Hepatitis C virus (HCV) is a major leading cause of chronic liver disease including cirrhosis and hepatocellular carcinoma and about 130 to 150 million people globally have chronic hepatitis C (CHC) infection.^1,2 According to the sequencing of HCV isolate, there are seven genotypes and 67 subtypes.³ While genotype 1, 2, and 3 are more prevalent and found around the world-wide, genotype 4, 5 and 6 are distributed in limited area. Genotype 4 and 5 are mainly distributed in the Middle East and Africa, and 6 in the Southern China and Southeast Asia including Singapore, Laos, Thailand, Vietnam, and Myanmar, where comprises up to 50% of all hepatitis C patients. On the other hand, HCV genotype 6 is rare in Korea where its prevalence is known as about 1%.^4,5

Since new oral direct-acting-agents (DAA) have been introduced, a treatment paradigm for HCV infection is changing. However, although some clinical trials demonstrated higher sustained virologic response (SVR) achievement in genotype 6 patients using DAA, those data included only small number of patients with genotype 6.^6,7 Moreover, DAA-based regimen is not likely to be available in many countries yet because of countries’ or personal socio-economic situations. For these reasons, peginterferon (PEG-IFN) and ribavirin are still affordable treatment regimens in real-world setting.

This study was conducted to find the clinical characteristics and treatment outcomes in the patients infected with HCV genotype 6 in Korea.

From April 2004 to December 2014, data of patients infected with genotype 6 were reviewed from one regional hospital and seven hospitals affiliated with medical colleges located in Gyeonggi-Incheon region, South Korea. Among them, seven patients were from the Gyeonggi-Incheon Peginterferon Alpha and Ribavirin Effect in CHC Treatment (KIPECT) study group.⁸

Baseline clinical and virologic characteristics were obtained by retrospective review of medical records. Data collection was performed with an Excel (Microsoft Corp., Redmond, WA, USA)-based case report form by physicians at each individual hospital. The study protocol was approved by the Institutional Review Boards for exemption from the requirement for informed consent at each hospital and was conducted in accordance with the principles of the Declaration of Helsinki.

All patients were Koreans and anti-HCV positive more than 6 months or clinically assessed as a CHC by using laboratory and radiologic findings. HCV genotyping was conducted by the reverse hybridization principle, nested polymerase chain reaction (PCR) was performed with biotinylated primers from the 5′ untranslated region, and the second-round product was genotyped with a second-generation line probe assay (INNO-LiPA HCV II; Innogenetics, Ghent, Belgium) or restriction fragment mass polymorphism (RFMP; Genematrix Inc., Seongnam, Korea), or direct sequencing method as used in each institution. Qualitative HCV RNA, when it performed, was conducted by an RNA PCR and hybrid method, Cobas Amplicor HCV test version 2.0 (Roche Molecular Systems, Branchburg, NJ, USA; detection limit, 50 IU/mL). Quantitative HCV RNA was measured by real-time PCR assay using the Abbott RealTime HCV assay (Abbott Molecular, Des Plaines, IL, USA; lower detection limit 12 IU/mL) or CobasAmpliPrep/CobasTaqMan HCV assay (Roche Molecular Systems, Pleasanton, CA, USA; lower detection limit 15 IU/mL).

When the patients were treated, either PEG-IFN α-2a or PEG-IFN α-2b plus ribavirin were used. The starting dosage and dose modification of PEG-IFN and ribavirin were determined based on the current guidelines suggested by the Korean Association for the Study of the Liver. However, according to the nature of this retrospective study, selection and discontinuation as well as dosing and treatment duration of PEG-IFN and ribavirin were not controlled, but reflected the clinical practice of the attending physicians.

The patients who developed anemia, neutropenia, and/or thrombocytopenia were generally managed with a dose reduction or permanent discontinuation of the PEG-IFN or ribavirin, as per the guidelines provided.

A rapid virological response (RVR) was defined as undetectable serum HCV-RNA at week 4. The complete early virologic response (cEVR) is based on week 12 data and is defined as an undetectable HCV RNA. A partial EVR (pEVR) refers to a 2 log₁₀ or greater decrease from baseline in HCV RNA at week 12, but persistent detectable HCV RNA. End of treatment response (ETR) was defined as undetectable HCV RNA at the end point of treatment. A SVR was defined as undetectable HCV RNA 24 weeks after completion of antiviral therapy maintained throughout the remaining documented follow-up period. Virological relapse was defined as achieving an ETR but subsequently becoming HCV RNA positive after cessation of treatment. An adherence to antiviral therapy was defined as the actual dose administered divided by the total dose first expected.

Comparison of treatment outcome according to the presence of RVR and the treatment duration was performed using two-sided Fisher exact test.

A total of 32 patients were enrolled. Included patients were aged between 34 and 57 (median, 44) years, and 63% were male. No specific risk for CHC was identified but two patients who received tattooing from the same unlicensed person. Three patients showed hepatitis B surface antigen (HBsAg) positive but negative HBV DNA on their serum (Table 1).

Most common subtype was 6c, documented in 26 patients (81%). There were 6a in three patients, mixed 6/6c in two patients, and 6 in one patient, respectively. Baseline median aspartate transaminase level was 80 (24 to 1,123) U/L and alanine transaminase level was 88 (21 to 1,019) U/L, and HCV RNA level was 1,405,000 (96,500 to 28,844,529) IU/mL. Based on laboratory and imaging findings, five patients showed clinical features suggestive liver fibrosis.

Twenty-five treatment-naïve patients were treated with 180 μg of PEG-IFN α-2a or 1.0 to 1.5 μg/kg of PEG-IFN α-2b with 800 to 1,200 mg of ribavirin. Treatment duration was determined by physicians’ discretion, and intended treatment duration was ranged 24 to 54 weeks (Table 2).

Overall, SVR was attained in 13 patients (52%). As follow-up loss occurred in three patients, a total of 22 patients conformed to the treatment protocol. Seven patients relapsed and two were null-responders including one who discontinued treatment for no early virologic response.

By treatment durations, four of 10 patients attained SVR following 24 weeks and nine of 12 patients following more than 48 weeks treatment. When excluding three patients who lost follow-up, the treatment outcome was slightly improved according to longer duration of treatment (p=0.192).

RVR was checked in 14 patients. Five of six patients who showed positive RVR had attained SVR whereas four of eight patients without RVR attained SVR. Two patients who did not achieve EVR were nonresponder. In three patients with pEVR, two were relapsed and one attained SVR (p=0.301).

Most common adverse event was flu-like syndrome, and dose reduction was necessary in 24% (6/25) of patients. There was no treatment discontinuation by adverse events.

HCV genotype 6 is geographically restricted in South East Asia and surrounding regions, where 30% to 50% of all hepatitis C patients are infected with genotype 6.^9–11 Diverse subtypes of genotype 6 are accumulated and newly isolated subtypes are almost always reported in this area. So, it is suggested that genotype 6 may have been long circulated or evolved within Southeast Asia, and transmitted to the adjacent countries.¹² Whereas most data about genotype 6 HCV were centered on these regions, there have been not many data in Korea probably due to low prevalence of HCV genotype 6. While Shin et al.¹³ recently reported the prevalence of genotype 6 as 10.5%, most other studies reported the prevalence as about 1.0%.^8,13–15 The affiliated hospital of Shin et al.¹³ is located where many immigrants from Southeast Asia and China and drug abusers exist, which may explain the higher prevalence than others.

Genotype 6 HCV is highly diverse with 23 subtypes and certain subtypes have different geographic predominance like genotypes; 6a in South China, Taiwan, and Hong Kong, 6n in Myanmar, 6f in Thailand, and 6g in Indodesia.^1,16 Until recently, subtype 6c is an overwhelming subtype regardless of genotyping methods in Korea.^13–15 Interestingly, clustered 6c has not been reported but in Korea since first isolated as a sole strain from a commercial blood donor in Thailand.¹⁷ We assume that 6c is a rare HCV variant and its spread within Korea might be related with migration of some hosts.

In this study, RFMP was most commonly used, that is known as accurate for HCV genotyping. On the other hand, INNO-LiPA HCV II (Innogenetics) has been criticized due to less complete ability to distinguish genotype 6 from 1, especially 6a from 1b.^15,18–21 Although a gold standard for genotyping and subtyping is direct sequencing assay, RFMP assay could distinguish mass differences between oligonucleotide fragment levels and be most reliable modality among commercial kits. We also suggest that further study is required to investigate the prevalence and distribution of each subtypes of genotype 6 with direct sequencing or combining two or more modalities.

To date, virological and clinical features of hepatitis C genotype 6 are known to be not significantly different to genotype 1, and 2/3.^1,16,22 However, Korean patients infected with genotype 6 tend to be younger and have chronic hepatitis rather than advanced stage such as hepatocellular carcinoma or cirrhosis.^11,23 These suggest that it has been not so long since a transmission into Korea and propagation of HCV genotype 6 occurred. We would prevent further amplification of genotype 6 infection in Korea by tracking human migration and transmission route of genotype 6 HCV.

Genotype is an important factor of response to treatment. Prior studies have suggested that the HCV genotype 6 has responded to the IFN-based treatment better than genotype 1 but less than genotype 2/3. The 48-week treatment may be more effective to achieve SVR than 24-week treatment in PEG-IFN and ribavirin combination. Those who achieve RVR may receive the shorter 24-week treatment.^23–27 Despite statistically insignificant with too small sample size, our results showed a similar trend to the prior data. In our study, SVR rates were 40% and 75% according to the 24- and more than 48-week of PEG-IFN and ribavirin treatment respectively and RVR showed high positive predictive value (83%).

Since the introduction of DAA, a treatment paradigm for CHC has been rapidly changing. Combination of PEG-IFN and ribavirin is not recommended as a standard treatment of genotype 1 anymore and plays a minimal role even in genotype 2/3.^5,28,29 However, unlike the major genotypes of HCV infection, data about minor genotypes such as genotype 6 are still limited.^6,30 In addition, a surpassing cost per SVR of DAA is a major hurdle in the real world.^31,32 Considering that a compulsory public health care system exists in Korea, where the lowest cost per cure is a more valuable, PEG-IFN and ribavirin could not be discarded yet. Therefore, we consider the results of our study are still meaningful for guide to treat patients infected with genotype 6 HCV.

This is a retrospective study. Nevertheless, a prospective study about genotype 6 HCV is hardly to be established due to low prevalence. To overcome inhomogeneity of enrolled patients and treatment regimen, the subjects were limited as Koreans.

In this study, we showed that Korean patients infected genotype 6 HCV tended to be younger and have relatively short infection duration comparing to those who in the prevalent area. In Korea, 6c is the most common subtype. When treated with PEG-IFN and ribavirin combination, the overall SVR rate was observed as 52% (13/25) in patients. As like other HCV infection, longer duration of treatment and attainment of RVR are favorable to achieve SVR in genotype 6 HCV infection. Although the treatment profile of our study is not an updated one, we believe that this is valuable data to have insights regarding the evolution and spread of genotype 6 HCV in Korea and manage patients infected with genotype 6 HCV.