sofosbuvir therapy in Mongolian chronic hepatitis C patients

+

MODEL

Journal of the Formosan Medical Association xxx (xxxx) xxx

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.jfma-online.com

Original Article

Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients Shih-Jer Hsu a,b, Sukhee Enkhzaya c,g, You-Yu Lin d, Tai-Chung Tseng e,f, Tulgaa Khosbayar h, Cheng-Hsueh Tsai f, Tzu-San Wang d, Damba Enkhtuya i, Dogsom Ivshinkhorol i, Nyamsuren Naranzul c, Badarch Jargalsaikhan j, Jazag Amarsanaa k, Oidov Baatarkhuu c,g,k,**,1, Jia-Horng Kao d,e,f,l,*,1 a

Department of Internal Medicine, National Taiwan University Hospital Yunlin Branch, Yunlin, Taiwan Hepatology Medical Center, National Taiwan University Hospital Yunlin Branch, Yunlin, Taiwan c Department of Infectious Disease, School of Medicine, Mongolian National University of Medical Sciences, Ulaanbaatar, Mongolia d Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan e Division of Gastroenterology, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan f Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan g Department of Comprehensive Laboratory, National Center for Communicable Diseases, Ulaanbaatar, Mongolia h Department of Molecular Biology and Genetics, School of BioMedicine, Mongolian National University of Medical Sciences, Ulaanbaatar, Mongolia i Happy Veritas Liver Diagnostic Center, Ulaanbaatar, Mongolia j Department of Obstetrics and Gynecology, School of Medicine, Mongolian National University of Medical Sciences, Ulaanbaatar, Mongolia k Mongolian Association for the Study of Liver Diseases, Mongolia l Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan b

Received 6 September 2019; received in revised form 30 September 2019; accepted 3 October 2019

* Corresponding author. Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine and Hospital, 7 ChungShan South Road, Taipei, 10002, Taiwan. Fax: þ886 2 23825962. ** Corresponding author. Department of Infectious Disease, School of Medicine, Mongolian National University of Medical Sciences, Zorig Street, Ulaanbaatar, 14210, Mongolia. E-mail addresses: [email protected] (O. Baatarkhuu), [email protected] (J.-H. Kao). 1 Profs. Oidov Baatarkhuu and Jia-Horng Kao share corresponding authorship. https://doi.org/10.1016/j.jfma.2019.10.003 0929-6646/Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Hsu S-J et al., Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.10.003

+

MODEL

2

S.-J. Hsu et al.

KEYWORDS Chronic hepatitis C; Resistance-associated substitution; Ledipasvir; Sofosbuvir; Mongolia

Background: Mongolia has the highest prevalence of hepatitis C virus (HCV) infection worldwide. Ledipasvir/sofosbuvir (LDV/SOF) was introduced to Mongolia since 2016 for HCV eradication. It has been reported that HCV resistance-associated substitutions (RASs) would affect the effectiveness of LDV/SOF in western chronic hepatitis C (CHC) patients. We thus investigated the effectiveness of LDV/SOF and the impact of RAS on the treatment outcome in Mongolian CHC patients. Methods: Patients with genotype (GT) 1b HCV infection were prospectively enrolled in Mongolia and treated with LDV/SOF for 12 weeks. The proportion of pre-treatment NS5A Y93H RAS in viral quasispecies was measured with next-generation sequencing. The endpoint of LDV/SOF effectiveness was sustained virological response at post-treatment week 12 (SVR12). Results: A total of 94 CHC patients were evaluated. The baseline Y93H proportion was <1% in 74 patients, 1e15% in 7, 15e50% in 2, and 50% in 11. All patients completed 12-week LDV/SOF treatment and the SVR rate was 90.4%. The rate of failure to achieve SVR12 for patients with Y93H < 1%, 1e15%, and 15% were 0%, 14.3%, and 61.5%, respectively (p for trend Z 0.001). In univariable analysis, older age, baseline alanine transaminase level <40 U/mL, and a higher proportion of Y93H were associated with treatment failure. In multivariable analysis, only a higher proportion of Y93H was associated with treatment failure (p Z 0.022). Conclusion: LDV/SOF therapy achieves a high SVR rate in Mongolian CHC GT1b patients without baseline Y93H RAS. A higher proportion of Y93H may severely undermine the effectiveness of LDV/SOF. Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Introduction Chronic hepatitis C virus (HCV) infection is a global health problem, affecting 71 million people worldwide.1 Chronic hepatitis C (CHC) is the leading cause of cirrhosis, hepatic decompensation, hepatocellular carcinoma (HCC), and liver-related death.2 There exist geographic variations of CHC disease burden across the world. Most notably, Mongolia has the highest prevalence of CHC in Asia countries and worldwide.1 Several epidemiology studies had shown the anti-HCV seroprevalence ranged 11e15.6% in Mongolia. Majority of cases were concentrated in those aged over 50 years and most were infected by HCV genotype (GT) 1b.3e5 The high HCV endemicity was reflected by the liver disease burden in Mongolia, which has the highest incidence of (and mortality from) HCC in the world.6 Near 60% of HCC cases in Mongolia was associated with HCV infection.5 The introduction of all-oral direct-acting antivirals (DAAs) revolutionized the management of CHC with high sustained virological response (SVR) rates and favorable tolerability. Ledipasvir/sofosbuvir (LDV/SOF) was among the first-wave all-oral DAAs introduced into Asia in 2014. This regimen has been proven effective and safe for GT1 CHC patients with SVR rates of 94%e99% in both pivotal trials and real-world evidence.7e11 Accumulating data suggested that DAA therapy was associated with a decrease in all-cause mortality and incidence of HCC.12,13 To diminish the disease burden of CHC, the Government of Mongolia launched the Hepatitis Prevention, Control, and Elimination (HPCE) Program in 2016 and LDV/SOF has been introduced at affordable prices; over 18,625 Mongolian CHC patients

had received DAA treatment by the end of August 2019.14 However, the information on the treatment outcome of Mongolian CHC patients is quite limited and has not been reported in any full paper. The effectiveness of LDV/SOF has been shown to be associated with cirrhosis status, ethnicity, concomitant proton-pump inhibitor usage, and drug compliance.10,15 Recent studies revealed HCV NS5A resistance-associated substitutions (RASs) may compromise the effectiveness of LDV/SOF in CHC GT1b patients.16,17 Among known NS5A RASs, the Y93H RAS is the most commonly identified in CHC GT1b patients and confers >1000-fold resistance to ledipasvir.16,17 In this collaborative study between Taiwan and Mongolia, we aimed to study the effectiveness of LDV/SOF in Mongolian CHC patients. Furthermore, we planned to investigate the prevalence of NS5A Y93H RAS using next-generation sequencing (NGS) and explored the impact of RAS on the effectiveness of LDV/SOF.

Materials and methods Patient cohort Patients with CHC were consecutively enrolled at Mongolia Happy Veritas Hospital and Mongolian National University of Medical Sciences from January 2016 to December 2018. Patients aged 18e75 years who had HCV GT1b infection and no prior experience of DAA treatment were eligible for the study. Patients with HCC or other malignancies, kidney disease with estimated glomerular filtration rate <30 ml/min/1.73 m2, clinical heart failure, Child-Pugh C decompensated liver

Please cite this article as: Hsu S-J et al., Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.10.003

+

MODEL

Impact of RAS on LDV/SOF in Mongolian HCV patients disease, epilepsy, uncontrolled diabetes mellitus, thyroid dysfunction, or co-morbidities with a life expectancy <6 months were excluded. The patients received a fixed-dose combination of LDV 90 mg and SOF 400 mg (Gilead Sciences, Foster City, CA, USA) once daily for 12 weeks after enrollment. The study was approved by the Ethics Committees of the National Taiwan University Hospital and the Mongolian National University of Medical Sciences. All patients provided informed consents before enrollment.

Data collection Demographics and clinical characteristics of patients were evaluated at baseline, including HCV viral load, blood biochemistry, complete blood count, HBsAg status, prior CHC treatment experience, and presence of clinical cirrhosis. Serum HCV RNA level was determined by HCV RNA quantification kit (Sunsure Co., Ltd., China) with a lower limit of quantification (LLOQ) of 20 IU/mL. To estimate the stage of liver fibrosis, the fibrosis index based on 4 factors (FIB-4) was calculated.18 The diagnosis of clinical cirrhosis was made by the treating physicians. The endpoint of LDV/ SOF effectiveness is the percentage of patients who achieved sustained virological response 12 (SVR12), defined as HCV RNA < LLOQ at 12 weeks post-treatment. The serum samples before treatment were collected and stored in 80  C until use.

HCV NS5A Y93H RAS analysis We designed a target-specific library for sequence analysis on Illumina MiSeq platform (Illumina, Hayward, CA, USA). A nested polymerase chain reaction (PCR) was first conducted to amplify the region using KOD -Plus-polymerase (Toyobo, Osaka, Japan) and the primer sequences are present in Supplementary Table 1. The primers used in the first round PCR were NS5A-F18 and NS5A-R546. The primers used in the second round PCR were HCV-specific primer pairs with 50 adapters (50 -TCGTCGGCAGCGTCN8 for forward primer [NS5A-F196] and 50 - GTCTCGTGGGCTCGG for reverse primer [NS5A-R396]). The PCR conditions were 94  C for 2 min, followed by 25 cycles of 94  C for 15 s, 60  C for 30 s, and 68  C for 40 s, then a final extension at 68  C for 7 min for the both rounds of PCR. To assess the PCR quality, 5 ml of PCR products were analyzed on a 1% agarose gel. The PCR products were purified by adding AMPure SPRI beads (SPRIselect reagent, Beckman Coulter, Indianapolis, IN, USA) at a 0.8-fold volume of PCR mixture. With the NS5A PCR products available, dual-index was added by using Nextera Index kit (Illumina, Hayward, CA, USA) in the third PCR. The PCR was performed using Phusion high-fidelity DNA polymerase (Thermo, Pittsburgh, PA, USA) with the following PCR parameters: 98  C for 3 min, followed by 10 cycles of 98  C for 30 s, 57  C for 30 s, and 72  C for 45 s, then a final extension at 72  C for 1 min. The PCR products were purified by adding AMPure SPRI beads at a 0.8-fold volume again. The dual-indexed PCR products were quantified by Quant-iT dsDNA high sensitivity assay kit (Life Technologies, Eugene, OR, USA) and mixed at equal copies of each fragment to prepare the library for MiSeq run.

3

Sequence determination by MiSeq desktop sequencer and analysis The library was quantified by using KAPA library quantification kit (KAPA Biosystems, Wilmington, MA, USA) and qualified by using Agilent DNA 1000 kit analyzing in Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). All samples were pooled in one run with the 15e20% of PhiX control DNA and sent to standard Illumina MiSeq paired-end sequencing at 2  300 base pairs (bps). NGS reads were trimmed to a uniform length of 209 bps. NGS reads shorter than 209 bps or with more than 50% of low-quality sequenced nucleotides (quality scores <20) were excluded from further analyses. High-quality NGS data sets were assembled to the HCV reference sequence with BLAST-based assembly pipeline (BBAP).19 Nucleotide frequencies and heterozygosities of each nucleotide site for all data sets were calculated according to the assembled results.

Statistical analysis The baseline characteristics were reported as median (interquartile range) or frequencies (percentages) as appropriate. Both HCV RNA level and Y93H proportion were logarithmically transformed for analysis. Logistic regression was adopted to analyze the factors associated with failure to achieve SVR12. As the number of not achieving SVR12 was small, we only included factors with p < 0.15 in univariable analysis into multivariable analysis. Statistical significance of all tests was defined as p < 0.05 by two-tailed tests. All analyses were performed using Stata statistical software (version 10.0; Stata Corp, College Station, TX, USA).

Results Baseline characteristics and treatment response A total of 120 consecutive CHC patients were enrolled. Among them, 10 patients had no adequate serum sample for RAS analysis. In another 16 patients, NS5A amplification by nested PCR was unsuccessful. Therefore, 94 patients remained for final analysis. The baseline characteristics of the 94 patients are shown in Table 1. Of these 94 patients, 31 (33.0%) were males with a median age of 51.0 years. Eighty-seven (92.6%) patients had baseline alanine transaminase (ALT) level S40 U/L. The median values of platelet count and FIB-4 index were 206.8 k/mL and 1.92, respectively. One (1.1%) patient had received interferon treatment previously. None of them had HCC nor clinical cirrhosis. In terms of viral factors, the median HCV RNA level was 7.15 log10 IU/mL. The Y93H proportion within viral population was <1% in 74 (78.7%) patients, 1e15% in 7 (7.4%), 15e50% in 2 (2.1%), and 50% in 11 (11.7%). Six (6.4%) patients had hepatitis B virus coinfection. All patients completed 12-week LDV/SOF treatment uneventfully and received evaluations for LDV/ SOF effectiveness. A total of 85 patients (90.4%) achieved SVR12.

Please cite this article as: Hsu S-J et al., Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.10.003

+

MODEL

4

S.-J. Hsu et al. Table 1 Baseline characteristics of 94 Mongolian chronic hepatitis C genotype 1b patients.

3.25. The FIB-4 index did not correlate with the SVR12 rate, in agreement with the analysis in Table 2.

Characteristics Male gender Age, years AST level, U/L ALT level, U/L Platelet count, k/mL FIB-4 Interferon-experienced Clinical cirrhosis HBV coinfection HCV RNA, log10 IU/mL Y93H, log10% Y93H, % <1 1e15 15e50 50 Heterozygosities of NS5A amplicon

31 (33.0) 51.0 (21.0) 84.8 (27.6) 85.3 (19.0) 206.8 (43.0) 1.92 (1.04) 1 (1.1) 0 (0) 6 (6.4) 7.15 (0.70) 0.60 (0.49) 74 (78.7) 7 (7.5) 2 (2.1) 11 (11.7) 0.028 (0.015)

Categorical variables are expressed as n (%); continuous variables are expressed as median (interquartile range). Abbreviation: ALT, alanine transaminase; AST, aspartate transaminase; FIB-4, fibrosis index based on 4 factors; HBV, hepatitis B virus; HCV, hepatitis C virus.

Impact of baseline Y93H and heterozygosities of NS5A amplicon on LDV/SOF effectiveness The relationship between the presence of baseline Y93H and the treatment outcome of LDV/SOF is shown in Fig. 1. All patients who had baseline Y93H < 1% achieved SVR12. Among 9 patients who failed to achieve SVR12, one had 4% of Y93H at baseline and the other 8 had Y93H > 90% (Fig. 1A). When categorizing the patients into <1%, 1e15% and 15% according to the Y93H proportion, we found that treatment failure rates were 0%, 14.3%, and 61.5%, respectively (p for trend Z 0.001, Fig. 1B). We also compared the heterozygosities of the NS5A amplicon between patients with and without SVR12. The data showed that there were comparable viral diversities in both groups (p Z 0.629) by ManneWhitney U test.

Predictors for the treatment failure In univariable analysis, we found that older age, ALT <40 U/mL, and a higher proportion of Y93H were associated with treatment failure (Table 2). Among them, a higher proportion of Y93H was associated with the highest risk of treatment failure with OR of 15.43 (95% confidence interval 2.88e82.70) per 1 log10 Y93H percentage increase. In multivariable analysis, only a higher proportion of Y93H was still associated with treatment failure (p Z 0.022), while the ALT and age were not independent predictors. To examine possible interactions between the fibrosis status and the clinical impact of Y93H RAS, we categorized the patients by FIB-4 index and Y93H proportion and analyzed the SVR12 rate in each subgroup (Fig. 2). There was no patient harboring Y93H  15% in the subgroup with FIB-4

Discussion Mongolia has a high disease burden of CHC and the highest incidence of HCC in the world.4,20 To halt the HCV endemicity, LDV/SOF has been introduced into Mongolia since 2016. In this collaborative study between Taiwan and Mongolia, we thus investigated the effectiveness of LDV/ SOF in 94 Mongolian GT1b CHC patients, with an overall SVR rate of 90.4%. Our findings were different from previous conference reports on Mongolian CHC patients, who achieved SVR rates more than 97% with LDV/SOF therapy.21e23 Moreover, our molecular analysis deciphered the failure to achieve SVR was associated with the presence of Y93H RAS, but not other baseline characteristics including FIB-4 score and viral load. In this study, a high rate of virological failure was noted in patients with baseline Y93H. The SVR rate in patients with Y93H (using 15% frequency cut-off) was only 38.5%, compared to 98.8% in patients without Y93H. Y93H is the most frequent NS5A RAS in CHC GT1b patients and confers >1000-fold reduction of in vitro susceptibility to LDV.16,24 Zeuzem et al. recently conducted a comprehensive analysis of more than 5000 HCV GT1 patients enrolled in LDV/ SOF pivotal trials, including 313 GT1b patients from Asia Pacific countries. They concluded the presence of Y93H or other NS5A class RASs adversely impacted the virological outcome of treatment-experienced GT1b CHC patients.17 In addition, several real-world cohort data from Japan also echoed these findings of Zeuzem et al. A multicenter study by Ogawa et al. showed that NS5A RASs undermined the virological effect of LDV/SOF for CHC GT1b cirrhosis patients (SVR rate 91% for patients with baseline NS5A RASs and cirrhosis).25 Kozuka et al. enrolled 313 CHC GT1b patients treated with LDV/SOF in a single center; they found the Y93H or L31M RAS compromised the virological outcome; the presence of multiple NS5A RASs was associated with the effectiveness of LDV/SOF (SVR rate 71% for patients with 3 RASs at baseline).26 Another study by Mawatari et al. showed that the co-existence of NS5A and NS5B RASs was associated with virological failure of LDV/ SOF.27 Taking these lines of evidence together, when treating CHC GT1b patients with LDV/SOF, the impact of Y93H should be considered in the presence of unfavorable characteristics such as cirrhosis, prior treatment failure, or multiple RASs. Of note, compared to previous studies, the even lower SVR rate of our patients with Y93H deserves further examinations. To this end, next-generation sequencing was implemented to tackle the puzzle of unsatisfactory treatment effectiveness. In our cohort, 8 out of 9 virological failures occurred in patients with a Y93H proportion above 90%. The SVR rates in patients with Y93H frequencies of <15%, 15e50%, and 50% were 98.9% (80/81), 100% (2/2), and 27.3% (3/11), respectively. Our data suggested that the genetic barrier of LDV/SOF against GT1b HCV harboring Y93H may be effective unless there is a high proportion of Y93H population in the quasispecies. Only a few studies described the relationship of virological outcome and RAS

Please cite this article as: Hsu S-J et al., Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.10.003

+

MODEL

Impact of RAS on LDV/SOF in Mongolian HCV patients

5

(A) With SVR12 (N = 85)

Without SVR12 (N = 9)

Y93H % <1 1-15 15-50 ≥50

(B) p = 0.001

Treatment failure rate (%)

100 80

61.5 60 40 14.3

20 0

0 <1 (n = 74)

1-15 (n = 7) Y93H propor on (%)

≥15 (n = 13)

Figure 1 Relationship between Y93H proportion and SVR12 rate (A) The distribution of Y93H proportion in patients with and without SVR12 (B) The treatment failure rate according to the Y93H proportion.

frequency using detailed cut-off levels. Mizokami et al. performed resistance analyses of Japanese GT1 CHC patients, either treatment-naı¨ve or -experienced, who received LDV/SOF  RBV for 12 weeks. Among 330 GT1b patients, a total of 63 patients harbored Y93H (using 1% cutoff). Of these patients, 17, 17, and 29 had Y93H at frequencies of <15%, 15e50%, and 50%, respectively. Eleven patients carried Y93H at a frequency of >99%. Of the 63 patients with Y93H, 33 and 30 received LDV/SOF and LDV/ SOF þ RBV, respectively; all of them achieved SVR12, except one with Y93H >99% who received LDV/SOF þ RBV had viral relapse.28 Zeuzem et al. analyzed SVR rates of LDV/SOF-containing regimens in patients with and without LDV-specific RASs by using various cut-off thresholds. The SVR rates of interferon-experienced GT1b patients with LDV RASs frequencies 0e15%, 15e50%, and 50% were 98%, 100%, and 85%, respectively. In addition, virological failures occurred only in patients harboring LDV RASs with >100fold resistance.17 On the basis of these data we may infer that Y93H had little impact on the SVR rate unless its proportion was much higher than conventional cut-off 15%, and

the impact seemed different between Japanese patients and those from other parts of the world, implying genetic background might play a role. Earlier studies showed that the beneficial IFNL4 rs12979860 CC genotype was associated with the presence of Y93H and higher HCV viral load.29,30 The distribution of IFNL4 genotypes in healthy Mongolian people was similar to other Asian people according to a previous report.31 The baseline characteristics of our cohort, compared with aforementioned studies, were generally favorable since our patients were younger, non-cirrhotic, and mostly interferon-naı¨ve. The prevalence of Y93H in our cohort, estimated in conventional 15% or higher cut-offs, was similar to those reported in other studies.17,28,32e34 However, in our study the median HCV viral load was 7.15 log10 IU/mL, numerically higher than 6.4 log10 IU/mL and 6.6 log10 IU/mL reported by Zeuzem et al. and Mizokami et al., respectively. High HCV viral load and NS5A RASs had been shown to compromise the effectiveness of 8-week regimen of LDV/SOF in GT1 CHC patients who were non-cirrhotic and previously untreated.9,16 Some unknown host factors including drug compliance may exist

Please cite this article as: Hsu S-J et al., Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.10.003

+

MODEL

6

S.-J. Hsu et al. Table 2

Univariable and multivariable analysis of factors associated with treatment failure.

Male (ref. female) Age, per 1 year increase ALT level S40 U/L (ref. <40) Platelet count, per k/mL increase FIB-4 S1.90 (ref. <1.90) HCV viral load, per 1 log10 IU/mL Increase Y93H, per 1 log10% increase

Crude OR (95% CI)

p-value

Adjusted OR (95% CI)

p-value

2.84 (0.70e11.43) 1.10 (1.02e1.17) 0.46 (0.08e0.26) 1.00 (0.98e1.02) 0.75 (0.19e2.97) 0.76 (0.41e1.43) 15.43 (2.88e82.70)

0.143

0.690

0.955

0.45 (0.01e22.32) 1.26 (0.97e1.42) 0.25 (0.00e16.41) e

e

0.677

e

e

0.397

e

e

<0.001

65.29 (1.82e2337.50)

0.022

0.008 0.001

0.080 0.26

Abbreviation: ALT, alanine transaminase; CI, confidence interval; FIB-4, fibrosis index based on 4 factors; HCV, hepatitis C virus; OR, odds ratio.

p <0.0001

SVR 12 rate (%)

100

100

98.5

80

Y93H <15% Y93H ≥15%

60 38.5

40 20 0

67 68

5 13

FIB-4 <3.25

13 13 FIB-4 ≥3.25

Figure 2 SVR12 rate according to FIB-4 index and Y93H proportion. The SVR12 rates in patients with FIB-4 <3.25 and FIB-4 3.25 were 88.9% and 100%, respectively (p Z 0.3519). In the subgroup of FIB-4 <3.25, patients with Y93H  15% had significantly lower SVR12 rate (p < 0.0001) compared to those with Y93H < 15%.

and interact with viral load and RAS to affect the treatment outcome of our cohort. There are a few limitations of this study. First, RASs other than Y93H were not investigated thus the possibility of co-existing NS5A or NS5B RASs, which may contribute to virological failure, could not be excluded. Nonetheless, several lines of evidence strongly indicated that Y93H, in comparison with other NS5A RASs, is more prevalent in CHC GT1b patients,16,17,28,34 and has a greater impact on the effectiveness of LDV/SOF therapy.16,17,28 In the study by Zeuzem et al., no LDV-specific RASs were observed at NS5A positions 26, 32, 38, 58, or 92 with prevalence >1%. There was no virological failure in patients harboring LDV-specific RASs with <100-fold resistance.17 The role of NS5B RAS in SOF-based DAA therapy has been shown to be minimal.16 Second, the prevalence of cirrhosis and HCC, which are negative factors for DAA efficacy,35 may be underestimated due to the limitations of diagnostic tools and surveillance strategies. Third, the drug compliance and usage of co-

medication were not evaluated. Fourth, due to small sample size, potential selection bias, and absence of cirrhosis case, the data of virological outcome and Y93H prevalence may be inaccurate; our findings may not be extrapolated to patients with advanced liver disease or cirrhosis. Nevertheless, our findings highlighted the role of Y93H should not be neglected while treating CHC GT1b patients with LDV/ SOF. Introduction of newer generations of DAA with higher genetic barriers would solve the problem of RAS. Otherwise, testing for RAS should be performed, especially for patients with advanced liver disease. In addition, combination of ribavirin or extending treatment duration of DAA should be considered in the presence of significant Y93H population. In summary, LDV/SOF therapy achieves a high SVR rate in Mongolian CHC GT1b patients without baseline Y93H RAS, and a high proportion of Y93H may severely undermine the effectiveness of LDV/SOF.

Financial support This work was supported by the grants from the Ministry of Science and Technology, Executive Yuan, Taiwan (MOST105-2923-B-002-004-MY3).

Declaration of Competing Interest J.-H. Kao has served as a consultant for Abbvie, Gilead Sciences, Merck Sharp and Dohme, and Roche and on speaker’s bureaus for Abbvie, Bristol-Myers Squibb, Gilead Sciences, Merck Sharp and Dohme; all other authors declare no competing interests.

Acknowledgments The authors would like to acknowledge the service provided by the Medical Microbiota Center of the First Core

Please cite this article as: Hsu S-J et al., Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.10.003

+

MODEL

Impact of RAS on LDV/SOF in Mongolian HCV patients Laboratory, National Taiwan University College of Medicine, Taipei, Taiwan.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jfma.2019.10.003.

References 1. Polaris Observatory HCV Collaborators. Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study. Lancet Gastroenterol Hepatol 2017;2: 161e76. 2. Kao JH. Hepatitis C virus infection in Taiwan: past, present, and future. J Formos Med Assoc 2016;115:65e6. 3. Baatarkhuu O, Batzaya M, Brandon S, Estes C, Chiang B, Amarsanaa J, et al. Disease burden of chronic hepatitis C virus infection in Mongolia: potential impact of attaining World Health Organization (WHO) 2030 goals. In: Proceedings from the 24th annual meeting of the Korean Association for the Study of the Liver; June 14-June 16, 2018; Incheon, South Korea; 2018. Abstract PE-051. 4. Baatarkhuu O, Kim DY, Ahn SH, Nymadawa P, Dahgwahdorj Y, Shagdarsuren M, et al. Prevalence and genotype distribution of hepatitis C virus among apparently healthy individuals in Mongolia: a population-based nationwide study. Liver Int 2008; 28:1389e95. 5. Baatarkhuu O, Gerelchimeg T, Munkh-Orshikh D, Batsukh B, Sarangua G, Amarsanaa J. Epidemiology, genotype distribution, prognosis, control, and management of viral hepatitis B, C, D, and hepatocellular carcinoma in Mongolia. Euroasian J Hepato-Gastroenterol 2018;8:57e62. 6. Znaor A, Chimed T, Laversanne M, Tudev U, Sanjaajamts E, Sandagdorj T, et al. The public health challenge of liver cancer in Mongolia. Lancet Gastroenterol Hepatol 2018;3:660e2. 7. Afdhal N, Zeuzem S, Kwo P, Chojkier M, Gitlin N, Puoti M, et al. Ledipasvir and sofosbuvir for untreated HCV genotype 1 infection. N Engl J Med 2014;370:1889e98. 8. Afdhal N, Reddy KR, Nelson DR, Lawitz E, Gordon SC, Schiff E, et al. Ledipasvir and sofosbuvir for previously treated HCV genotype 1 infection. N Engl J Med 2014;370:1483e93. 9. Kowdley KV, Gordon SC, Reddy KR, Rossaro L, Bernstein DE, Lawitz E, et al. Ledipasvir and sofosbuvir for 8 or 12 weeks for chronic HCV without cirrhosis. N Engl J Med 2014;370: 1879e88. 10. Terrault NA, Zeuzem S, Di Bisceglie AM, Lim JK, Pockros PJ, Frazier LM, et al. Effectiveness of ledipasvir-sofosbuvir combination in patients with hepatitis C virus infection and factors associated with sustained virologic response. Gastroenterology 2016;151:1131e1140 e5. 11. Hong CM, Liu CH, Su TH, Yang HC, Chen PJ, Chen YW, et al. Real-world effectiveness of direct-acting antiviral agents for chronic hepatitis C in Taiwan: real-world data. J Microbiol Immunol Infect 2018. https://doi.org/10.1016/j.jmii.201 8.09.005. 12. Carrat F, Fontaine H, Dorival C, Simony M, Diallo A, Hezode C, et al. Clinical outcomes in patients with chronic hepatitis C after direct-acting antiviral treatment: a prospective cohort study. Lancet 2019;393:1453e64. 13. Backus LI, Belperio PS, Shahoumian TA, Mole LA. Impact of sustained virologic response with direct-acting antiviral treatment on mortality in patients with advanced liver disease. Hepatology 2019;69:487e97. 14. Ministry of Health, Mongolia. Report on Healthy Liver Program. 2019. Ulaanbaatar, Mongolia.

7 15. Backus LI, Belperio PS, Shahoumian TA, Loomis TP, Mole LA. Real-world effectiveness and predictors of sustained virological response with all-oral therapy in 21,242 hepatitis C genotype-1 patients. Antivir Ther 2017;22:481e93. 16. Sarrazin C, Dvory-Sobol H, Svarovskaia ES, Doehle BP, Pang PS, Chuang SM, et al. Prevalence of resistance-associated substitutions in HCV NS5A, NS5B, or NS3 and outcomes of treatment with ledipasvir and sofosbuvir. Gastroenterology 2016; 151:501e512 e1. 17. Zeuzem S, Mizokami M, Pianko S, Mangia A, Han KH, Martin R, et al. NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: prevalence and effect on treatment outcome. J Hepatol 2017;66:910e8. 18. Vallet-Pichard A, Mallet V, Nalpas B, Verkarre V, Nalpas A, Dhalluin-Venier V, et al. FIB-4: an inexpensive and accurate marker of fibrosis in HCV infection. comparison with liver biopsy and fibrotest. Hepatology 2007;46:32e6. 19. Lin YY, Hsieh CH, Chen JH, Lu X, Kao JH, Chen PJ, et al. De novo assembly of highly polymorphic metagenomic data using in situ generated reference sequences and a novel BLASTbased assembly pipeline. BMC Bioinf 2017;18:223. 20. International Agency for Research on Cancer. Estimated agestandardized incidence rates (World) in 2018, liver, both sexes, all ages. Available from: http://gco.iarc.fr/today/ online-analysis-map. [Accessed 3 September 2019]. 21. Baatarkhuu O, Naranzul N, Enkhtuya D, Saruul B, Gantuul C, Gegeebadrakh B, et al. Efficacy and safety of ledipasvir/sofosbuvir in 5052 Mongolian patients infected with hepatitis C virus genotype 1: real-life data from multi-center prospective cohort. Hepatol Int 2019;13(Suppl 1):S87. 22. Nemekhbaatar L, Oidov B, Palam L, Davaadorj T, Damba E, Jenskhan A, et al. Outcome of HCV treatment using ledipasvir/sofosbuvir combination in Mongolian population. Hepatol Int 2017;11(Suppl 1):S159. 23. Naranzul N, Baatarkhuu O. Sofosbuvir and ledipasvir fixed-dose combination without ribavirin in treatment-naive patients with hepatitis C virus infection in Mongolia. Hepatol Int 2018; 12(Suppl 2):S497. 24. Cheng G, Tian Y, Doehle B, Peng B, Corsa A, Lee YJ, et al. In vitro antiviral activity and resistance profile characterization of the hepatitis C virus NS5A inhibitor ledipasvir. Antimicrob Agents Chemother 2016;60:1847e53. 25. Ogawa E, Furusyo N, Nomura H, Dohmen K, Higashi N, Takahashi K, et al. NS5A resistance-associated variants undermine the effectiveness of ledipasvir and sofosbuvir for cirrhotic patients infected with HCV genotype 1b. J Gastroenterol 2017;52:845e54. 26. Kozuka R, Hai H, Motoyama H, Hagihara A, Fujii H, UchidaKobayashi S, et al. The presence of multiple NS5A RASs is associated with the outcome of sofosbuvir and ledipasvir therapy in NS5A inhibitor-naive patients with chronic HCV genotype 1b infection in a real-world cohort. J Viral Hepat 2018;25:535e42. 27. Mawatari S, Oda K, Tabu K, Ijuin S, Kumagai K, Fujisaki K, et al. The co-existence of NS5A and NS5B resistance-associated substitutions is associated with virologic failure in Hepatitis C Virus genotype 1 patients treated with sofosbuvir and ledipasvir. PLoS One 2018;13:e0198642. 28. Mizokami M, Dvory-Sobol H, Izumi N, Nishiguchi S, Doehle B, Svarovskaia ES, et al. Resistance analyses of Japanese hepatitis C-infected patients receiving sofosbuvir or ledipasvir/sofosbuvir containing regimens in phase 3 studies. J Viral Hepat 2016;23:780e8. 29. Akamatsu S, Hayes CN, Ochi H, Uchida T, Kan H, Murakami E, et al. Association between variants in the interferon lambda 4 locus and substitutions in the hepatitis C virus non-structural protein 5A. J Hepatol 2015;63:554e63. 30. Peiffer KH, Sommer L, Susser S, Vermehren J, Herrmann E, Doring M, et al. Interferon lambda 4 genotypes and resistance-

Please cite this article as: Hsu S-J et al., Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.10.003

+

MODEL

8 associated variants in patients infected with hepatitis C virus genotypes 1 and 3. Hepatology 2016;63:63e73. 31. Malov IV, Malov SI, Savilov ED, Gantulga D, Stepanenko LA, Ogarkov OB, et al. Population polymorphism of IFNL3 and IFNL4 genes of type 3 interferon associated with spontaneous clearance of hepatitis C virus in representatives of Caucasian and Mongoloid races. Bull Exp Biol Med 2016;161:404e7. 32. Wang Y, Rao HY, Xie XW, Wei L. Direct-acting antiviral agents resistance-associated polymorphisms in Chinese treatmentnaive patients infected with genotype 1b hepatitis C virus. Chin Med J 2015;128:2625e31. 33. Ikeda H, Watanabe T, Okuse C, Matsumoto N, Ishii T, Yamada N, et al. Impact of resistance-associated variant dominancy on

S.-J. Hsu et al. treatment in patients with HCV genotype 1b receiving daclatasvir/asunaprevir. J Med Virol 2017;89:99e105. 34. Huang CF, Hung CH, Cheng PN, Bair MJ, Huang YH, Kao JH, et al. An open-label, randomized, active control trial of 8 versus 12 weeks of elbasvir/grazoprevir for treatment-naive chronic hepatitis C genotype 1b patients with mild fibrosis (EGALITE): impact of baseline viral loads and NS5A resistanceassociated substitution. J Infect Dis 2019;220:557e66. 35. Ji F, Yeo YH, Wei MT, Ogawa E, Enomoto M, Lee DH, et al. Sustained virologic response to direct-acting antiviral therapy in patients with chronic hepatitis C and hepatocellular carcinoma: a systematic review and meta-analysis. J Hepatol 2019; 71:473e85.

Please cite this article as: Hsu S-J et al., Resistance-associated substitution and ledipasvir/sofosbuvir therapy in Mongolian chronic hepatitis C patients, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.10.003