated with an NS5A inhibitor, the prevalence of NS5A-RASs was very high in the 14 with genotype-1a and the 27 with genotype-1b (78.6% and 92.5%, respectively). In patients having failed sofosbuvir, the prevalence of NS5B-RASs was more frequently identified in the 45 with genotype-1b than in the 10 with genotype-1a (37.7% versus 10%). The prevalence of NS5B-RASs in patients having failed dasabuvir was high in both genotypes, 66.6% in the 6 with genotype-1a and 45.5% in the 11 with genotype-1b.

The 6 patients re-treated with genotype-1a less frequently (50%) showed sustained virological response (SVR) than the 18 with genotype-1b (88.8%; P=0.07). SVR was more frequent in the 21 patients with an effective second-line direct-acting antiviral (DAA) regimen than the 3 without (90.4% versus 0%; P<0.005).

Conclusions

The prevalence of RASs was high in our real-world population. NS3, NS5A and NS5B sequencing seems mandatory in the choice of DAA re-treatment.

Introduction

HCV infection affects more than 70 million people worldwide. In the past, pegylated interferon-alpha (PEG-IFN) plus ribavirin (RBV) were the standard treatment for HCV infection. This therapy provided a sustained clearance of HCV (sustained virological response [SVR]) in about 50% of the patients infected with HCV genotype-1, the most frequent HCV genotype in Western countries, in almost 70% of those with HCV genotype-2 and around 60% of those with HCV genotype-3. Moreover, the frequency and severity of the side effects (flu-like symptoms, depression, cytopenia and haemolytic anaemia) made this therapy arduous for many patients [1–5].

The treatment of HCV infection has been revolutionized by the recent development of potent direct-acting antivirals (DAAs). IFN-free treatment with DAAs provides excellent chances for sustained HCV elimination and can prevent the progression of liver disease [6,7].

In spite of the excellent efficacy of DAAs, some patients (around 5%) still fail to eradicate HCV. The failure to DAA was associated with the emergence of resistance-associated substitutions (RASs) within the viral quasispecies [8], and many questions about their prevalence and relevance in re-treatment remain unanswered [9]. Genotype-1 is the most frequent HCV genotype in Italy and worldwide. Few data are available in the literature on the virological characteristics and efficacy of re-treatment of HCV genotype-1 patients with a failure to a DAA regimen.

In this paper the authors analyse the virological patterns and RASs in the HCV genotype-1 patients failing an IFN-free regimen enrolled in a real-world clinical setting. In addition, the authors analysed the efficacy of re-treatment in terms of SVR.

Methods

All 73 consecutive HCV genotype-1 patients failing an IFN-free regimen were enrolled at the Laboratory of Infectious Diseases of the University of Campania Luigi Vanvitelli, Naples from October 2015 to September 2017. All patients had been followed in 19 clinical centres in Campania, southern Italy and had been treated with a DAA regimen according to the HCV genotype, international guidelines and local availability. These centres had cooperated in several clinical investigations using the same clinical approach [10–12].

At failure, these patients were referred to the laboratory of infectious diseases of the University of Campania Luigi Vanvitelli for a genotype resistance test for RASs in the NS3, NS5A and NS5B HCV regions.

The patients who were HCV-RNA-negative at week 12 after stopping treatment were defined as having an SVR. Patients that showed a virological reactivation once treatment was discontinued were defined as relapsers. The patients who did not obtain HCV RNA negativity during treatment were defined as non-responders. The patients were defined as having a viral breakthrough when, after an initial HCV-RNA-negative result, they had a subsequent recurrence of HCV RNA during DAA treatment. The patients with initial liver 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, porto-systemic encephalopathy, esophageal varices and ultrasound evidence characterizing liver cirrhosis.

From each patient at the time of enrolment (at failure to DAA regimen), a serum sample was obtained and stored at −80°C until used for this investigation.

For all the failed patients, the physician had the data on the presence of RASs in NS3, NS5A and NS5B regions. However, the choice of the second-line DAA regimen was made by the physician, according to the international guidelines [13] and/or local availability and/or considering the presence of RASs in NS3, NS5A and NS5B regions.

In accordance with the international guidelines and with the Helsinki Declaration of 1975 and revised in 1983 and with the guidance set out by the Ethics Committee of the Azienda Ospedaliera of the University of Campania Luigi Vanvitelli 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. 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 6,000 automated analyzer using c501 biochemistry modules (Roche Diagnostics Ltd, Rotkreuz, Switzerland).

Viral RNA was extracted from 140 ml 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 [14]; 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, Loos, France), following the manufacturer's instructions.

NS3, NS5A and NS5B Sequencing Methods

On the serum collected at 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 [15]. 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, Foster City, CA, USA). The sequences were submitted to bio-technology 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 programme and by comparison with the reference sequence, it was possible to establish the genotypes and possible contaminations of the samples. The mutations and quasi-species were identified by seqscape programme software (Applied Biosystems) with a >20% tolerance for improper sequencing. Finally, we interpreted the resistance mutations based on the reference sources. More specifically, the list of RASs analysed, known to confer reduced susceptibility (increase of > twofold in 50% effective drug concentrations [EC₅₀]) of different HCV genotypes/subtypes to currently available HCV DAAs, was as follows:

NS3-RASs (reduced sensitivity to asunaprevir; boceprevir; paritaprevir, simeprevir, telaprevir, grazoprevir, glecaprevir, danoprevir, voxilaprevir) at positions: V36A/G/M, F43I/L/S, T54S, V55A, Y56H, Q80K/R, S122G/I/N/R/T, R155G/K/Q/T/W, A156S/G/T/V, D168A/C/E/G/H/I/N/T/V/Y, any V/I170T/I, M175L.

NS5A-RASs (reduced sensitivity to daclatasvir, ledipasvir, ombitasvir, elbasvir, velpatasvir, pibrentasvir, ravidasvir, ruzasvir, GSK-2336805, odalasvir, samatasvir) at positions: any 24G/N/R, any 28A/G/M/S/T/V, any 30D/E/G/H/L/Q/K/R/S/T, any 31I/F/M/V, any 32L/F, S38F, Q54H, any 58D/P/S, any 92K/R/T, Y93C/F/H/N/R/S/T/W.

NS5B nucleotide inhibitor RASs (reduced sensitivity to sofosbuvir): L159F, K211R, E237G, S282T, C316H/N, L320F, V321A, A421V, E440G.

NS5B non-nucleotide inhibitor RASs (reduced sensitivity to dasabuvir and beclabuvir, GS-9669, ABT-072): L314H, C316H/N/Y, S368T, A395G, M414I/T/V, L419I/L/M/P/S/T/V, A421V, R422K, M423A/M/I/T/V, N444K, E446K/Q, Y448C/H/R, I482L, A486A/I/M/T/V, V494A, L497F, A553T/V, G554S/D, S556G/N/R, G558R, D559G/N, Y561H, S565F.

Statistical analysis

We used the Kolmogorov-Smirnov test to check for a Gaussian distribution of quantitative variables. In cases of Gaussian distribution, we used for comparisons the Student t-test for unpaired variables. In cases of non-Gaussian distribution, we used for comparisons the Mann-Whitney U-test. The χ² 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).

Results

The demographic, clinical and virological data at DAA failure of the 73 patients enrolled are summarized in Table 1. 17 patients had HCV genotype-1a and 56 genotype-1b. Forty-nine patients (67.1%) were males, and the median age was 67 years (range 42–81). All patients were HIV-negative. All but two patients at the time of virological failure had the same genotype as that reported before the start of the first-line of therapy with DAAs. The majority of patients (57, 78.1%) had cirrhosis. According to the type of virological failure, 63 patients (86.3%) were relapsers, 4 (5.5%) were non-responders and 6 (10.9%) experienced a virological breakthrough. The DAAs used in the failed regimens are shown in Table 1. The median timing of the resistance test from the end of the first-line DAA regimen was 7 months (range, 1–22).

Table 1

Demographic, virological and clinical characteristics of the patients enrolled and type of treatment

Number of patients	73
Median age, years (range)	67.5 (42–81)
Males, n (%)	49 (67.1)
Median HCV RNA, IU/ml (range)	9.75E+06 (2,370–4.65E+09)
Patients with cirrhosis, n (%)	57 (78.1)
Patients with HCC, n (%)	4 (5.4)
Relapser, n (%)	63 (86.3)
Non-responder, n (%)	4 (5.5)
Breakthrough, n (%)	6 (10.9)
HCV genotype-1a, n (%)	17 (23.3)
treated with:
Sofosbuvir plus simeprevir ± ribavirin, n (%)	3 (17.6)
Sofosbuvir plus ledipasvir ± ribavirin, n (%)	7 (41.2)
Paritaprevir/r, ombitasvir, dasabuvir ± ribavirin, n (%)	6 (35.3)
Simeprevir plus daclatasvir, n (%)	1 (5.9)
HCV genotype-1b, n (%)	56 (76.7)
treated with:
Sofosbuvir plus ribavirin, n (%)	16 (28.6)
Sofosbuvir plus simeprevir ± ribavirin, n (%)	13 (23.2)
Sofosbuvir plus ledipasvir ± ribavirin, n (%)	16 (28.6)
Paritaprevir/r, ombitasvir, dasabuvir ± ribavirin, n (%)	11 (19.6)
Median duration of DAA, months (range)	12 (2–24)
Median timing of resistance test at the end of treatment, months (range)	7 (1–22)

DAA, direct-acting antiviral; HCC, hepatocellular carcinoma.

Figure 1 shows the RAS distribution among the different HCV subtypes according to the DAA regimen used. In the 34 patients having failed an NS3 inhibitor, the prevalence of NS3-RASs was higher in the 10 with genotype-1a than in the 24 with genotype-1b (80% versus 41.6%; P=0.06). Instead, in the 41 patients treated with an NS5A inhibitor, the prevalence of NS5A-RASs was very high and similar in the 14 with genotype-1a and the 27 with genotype-1b (78.6% and 92.5%, respectively). In the 55 patients having failed sofosbuvir, the prevalence of NS5B-RASs was more frequently identified in the 45 with genotype-1b than in the 10 with genotype-1a (37.7% versus 10%) with no significance at the statistical analysis, P=0.13). In any case, no patient harboured the RAS 282T, which confers clinically relevant resistance. The prevalence of NS5B-RASs in the patients having failed dasabuvir was high in both genotypes, 66.6% in the 6 with genotype-1a and 45.5% in the 11 with genotype-1b. The distribution of the ‘clinically relevant’ RASs according to the EASL guidelines [13] and reference sources [15,16] is shown in Additional file 1.

Figure 1

Prevalence of RASs in the three HCV regions (NS3, NS5A and NS5B) among the different HCV-1 subtypes and according to the DAA regimen used

Out of the 73 patients enrolled, 24 (33%) patients were re-treated. Table 2 shows the clinical and virological data of the 24 re-treated patients.

Table 2

Clinical and virological data of the 24 re-treated patients

Patients	Reported genotype/identified genotype	First-line DAAs	Duration of therapy, weeks	Response at 12 weeks after therapy	RASs NS3A	RAS NS5A	RAS NS5B	Second-line DAAs	Duration of therapy, weeks	Response at 12 weeks after therapy
1	1b	Sim+sof	12	Relapser	NO	Y93H^a	L159F^b C316N^b	3D+ribavirin	12	SVR
2	2/1b	Sof+riba	24	Relapser	S122[S-G]^a	NO	NO	Sof+led	24	SVR
3	1/1a	Led+sof	24	Relapser	NO	Y93H^a	NO	Sim+sof+riba	12	SVR
4	2/1b	Sof+riba	24	Relapser	NO	L28M	NO	Sof+led	24	SVR
5	1b	Sof+sim	12	Breakthrough	NO	NO	NO	Sof+led+riba	24	SVR
6	1b	Sof+riba	24	Relapser	S122T/S^a	NO	C316N^b	Sof+led+riba	12	SVR
7	1b	Sof+sim+riba	a 24	Relapser	D168E^a	NO	L159F^b C316N^b	Sof+led	24	SVR
8	1b	Sim+sof	12	Relapser	NO	NO	NO	3D+ribavirin	12	SVR
9	1b	Sof+led+riba	a 12	Relapser	V170[V-A]^a	L28M^a-Y93H^a	NO	Sof+sim+riba	12	Relapser
10	1a	Sof+sim+riba	a	Non-responder	T54S-^aR155K^a	L31M	NO	Sof+dacl		Relapser
11	1a	Sim+dacl		Non-responder	D168E^a, Q80R^a	L31M^a, Q54H^a, Y93C^a		Sof+velp+riba	24	SVR
12	1b	Sof+led	24	Relapser	NO	Y93H^a	S556^b [S-G]	Sof+sim+riba	24	SVR
13	1a	Sof+sim	24	Relapser	R155K^a	NO	NO	Sof+led+riba	24	SVR
14	1b	Sof+led+riba	a 12	Relapser	NO	L31I^a	NO	Sof+sim	24	SVR
15	1b	Sof+led	12	Relapser	NO	Y93H^a, L31[M-L]^a	L159F^b C316N^b	3D+ribavirin	12	SVR
16	1b	Sof+riba	24	Relapser	NO	NO	NO	Sof+dacl	24	Breakthrough
17	1b	Sof+sim	12	Relapser	NO	NO	NO	Sof+led+riba	12	SVR
18	1b	Sof+sim		Non-responder	NO	NO	NO	Sof+led	24	SVR
19	1a	Sof+led	24	Relapser	NO	L31M^a	M414[M-I]	Sof+dacl+riba	24	Relapser
20	1b	Sof+riba	48	Relapser	NO	NO	NO	Sof+led+riba	24	SVR
21	1b	3D	12	Relapser	NO	Y93H^a	E440G-S556^b [S-G]	Sim+sof+riba]	12	SVR
22	1a	3D	6	Breakthrough	NO	M28V^a	A421V-S556G^b	Sof+velp	24	SVR
23	1b	Sof+riba	12	Relapser	S122[S-G]^a	NO	C316N^b	Sof+led	24	SVR
24	1b	Sof+sim	12	Relapser	NO	NO	NO	3D+ribavirin	24	SVR

‘Clinically relevant’ resistance-associated substitutions (RASs; according to [13] and [15]).

RASs conferring low level of resistence to sofosbuvir. DAA, direct-acting antiviral; dacl, daclatasvir; led, ledipasvir; riba, ribavirin; sim, simeprevir; sof, sofosbuvir; velp, velptasvir; 3D, ombitasvir, dasabuvir, paritaprevir-ritonavir.

The demographic, clinical and virological data of the 24 re-treated patients are summarized in Table 3 according to the different HCV subtypes. No association was observed between different first-line DAA regimens or specific RASs and the outcome of the second-line DAA regimen. Table 4 shows the outcome of the 24 re-treated patients according to the first-line DAA and second-line regimen. The 6 patients re-treated with genotype-1a less frequently (67%) showed an SVR than the 18 with genotype-1b (89%), but the difference was not significant to the statistical analysis (P=0.2). Considering as effective a DAA regimen that was active in at least two HCV regions according to NS3, NS5A and NS5B genotyping (absence of RASs of at least fivefold in EC₅₀) [14], SVR was more frequently obtained in the 21 patients with an effective second-line DAA regimen than in the 3 without (90.4% versus 0%; P<0.005). In the first group, patient number 16 showed a breakthrough to a daclatasvir-based retreatment, although no RAS in NS5A was identified before re-treatment, suggesting a low compliance. On the other hand, patients number 10 and number 19 experienced a relapse to daclatasvir-based re-treatment and patient number 9 a relapse to a simeprevir-based regimen, because of the presence of significant NS5A-and NS3-RASs, respectively (Table 2).

Table 3

Epidemiological, clinical and virological characteristics of HCV genotype-1a and 1b retreated patients

Genotype	1a	1b
Patients, n	6	18
Median age, years ±sd	61.5 ±11.2	63 ±16.9
Males, n (%)	5 (83)	11 (61)
Patients with cirrhosis, n (%)	6 (100)	17 (94)
Patients with HCC, n (%)	0	1 (5)
Patients with RAS, n (%)	6 (100)	11 (61)
Patients with RAS in ≥2 HCV genome regions, n (%)	3 (50)	8 (44)
Previous therapy
Simeprevir + sofosbuvir ± ribavirin, n (%)	2 (32)	7 (39)
Sofosbvir + ledipasvir ± ribavirin, n (%)	3 (50)	2 (22)
Ombitasvir/paritaprevir/ritonavir + dasabuvir ± ribavirin, n (%)	1 (17)	1 (6)
Sofosbuvir + ribavirin, n (%)	/	6 (33)
Median duration of first DAA therapy, weeks (range)	18 (6–24)	12 (12–48)
Patients without clinically relevant RAS, n/total n (%)	/	5/18 (27.7)
Median duration of second DAA therapy in weeks (range)	24 (12–24)	24 (12–24)
Response
SVR 12, n/total n (%)	4/6 (67) A	16/18 (88.9) B
Relapse at 12 weeks, n/total n (%)	1/6 (16.5)	2/18 (11.1)
Breakthrough, n/total n (%)	1/6 (16.5)	/

A versus B, P=0.2. DAA, direct-acting antiviral; HCC, hepatocellular carcinoma; RAS, resistance-associated substitution; SVR, sustained virological response; /, none.

Table 4

Outcome of the 24 re-treated patients according to the first-line DAA and second-line regimen

Genotype	Failed DAA regimen	Number of patients	Second-line regimen	SVR, n/total n (%)
1a	Sof+led±riba	2	Sof+sim+riba; sof+dacl	1/1 (100); 0/1
1a	Sim+dacl	1	Sof+vel+riba	1/1 (100)
1a	3D	1	Sof+vel	1/1 (100)
1a	Sim+sof±riba	2	Sof+led+riba; sof+dacl	1/1 (100); 0/1
1b	Sim+sof±riba	7	3D; sof+led±riba	3/3 (100); 4/4 (100)
1b	Sof+led±riba	4	Sof+sim±riba; 3D	2/3 (66); 1/1 (100)
1b	3D	1	Sof+sim+riba	1/1 (100)
1b	Sof+riba	6	Sof+led±riba; sof+dacl	5/5 (100); 0/1

DAA, direct-acting antiviral; dacl, daclatasvir; led, ledipasvir; riba, ribavirin; sim, simeprevir; sof, sofosbuvir; SVR, sustained virological response; vel, velpatasvir; 3D, ombitasvir, dasabuvir, paritaprevir-ritonavir.

Discussion

In this real-world clinical setting study the clinical, demographic and virological characteristics of 73 patients with HCV genotype-1 infection experiencing failure to DAA therapy were described. The results obtained from the present study demonstrated that the first-line therapy influenced the development of RASs particularly for genotype-1a. In fact, the prevalence of NS3-RASs in patients treated with an NS3 inhibitor was higher in the patients with HCV genotype-1a than in the patients with HCV genotype-1b. Moreover, the rate of NS5A RASs was very high (78–92%) in the patients treated with an NS5A-based DAA regimen, with no difference between the two HCV subtypes; the rate of NS5B RASs was high (45–66%) in the patients treated with dasabuvir and low (10–37%) in those treated with sofosbuvir, confirming the high genetic barrier of the latter drug. However, it should be taken into account that the prevalence of the RASs identified in the different HCV regions could have been influenced by the fact that the median time of the resistance test from the end of treatment was long (median of 7 months, range 1–22). In fact, it is known that the persistence of RASs typically varies widely, from very short (282T in NS5B) to very long (NS5A RASs), with NS3 RASs usually intermediate.

In any case, these data confirmed that NS5A and non-nucleoside inhibitors of the NS5B region have relatively low barriers to resistance, while sofosbuvir has a high genetic barrier [17–22]. Moreover, our data con-firmed that the genetic barrier of the second-generation NS3 inhibitors was different in HCV sub-genotype-1a and −1b, being high in genotype-1b and low in −1a [15,23–25]. This difference was already known regarding the first-generation NS3 inhibitor: for example, two nucleotide changes were required to generate an amino acid change in position 155 in subtype-1b isolates [26,27], while only one for subtype-1a. Differences between the HCV-1 sub-genotypes were present also for the clinical impact of RASs in the NS5A region [27,28]. For example, in genotype-1b, RASs in positions 31 and 93 were associated with a limited impairment of the efficacy of third-generation NS5A-inhibitors (velpatasvir or pibrentasvir), except in the presence of other secondary resistance mutations (for example at position R30 and P58). Instead, in genotype-1a, changes at positions 28, 30, 31 and 93 were associated with high resistance also to the more recent NS5A inhibitors [15,25,29–33].

These data suggest that the patients with HCV genotype-1a more frequently show RASs after a failure to a DAA regimen and, thus, they are more difficult to retreat compared to those with genotype-1b.

Another interesting observation is that according to our finding, the background level of RASs for NS3 is relatively similar for patients with genotype-1b who failed treatment with an NS3 inhibitor and those who failed with a non-NS3 inhibitor DAA. This is probably due to the high prevalence of NS3-RASs also in naive patients. For example, Li et al. [34] observed the presence of at least one RAS in the NS3 region in 85.48% of 62 DAA treatment-naive HCV-1b patients.

Of the 73 patients enrolled, 24 were re-treated with a second-line DAA-regimen. The rate of SVR was high (83%), higher in genotype -b (89%) than in genotype-1a (67%).

Although the number of re-treated patients was low, the data seems to be interesting since few data are available in the literature and the recommended re-treatment approaches come prevalently from expert opinion based on the analysis of trials or of small case series. In this regard the results are interesting from the POLARIS-4 trial evaluating the efficacy of the combination sofosbuvir/velpatasvir/voxilaprevir in 182 retreating genotype-1–4 patients who had failed a prior non-NS5A DAA regimen; the patients re-treated with this combination for 12 weeks showed a high SVR12 rate (98%) [35]. The placebo-control Phase 3 POLA-RIS-1 trial, instead, evaluated the efficacy of a 12-week course of the same combination in 263 patients with HCV genotype-1–6 infection who had failed a previous DAA regimen containing an NS5A inhibitor. Overall, the SVR rate was 93% in patients with cirrhosis and 99% in those without [35]. Moreover, the MAGELLAN-1-part 2 randomized, open-label, Phase 3 study evaluating the efficacy and safety of 12 or 16 weeks of the combination glecaprevir/pibrentasvir in 50 patients with chronic HCV infection who experienced a virological failure to an NS3/4A protease- and/or an NS5A inhibitor-containing therapy is also interesting. An SVR was achieved by 89% and 91% of patients who, respectively, received 12 or 16 weeks of this combination [36]

Another interesting study is that performed by de Lédinghen et al. [37] evaluating 26 patients with NS3 or NS5A RASs detectable at the time of virological failure. These patients were randomized into two groups to receive 16 or 24 weeks of a combination of sofosbuvir + grazoprevir/elbasvir + ribavirin. 25 of 26 patients achieved an SVR (0.96 [95% CI 0.80,0.99]).

Therefore, considering that in the literature the data on re-treatment are few, in our opinion the observations on our 24 re-treated patients could make a contribution to the knowledge on this topic although in the present study the associations of NS3, NS5A and NS5B combinations (sofosbuvir + velpatasvir + voxilaprevir or sofosbuvir + pibrentasvir + glecaprevir) were not used as the second-line DAA regimen. Moreover, the most interesting data of the present study were those regarding the re-treatment schedule considering the RAS data: the rate of SVR was very high (up to 91%). Thus, the identification of RASs in all three HCV regions after DAA failure is mandatory for the efficacy of the re-treatment schedule in patients with genotype-1. Moreover, because of the low number of patients, the comparison between different first-line DAA regimens or specific RASs and the outcome to the second-line DAA regimen was not possible.

The lack of a serum sample before the start of the first DAA regimen did not allow us to perform the phylogenetic analysis to exclude a re-infection.

In conclusion, in this paper we characterized the virological characteristics of genotype-1 patients failing a first-level DAA regimen. In addition, we observed a high SVR rate when the re-treatment regimen was chosen according to the sequencing data of NS3, NS5A and NS5B, suggesting a close interaction between the clinical and laboratory centres in the management of these patients.

Footnotes

Acknowledgements

MP, M Starace and NC were involved in study concept and design, drafting of the manuscript, critical revision of the manuscript for important intellectual content; statistical analysis; administrative, technical, or material support; study supervision. CM, SDP, MM, LO, VM, VS, EC, DP, GS, M Stanzione, IG, GB, SM, A Masiello, ASM, CC, AF, MP, AGL, A Marrone and were involved in acquisition of data, analysis and interpretation of data and in critical revision of the manuscript; ES, GBG and NC were involved in acquisition of data and critical revision of the manuscript.

The authors declare no competing interests.

Additional file 1: A table showing ‘clinically relevant’ RASs according to HCV genotype and reference sources can be found at

References

Coppola

, Pisaturo

, Tonziello

, Sagnelli

, Angelillo

I.F.

Efficacy of pegylated interferon α-2a and α-2b in patients with genotype 1 chronic hepatitis C: a meta-analysis. BMC Infect Dis 2012; 12: 357.

Italian Association for the Study of the Liver; Italian Society of Infectious, Tropical Diseases; Italian Society for the Study of Sexually Transmitted Diseases. Practice guidelines for the treatment of hepatitis C: recommendations from an AISF/ SIMIT/SIMAST ExpertOpinion Meeting. Dig Liver Dis 2010; 42: 81–91.

Calvaruso

, Craxì

2011 European Association of the Study of the Liver hepatitis C virus clinical practice guidelines. Liver Int 2012; 32: 2–8.

Ghany

M.G.

, Strader

D.B.

, Thomas

D.L.

, Seeff

L.B.

, American Association for the Study of Liver Diseases. Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 2009; 49: 1335–1374.

Coppola

, Pisaturo

, Sagnelli

, Angelillo

I.F.

Peg-interferon plus ribavirin with or without boceprevir or telaprevir for HCV genotype 1: a meta-analysis on the role of response predictors. PLoS ONE 2014; 9: e94542.

Heimbach

J.K.

Overview of the Updated AASLD Guidelines for the Management of HCC. Gastroenterol Hepatol (N Y) 2017; 13: 751–753.

European Association for the Study of the Liver. EASL Recommendations on Treatment of Hepatitis C 2018. J Hepatol 2018; 69: 461–511.

Pawlotsky

J.M.

Treatment failure and resistance with direct-acting antiviral drugs against hepatitis C virus. Hepatology 2011; 53: 1742–1751.

Pawlotsky

J.M.

Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-free regimens. Gastroenterology 2016; 151: 70–86.

10.

Coppola

, Onorato

, Iodice

Occult HBV infection in HCC and cirrhotic tissue of HBsAg-negative patients: a virological and clinical study. Oncotarget 2016; 7: 62706–62714.

11.

Coppola

, De Pascalis

, Messina

ITPase activity modulates the severity of anaemia in HCV-related cirrhosis treated with ribavirin-containing interferon-free regimens. Antivir Ther 2017; 22: 551–558.

12.

Macera

, Stanzione

, Messina

Interferon-free regimens in hepatitis B surface antigen/anti-hepatitis C virus patients: the need to control hepatitis B virus replication to avoid hepatitis B virus reactivation. Clin Gastroenterol Hepatol 2017; 15: 1800–1802.

13.

EASL Recommendations on Treatment of Hepatitis C. 2016. J Hepatol 2017; 66: 153–194.

14.

Coppola

, Zampino

, Cirillo

TM6SF2 E167K variant is associated with severe steatosis in chronic hepatitis C, regardless of PNPLA3 polymorphism. Liver Int 2015; 35: 1959–1963.

15.

Starace

, Minichini

, De Pascalis

Virological patterns of HCV patients with failure to interferon-free regimens. J Med Virol 2018; 90: 942–950.

16.

Di Maio

V.C.

, Cento

, Lenci

Multiclass HCV resistance to direct-acting antiviral failure in real-life patients advocates for tailored second-line therapies. Liver Int 2017; 37: 514–528.

17.

Kati

, Koev

, Irvin

In vitro activity and resistance profle of dasabuvir, a nonnucleoside hepatitis C virus polymerase inhibitor. Antimicrob Agents Chemother 2015; 59: 1505–1511.

18.

Marascio

, Pavia

, Strazzulla

Detection of natural resistance-associated substitutions by ion semiconductor technology in HCV1b positive, direct-acting antiviral agents-naive patients. Int J Mol Sci 2016; 17: E1416.

19.

Patino-Galindo

J.A.

, Salvatierra

, Gonzalez-Candelas

, Lopez Labrador

F.X.

Comprehensive screening for naturally occurring hepatitis C virus resistance to direct-acting antivirals in the NS3, NS5A, and NS5B genes in worldwide isolates of viral genotypes 1 to 6. Antimicrob Agents Chemother 2016; 60: 2402–2416.

20.

Coppola

, Minichini

, Starace

, Sagnelli

Clinical impact of the hepatitis C virus mutations in the era of directly acting antivirals. J Med Virol 2016; 88: 1659–1671.

21.

Yang

, Xing

, Feng

Prevalence of NS5B resistance-associated variants in treatment-naive Asian patients with chronic hepatitis C. Arch Virol 2018; 163: 467–473.

22.

Costantino

, Spada

, Equestre

Naturally occurring mutations associated with resistance to HCV NS5B polymerase and NS3 protease inhibitors in treatment-naive patients with chronic hepatitis C. Virol J 2015; 12: 186.

23.

Kuntzen

, Timm

, Berical

Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naive patients. Hepatology 2008; 48: 1769–1778.

24.

Halfon

, Locarnini

Hepatitis C virus resistance to protease inhibitors. J Hepatol 2011; 55: 192–206.

25.

Dietz

, Susser

, Vermehren

Patterns of resistance-associated substitutions in patients with chronic HCV infection following treatment with direct-acting antivirals. Gastroenterology 2018; 154: 976–988.e4.

26.

Sarrazin

, Zeuzem

Resistance to direct antiviral agents in patients with hepatitis C virus infection. Gastroenterology 2010; 138: 447–462.

27.

McCown

M.F.

, Rajyaguru

, Kular

, Cammack

, Najera

GT-1a or GT-1b subtype-specific resistance profiles for hepatitis C virus inhibitors telaprevir and HCV-796. Antimicrob Agents Chemother 2009; 53: 2129–2132.

28.

Sarrazin

, Zeuzem

Resistance to direct antiviral agents in patients with hepatitis C virus infection. Gastroenterology 2010; 138: 447–462.

29.

Fridell

R.A.

, Qiu

, Wang

, Valera

, Gao

Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob Agents Chemother 2010; 54: 3641–3650.

30.

Vermehren

, Sarrazin

The role of resistance in HCV treatment. Best Pract Res Clin Gastroenterol 2012; 26: 487–503.

31.

Wang

, Huang

, Valera

Hepatitis C virus RNA elimination and development of resistance in replicon cells treated with BMS-790052. Antimicrob Agents Chemother 2012; 56: 1350–1358.

32.

Malta

, Gaspareto

K.V.

, Lisboa-Neto

, Carrilho

F.J.

, Mendes-Correa

M.C.

, Pinho

J.R.R.

Prevalence of naturally occurring NS5A resistance-associated substitutions in patients infected with hepatitis C virus subtype 1a, 1b, and 3a, co-infected or not with HIV in Brazil. BMC Infect Dis 2017; 17: 716.

33.

Zeuzem

, Mizokami

, Pianko

NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: prevalence and effect on treatment outcome. J Hepatol 2017; 66: 910–918.

34.

, Chen

Z. W.

, Li

, Ren

, Hu

Prevalence of hepatitis C virus-resistant association substitutions to direct-acting antiviral agents in treatment-naive hepatitis C genotype 1b-infected patients in western China. Infect Drug Resist 2017; 10: 377–392.

35.

Bourliere

, Gordon

S.C.

, Flamm

S.L.

Sofosbuvir, velpatasvir, and voxilaprevir for previously treated HCV infection. N Engl J Med 2017; 376: 2134–2146.

36.

Poordad

, Pol

, Asatryan

MAGELLAN-1, Part 2: glecaprevir and pibrentasvir for 12 or 16 weeks in patients with chronic HCV genotype 1 or 4 and prior direct-acting antiviral treatment failure. Gastroenterology 2017; 152: S1057.

37.

de Lédinghen

, Laforest

, Hézode

Retreatment with sofosbuvir plus grazoprevir/elbasvir plus ribavirin of patients with hepatitis C virus genotype 1 or 4 who previously failed an NS5A- or NS3-containing regimen: The ANRS HC34 REVENGE Study. Clin Infect Dis 2018; 66: 1013–1018.

Patients with HCV Genotype-1 who have Failed a Direct-Acting Antiviral Regimen: Virological Characteristics and Efficacy of Retreatment

Abstract

Background

Methods

Results

Conclusions

Introduction

Methods

NS3, NS5A and NS5B Sequencing Methods

Statistical analysis

Results

Discussion

Footnotes

Acknowledgements

References