3.2 Analysis of RAVs to NS3/4A protein protease inhibitors (PIs)
The success rate of amplification of NS3 was 81.08% (60/74). The mutation rate was 38.33% (23/60). There were 11 cases (18.33%, 11/60) with the main mutation A156S associated with resistance to Asunaprevir, Boceprevir, Paritaprevir, Simeprevir, and Telaprevir. There were 4 cases (6.67%, 4/60) with the mutation T54S associated with resistance to Boceprevir and Telaprevir and 1 case (1.67%, 1/60) with mutation D168Y associated with resistance to Asunaprevir, Paritaprevir, and Simeprevir. The frequency of V170I was 16.7% (9/60) in HCV genotype 1b. Only 1 case (1.67%, 1/60) with the mutation V55R was found in HCV genotype 1b infected patient when 2 cases (3.33%, 2/60) with the mutation Q80L were found in HCV genotype 1b infected patients (Table 3).[23,26–31]
3.3 Analysis of RAVs to NS5A
The success rate of amplification of NS5A was 79.7% (59/74). The mutation rate was 100% (59/59). There were 34 cases (57.6%, 34/59) with Q30R mutation associated with resistance to Daclatasvir, Ombitasvir, and Ledipasvir, while there were 51 patients (86.4%, 51/59) with H58P associated with resistance to Daclatasvir and 3 cases (5.08%, 3/59) were detected Y93H mutation associated with resistance to Daclatasvir, Ombitasvir, and Ledipasvir. Other mutation sites such as M28L, H54Q, H58T, H58S, H58R, Y93T, and Y93A that were not been proven to correlate with the drug-resistant properties in previous studies (Table 4).[17,26,30,32–35]
3.4 Analysis of RAVs to NS5B
Due to the high difficulty to amplify the fragments of NS5B, the fragments of NS5B were divided into 3 fragments, and then amplification was performed. The first fragment contained A15 and S96. The second fragment contained C223, S282, C316, V321, S365, and S368. The third fragment contained M414, L419, M423, Y448, I482, V494.
The success rate of amplification for the first fragment was 93.2% (69/74). Among the successfully amplified samples, no patients had the drug resistance mutation. For the amplification of the second fragment, the success rate was 81.08% (60/74), while the mutation rate was 100% (60/60). These mutations are associated with resistance to Dasabuvir, Tegobuvir, and HCV796. For the amplification of the third fragment, the success rate was 68.92% (51/74), while the mutation rate was 47.05% (24/51). These mutations were associated with resistance to Dasabuvir, Tegobuvir, HCV796, JTK-109, and Deleobuvir (Table 5).[36–38]
3.5 Analysis of multiple RAVs
We also found that 59 patients have 2 or more than 2 RAVs that will result in highly resistant toward DAAs and resistant to multiple DAAs. There were 12 cases which had 3 to 5 RAVs, which will result in resistant toward NS3/4A inhibitor, NS5A inhibitor, and NS5B inhibitor. There were 38 cases that had 2 to 5 RAVs, which will result in resistant toward NS5A inhibitor and NS5B inhibitor. Two cases had RAVs that will result in resistant toward NS3/4A inhibitor and NS5B inhibitor. At the same time, we found that 4 cases had Q30R+ H58P that results in resistance to NS5A inhibitor: one case with C316N + V499A, one case with C316N + M423I, and one case with C316N + I482T, which is resistant to NS5B inhibitor (Table 6). There was linkage disequilibrium in our patients.
The development of DAAs represents a significant advancement in HCV antiviral therapy. Despite the antiviral potency of the majority of DAA being extraordinary, the ability of HCV to rapidly evolve in the setting of drug pressure and the presence of baseline natural polymorphisms associated with resistance to drugs must be considered as possible challenge to the success of these therapies. Clinical trials had already showed that the RAVs could lead to treatment failure and these RAVs could be found in DAAs treatment-naive HCV patients.
It had been reported that HCV NS3 was a multifunctional antiviral target exhibiting large gene polymorphisms. It had been detected that the main sites (R155 and A156) had less variation, whereas the second sites (V36, T54, Q80, D168, V170) had variation more frequently. Our study showed that 26.67% (16/60) patients infected with HCV genotype 1b had the drug resistance mutations to NS3/4A PIs before any direct antiviral treatments. The prevalence of 26.67% (16/60) for PI resistance proven patients observed in the present study was higher than the results of previous studies.[23,24,26] As compared with the previous studies, main mutations such as R155, A156T, Q80K, those that may result in high drug resistance, were not detected in our study, while D168Y that may result in high drug resistance was detected in our study. The Q80K variant was associated with different levels of resistance to some approved NS3 PIs (asunaprevir, paritaprevir, and simeprevir). Patients harboring the Q80K had lower SVR rates to simeprevir than those who did not. SVR rates in simeprevir-based treatment-naïve HCV genotype 1a infected patients with and without the Q80K variant were 58% versus 84%[23,28] and guidelines recommended to screen for presence of Q80K before beginning the simeprevir drug therapy and to consider alternative therapy if the Q80K are detected.[1,7,8] Variations A156S and T54S were found in 11 cases and 4 cases, while some invalid variations were found, including V170I, V55R, and Q80L, which have not been proved to be drug resistant.
NS5A PIs, a new type of direct-acting antivirals, interfered HCV replication cycle mainly through directly inhibiting NS5A. Compared with the majority of NS3 RAVs, variants conferring resistance to NS5A inhibitors are generally more frequently detected as natural variants in HCV genotype 1 infected DAA-naive patients. As a single drug resistance mutation, the rate of natural occurrence was estimated between 0.3% and 2.8% in different studies by population sequencing.[26,30,32,39] In our study, the rate of natural occurring mutations was higher. For instance, the mutation rate of Q30R was 57.63% (34/59), while H58P was 86.44% (51/59). Y93H that confers medium to high-level resistance to the 3 approved NS5A inhibitors (Daclatasvir, Ombitasvir, Ledipasvir) seems to be less frequent in Chinese HCV genotype 1b infected patients (5.08%) than the European (15.0%) and the US (9.3%). Four patients had dual combinations of mutations Q30R+H58P that had never been investigated and their level of resistance was unknown.
The HCV NS5B is the last nonstructural gene sequence of HCV, and is located in the end part genome of the HCV. The variation of NS5B amino acid sequence can influence DAAs antiviral capacity and resistance of genetic barrier. The RAVs C316N was detected in all of our patients who were never treated by the DAAs. Whereas main mutations such as S282T that may result in high drug resistance were not detected in our study. S282T that confers high-level resistance to sofosbuvir may result in virologic relapse and sofosbuvir-containing regimens treatment failure.[9,26] The RAVs of non-nucleoside inhibitors of NS5B were more frequent than that of nucleoside inhibitors of NS5B. In our patients, no RAVs to NS5B nucleoside inhibitors was detected, whereas 100% patients had the RAVs of non-nucleoside inhibitors, including C316N, S365A, M414L, M423I, Y448H, I482T/V, V494L, P495S, V499A. Interestingly, HCV genotype 1b isolates harboring C316N were more frequently observed in Chinese patients (100%) in comparison to in Europe (32%) and the United States (5%). Shindo et al report that 13.4% of HCV genotype 1b patients with resistance-proven mutations to PIs were reported in in Japan, while we found 31.1% patients in our study harboring RAVs to PIs.
In addition, we detected that some patients harbor one or more RAVs. Patients who carry combinations of multiple resistance mutations in both or triple the NS3/4A, NS5A, and NS5B genes might increase the possibility of failure in the antiviral treatment with multiple DAA-containing regimens.
This study had certain limitations. The study was held only in our center. The samples of our study were not so large and not all of our cases were successfully amplified.
In conclusion, DAAs RAVs do exist in untreated Chinese patients and the characteristics were different from that in Europe and the United States. R155, A156T, Q80K, and S282T that confer high-level drug resistance were not detected in our study. These results may be associated with the different race and the different HCV genotype epidemiology in our region. A new era of DAAs is now dawning in China; all clinicians should bear in mind that RAVs can pre-exist in HCV1b-infected patient; although the degree of resistance might not be strong, clinicians still need to consider this upon the introduction of DAA-based antiviral therapy. In certain situations, resistance testing might help to select the most optimized treatment option.
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direct-acting antiviral agents (DAAs); hepatitis C virus genotype 1b; NS3/4A; NS5A and NS5B genes; resistance-associated variants (RAVs)
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