High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays

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Journal of the Formosan Medical Association xxx (xxxx) xxx

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Original Article

High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays Jyh-Jou Chen a,*, Hung-Da Tung a, Pei-Lun Lee a, Hsing-Tao Kuo b, Ming-Jen Sheu b, Chun-Ta Cheng a, Tang-Wei Chuang a, Hsu-Ju Kao a, Na-Mi Lu c, Li-Ching Wu d,e, Chuan Lee f a Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chi-Mei Medical Center, Liouying, Tainan, Taiwan b Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chi-Mei Medical Center, Yongkang, Tainan, Taiwan c Department of Pathology, Chi-Mei Medical Center, Liouying, Tainan, Taiwan d Department of Clinical Pathology, Chi-Mei Medical Center, Yongkang, Tainan, Taiwan e Institute of Biomedical Science, National Sun Yat-sen University, Kaohsiung, Taiwan f Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chi-Mei Hospital, Chiali, Tainan, Taiwan

Received 26 March 2019; received in revised form 12 July 2019; accepted 18 July 2019

KEYWORDS Genotype 6; Hepatitis C; Genotype assay; Direct acting antiviral

Background/Purpose: Abbott RealTime Genotype II assay can effectively identify hepatitis C virus (HCV) genotypes (GTs), but some GT 6 subtypes might not be differentiated from GT 1. Abbott RealTime Genotype II PLUS and sequencing might be needed to resolve these ambiguous results. Unlike the high prevalence of GT 6 in Southeast Asia, GT 6 had rarely been reported in Taiwan except in intravenous drug abusers (IDU). But the prevalence of GT 6 in Taiwan might be underestimated. We conducted this study to determine the GTs in a HCV endemic area in Southern Taiwan. Methods: A total of 1147 patients with hepatitis C viremia for direct acting antivirals (DAA) treatment at the Chi Mei medical system in Tainan were enrolled. Genotype was determined using a working flow consisted of Abbott GT II, PLUS assays and 50 untranslated region (50 UTR)/ core sequencing.

Abbreviations: 50 UTR, 50 untranslated region; DAA, direct acting antiviral; GT, genotype; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IDU, injection drug users; LiPA, line probe assay; NS5B, nonstructural protein 5B; PCR, polymerase chain reaction; RT, reverse transcription. * Corresponding author. No. 201, Taikang, Vil. Taikang, Dist. Liouying, Tainan City, 736, Taiwan. Fax: þ886 6 2828928. E-mail address: [email protected] (J.-J. Chen). https://doi.org/10.1016/j.jfma.2019.07.021 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: Chen J-J et al., High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.07.021

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J.-J. Chen et al. Results: Among the 1147 patients, 883 (77.0%) obtained GT results by GT II, 264 (23.0%) samples with ambiguous results by GT II assay received further tests, including 194 (73.5%) with PLUS assay and 70 (26.5%) with 50 UTR/core sequencing. Nearly three-quarters (73.5%) of ambiguous results by GT II assay were GT 6. Overall, 18.3% of samples were GT 6. Phylogenetic study of 11 samples of GT 6 subtypes showed 7 (63.6%) were 6 g. Conclusions: GT 6 is the major factor for high ambiguous rate by GT II. Unexpected high prevalence of GT 6 (18.3%) in Southern Taiwan, especially subtype 6 g, closely related to Indonesian strains, is first reported. 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 Taiwan is a hepatitis C virus (HCV) endemic country with an overall prevalence of positive HCV antibody (anti-HCV) of about 4e5% in adults but with considerable geographic variation.1e12 Previous studies had reported that genotypes (GT) 1 and 2 were the major HCV GTs in Taiwan.4e12 However, most of these studies were performed decades ago, earlier genotyping methods cannot detect GT 6. A recent report in Southeastern Taiwan revealed that the prevalence of patients infected with HCV GT 6 increased to 5.2%,13 and the prevalence of HCV GT 6 among injection drug users (IDU) in Taiwan were reported to be up to 26%.14 These findings are higher than the previous reports in Taiwan.4e12 We also encountered patients whose relapsed genotype changed from GT 1 to 6 using different genotyping method. The Abbott RealTime HCV Genotype II assay uses realtime polymerase chain reaction (PCR) to amplify and detect portions of the 50 untranslated region (50 UTR) and nonstructural protein 5B(NS5B). The assay for GT 1 to 6 were detected using probes targeting the 50 UTR amplicons, and subtypes 1a and 1b were detected using probes targeting the NS5B amplicons. Previous studies have described that the Abbott GT II assay could effectively identify HCV GTs; however, some studies have shown that it could not discriminate some GT 6 subtypes from GT 1 or differentiate subtype 1a from 1b in 4.3e9.1% samples.15 In our institutes, up to 16.6% samples were genotyped as GT 1 without subtype designation, significantly higher than earlier reports.16 Although direct sequencing is an alternative method and gold standard to resolve those with ambiguous results but it is time and labor consuming. The Abbott RealTime Genotype II PLUS assay was developed to identify subtypes 1a and 1b and GT 6 and could be a useful approach to resolve ambiguities.17 Using Abbott GT II assay and GT II PLUS assay were reported to accurately evaluate the prevalence of GT 6 HCV infection.18,19 In the era of using DAAs to treat chronic hepatitis C, differentiating GTs before treatment is extremely important in selecting the appropriate treatment agents and duration, especially for the genotype-restricted regimens such as daclatasvir/asunaprevir and elbasvir/grazoprevir, both were not indicated for GT 6. Since 24 January 2017, paritaprevir/ritonavir/ombitasvir þ dasabuvir and daclatasvir/asunaprevir were reimbursed for GT 1 and 1b patients respectively with previous Interferon/ribavirin

failure and fibrosis score S3 by the National Health Insurance in Taiwan,20 the inclusion criteria was broadened to naı¨ve patients in May later. Hence, genotype determination is crucial and necessary before commencing DAA treatment. This study was designed to evaluate the Abbott GT assays and with supplementary sequencing on HCV genotyping and prevalence of genotypes in a highly endemic area of HCV infection in southern Taiwan.

Materials and methods A total of 1147 (514 men and 633 women; mean age, 65.0  11.9 years) patients with hepatitis C viremia who were followed up in Chi Mei medical system (i.e., Yunkang, Liouying, and Chiali campuses) for DAA treatment in Tainan, Taiwan, between July 2016 and June 2017 were included in this study. The working flow of determination of genotypes of HCV was shown in Fig. 1. In brief, the Abbott GT II(Des Plaines, IL, USA) was used to analyze the presence of HCV GTs, and then PLUS assay (Des Plaines, IL, USA) was used when GT II

Figure 1 VL: viral load; LOD: limit of detection; RUO: research use only.

Please cite this article as: Chen J-J et al., High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.07.021

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High GT 6 HCV prevalence in Southern Taiwan revealed ambiguous results. Genotyping by 50 UTR/core sequencing was conducted to check genotypes when PLUS assay still could not determine the accurate genotypes. The procedures were conducted following the standard manufacturer’s instructions. Although GT II PLUS assay were reported to accurately to detect GT 6 HCV infection. As sequencing of HCV is a relatively accurate method to determine genotype 6. To verity the detection rate of genotype 6 by GT II PLUS assay, forty-one patients (15 men and 26 women; mean age, 65.2  13.0 years) with Abbott GT II shown genotype 1 but no subtype designation or “indeterminate” result or HCV not detected results between 1 Jan 2017 and 30 June 2017 were selected for confirmation study using 50 UTR/core, E1 and NS 5B sequencing. The accuracy of genotype 6 was verified by phylogenetic analysis of E1 and NS5B in 11 patients.

Genotyping by Abbott GT II and PLUS assays For analysis GTs with the Abbott GT II and PLUS assays, RNA was extracted from the 500 uL serum on the Abbott m2000sp system using the Abbott mSample Preparation System kit (Des Plaines, IL, USA) according to the manufacturer’s instruction. Reverse transcription (RT)-PCR master mixes were set up using the Abbott m2000sp and Abbott RealTime HCV Genotype II Amplification Reagent Kit or the RealTime HCV Genotype PLUS Amplification Reagent Kit (Des Plaines, IL, USA). RT-PCR reactions were performed using the Abbott m2000rt instrument.

Genotyping by 50 UTR/core sequencing HCV RNA was extracted from 200 uL patient serum, nested PCRs were performed using Invitrogen kit to amplify the 50 UTR and core regions. For cDNA synthesis and the nested PCR, in the first PCR round the outer primers corresponded to HCV-1 sequence 268 to 251 (50 AGCGTCTAGCCATTGGCGT) and antisense primer core 391e410, (50 ATGTACCCC ATGAGGTCGGC) were used, numbered according to Bukh et al.21 Nested PCR primers were 199 to 183 (50 GTGGTCTGCGGAACCGG) and antisense core 364e383 (50 CAC/TGTA/GAGGGTATCGATGAC).22,23 PCR products lengths were 681 and 589 bps respectively which were purified and sequenced for analysis. Sequences generated were aligned using Basic Local Alignment Search Tool (BLAST) of NCBI database to determine genotypes.

Phylogenetic analysis by E1 and NS5B sequencing To verify whether GT 6 was accurately detected by current practices in our laboratory, 11 samples that were genotyped with GT 6 were randomly selected and sequenced using E1 and NS5B genes. For NS5B gene analysis, part of NS5B gene (330 bp) and E1 gene (750 bp) were amplified by PCR. The PCR products were then sequenced for following phylogenetic tree analysis to determine HCV GTs. MEGA software version 6.0 was used to construct the phylogenetic tree using the neighbor-joining method based on the Kimura 2-parameter distance.

3 The study was approved by ethical committee of Chi Mei Medical Center and informed consent was obtained from each patient.

Statistical analysis The significance of possible associations between discrete variables was compared using Chi-square test. The level of statistical significance was set at two-tailed P < 0.05.

Results Among the 1147 patients, 883 (77.0%) were successfully genotyped using GT II assay, including 46 (5.2%) GT 1a, 380 (43.0%) GT 1b, 428 (48.5%) GT 2, 4 (0.5%) GT 3, 4 (0.5%) GT 4, 16 (1.8%) GT 6, and 5 (0.6%) mixed type (Table 1). A total of 264 (23.0%) patients showed ambiguous results, including 191 (73.3%) unsubtyped GT 1, 60 (22.7%) undifferentiated results, and 13 (4.9%) others (mixed or undetected) (Table 2). One hundred and ninety-four (73.5%) of the samples with ambiguous results were successfully genotyped using GT II PLUS assay, including 31 (16.0%) were GT 1 (3 GT 1a, and 28 GT 1b), 157 (80.9%) GT 6, and 6 (3.1%) mixed types (Table 1). When using the Abbott GT II and PLUS assays to determine HCV GTs, 1077 of 1147 (93.9%) samples could be successfully genotyped, but there still were 70 (6.1%) samples need complement with 50 UTR/core sequencing to determine the genotypes, including 2 GT 1a, 12 GT 1b, 19 GT 2 and 37 GT 6. Overall, a total of 471 (41.1%), 447 (39.0%), 210 (18.3%), 11 (1.0%) patients had GT 1, GT 2, GT 6, and mixed type respectively (Table 1). Among the 51 patients with GT 1a (4.4%), 46 (90.2%) patients were detected using GT II assay and 5 (9.8%) with PLUS assay and sequencing; among the 420 (36.7%) patients with 1b, 380 (90.5%) and 28 (6.7%) patients were detected using GT II and PLUS assays, respectively; and the rest 12 (2.9%) were detected by sequencing. Among the 447 patients with GT 2 (39.0%), 428 (95.7%) and 19 (4.3%) were detected using GT II and sequencing respectively. All GT 3 and GT 4 were detected using GT II assay. Among the 210 (18.3%) GT 6 patients, 16 (7.6%) and 157 (74.8%) were Table 1 Genotypes determined using working flow with Abbott GT II, PLUS assays and sequencing. Genotypes

GT II

PLUS

Sequencing Total

GT 1(T) GT 1a GT 1b GT 2 GT 3 GT 4 GT 6 Mixed types(T) GT 1b & 6 GT 1b & 2 GT 1b, 2, 4 GT 1a & 2

426 46 380 428 4 4 16 5

90.4% 90.2% 90.5% 95.7% 100% 100% 7.6% 45.5%

31 3 28 0 0 0 157 6

6.6% 5.9% 6.7% 0.0% 0.0% 0.0% 74.8% 54.5%

14 2 12 19 0 0 37 0

3.0% 3.9% 2.9% 4.3% 0.0% 0.0% 17.6% 0.0%

471 51 420 447 4 4 210 11

41.1% 4.4% 36.6% 39.0% 0.3% 0.3% 18.3% 1.0%

0 2 1 2

0.0% 100% 100% 0.2%

6 0 0 0

100% 0.0% 0.0% 0.0%

0 0 0 0

0.0% 0.0% 0.0% 0.0%

6 2 1 2

0.5% 0.2% 0.1% 0.2%

883 77.0% 194 16.9% 70 6.2%

1147

Please cite this article as: Chen J-J et al., High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.07.021

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J.-J. Chen et al. Table 2

Genotypes determined by GI II PLUS assay in ambiguous samples by GT II.

GT 1a GT 1b GT 6 Mixed types Subtotal Undetermined Total

Unsubtyped GT 1

Indeterminate

Others

2 26 122 5 155 36 191

0 1 27 1 29 31 60

1 1 8 0 10 3 13

1.3% 17.0% 79.7% 2.0%

72.3%

detected using GT II and PLUS assay respectively. The rest 37 (17.6%) were detected by sequencing (Table 1), nearly 80% of (122/155, 78.7%) GT 1s without subtype were GT 6 using GT II PLUS assay. Among the 11 patients who showed mixed GTs in the final report, 6 with GT 6 (all with GT 1b) were all detected using the GT II PLUS assay but not GT II or sequencing. When analyzing the age of patients with genotype 6 infection, about 90% of patients were aged >50 years, and GT 6 were more common in patients aged >70 years (P Z 0.0014) (Table 3). The process to verify Abbott GT II PLUS assay results with 50 UTR/E1, core and NS5B and sequencing shown PLUS assay were 100% concordant with sequencing (Tables 4 and 5) Nearly 80% (23/29) of GT1 without subtype samples were GT6, of them, 21 could be detected by GT II PLUS assay. Twenty-seven percent (11/41) samples that cannot be determined genotype by GT II PLUS assay, the genotype can be determined by sequencing in 82% (9/11). (Amplification

70 years

Mixed type 6 0.8% 3 GT 1 314 43.4% 161 GT 2 286 39.6% 161 GT 3 4 0.6% 0 GT 4 2 0.3% 2 GT 6 111 15.4% 97 Total 723 424

0.7% 38.0% 38.0% 0.0% 0.5% 22.9%

Total 9 475 447 4 4 208 1147

0.8% 41.4% 39.0% 0.3% 0.3% 18.1%

4.9%

1.5% 14.4% 80.9% 3.1% 73.5% 26.5%

Table 5 Comparison of genotyping results between Abbott GT II PLUS and sequencing. PLUS

Sequencing GT1a GT1b GT2a GT6 ND

GT1a

GT1b

GT6

ND

1

4

25

11

1 4 25

0 1 5 3 2

ND: not detected.

0.8207 0.0700 0.6163

Discussion

0.5885 0.0014

GT II 1

Indeterminate

ND

Total

29

11

1

41

1 4 21 3

0 0 4 7

0 0 0 1

1 4 25 11

ND: not detected.

3 28 157 6 194 70 264

P-value

Table 4 Comparison of genotyping results between Abbott GT II and GT II PLUS.

PLUS 1a 1b 6 ND

22.7%

10.0% 10.0% 80.0% 0.0%

failed in two samples with extremely low viral load, 77 and < 12 IU respectively.) The preliminary phylogenetic analysis on 11 patients with GT 6 using maximum likelihood method demonstrated that the most common subtype was 6 g (n Z 7), followed by 6a (n Z 3) and 6w (n Z 1). Seven GT 6 g clustered with HK6554, JK046 and JK065 strains, while the only GT 6w was closely related two D140 and D370 strains in injections drug users reported earlier in Taiwan (Fig. 2(a) and (b)).14

Table 3 The ratio of genotypes between elder (70 years old) and younger patients. <70 years

0.0% 3.4% 93.1% 3.4%

Total

In this study, HCV genotypes could be determined in nearly 94% (1077/1147) of HCV samples using the Abbott GT II and PLUS assays. Among the group with ambiguous results by GT II assay, GT 6 was the major GT determined (194/264, 73.5%), either by PLUS assay (89.9%, 157/194) or by sequencing (52.9%, 37/70) respectively. Previous studies have shown that Abbott GT II failed to subtype GT 1 samples in 4.3e9.1%.16,17,24 GT 6 may also be misidentified as GT 1 due to the high genetic identity in the 50 UTR among 1b subtypes and some GT 6 strains.25 Only 2 of 14 GT 6 samples could be identified as GT 6 using GT II assay.,15 in contrast, GT II PLUS identified 35 of 42 GT6 samples as GT 6.,17 suggesting improved GT 6 detection rate using GT II PLUS. However, no subtype 6 g or 6w were included in both studies. Hence, in high HCV GT 6 or some rare subtype prevalence area, GT II assay that is designed to detect genotype 6a and 6b only, might not be sufficient to discriminate GT 6 from GT 1 and some rare subtypes, and these explained why lower rate of genotype 6 detection by

Please cite this article as: Chen J-J et al., High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.07.021

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Figure 2 Phylogenetic trees of 11 variants based on 330-bp NS 5b(A) and 750-bp E1(B) were labeled in green lines (CMH-). The bar at lower left indicates the genetic divergence. Reference HCV sequenced are indicated with an isolate name followed by a Genbank accession number. Please cite this article as: Chen J-J et al., High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.07.021

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6 GT II assay in our patients. Those with unsubtyped GT 1 should be proceeded to PLUS assay and/or sequencing. In our study, 78.7% (122/155) of unsubtyped GT 1 were GT 6, only 18.1% were GT 1. Line probe assay (LiPA) 2.0 had also been reported that failed to subtype GT 1 samples in 2.2e7.4% and a potentially high rate (up to 52%) of mistyping GT 6 samples as GT 1,26 suggesting that true GT 6 prevalence might be underestimated. The PLUS assay was developed to discriminate 1a, 1b and genotype 6. It incorporates probes to detect 6c-6l with improved detection rate up to 83% (35/42),17 hence it could be useful to resolve most of the ambiguous results or GT 1 without subtype. Our verify study about the PLUS assays of genotypes could 100% concordant with sequencing method. But there were still about 6% samples cannot be identified genotype by GT II with PLUS assays. When to resolve the higher rate uncertain genotype by Abbott GT II assay, we incidentally discovered the high genotype 6 prevalence, esp. 6 g, in southern Taiwan. From the results of our study, HCV GTs determination with working flow included GT II, PLUS assays and sequencing could be useful in an area with high prevalence of genotype 6 or high ambiguous result rate of GT II assay, especially GT 1 without subtype. HCVs are classified into 7 genotypes designated as GT 1e7, which are further classified into 67 subtypes,27 and up to 86 subtypes were identified until June 2107.28 Genotype 6 was the most diverse genotype and comprised of up to 29 subtypes from a to xf and 19 unassigned subtypes and possibly new subtypes to be identified.29 In fact, the earlier HCV isolates from East Asia were so divergent that they were initially classified as genotypes 7, 8, 9, and 11.30 These strains have been reclassified as individual subtypes within GT 6.31 Unlike the global GT 1, 2 and 3 HCV infections, genotype 6 isolates have been found exclusively in Southeast Asia and South China or immigrants from this region.32 But in Taiwan, GT 6 was seldom reported except in IDU and patients with human immunodeficiency virus (HIV) infection, 48 of 144 HIV patients with mono-infected HCV were GT 6, including 43 with 6a, 4 with 6n and one with 6w, excluding one with recombinant 2b6w.14 In contrast, only 3 samples were GT 6a in our limited phylogenetic data, and the predominant subtype was 6 g (7/11), suggesting different modes or routes of infection between IDU/HIV and our patients. GT 6 g samples in our study were closely related to Jakarta Indonesia strain JK 046 and 065 (Fig. 2). Interestingly, both Jakarta and Tainan were the capitals of colony by Netherlands in Seventeenth century. Colonial history and slave trade had been reported to shape the GT 2 diversity in Amsterdam as well as in West Africa.33,34 Such a phylogenetic, geographic distribution, evolution and colonial history correlation of GT 6 g strains between Jakarta, Indonesia and Tainan, Taiwan need more data to clarify. Genotype 6 is mainly observed in Southeast Asia countries with more than 20 subtypes identified (for detail, see ref. 35). Such an endemic with highly divergent subtypes was suggestive of a long term continuous infection in these areas for over 1000 years.32 However, subtype 6 g is the only GT 6 subtype reported in Jakarta, Indonesia. A recent study from Hainan island of China, which is in the vicinity of Vietnam, found a unique ecosystem of Austronesian descendants with HCV infection maintained for 600 years with

J.-J. Chen et al. a novel GT 6 virus that closely related to subtype 6 g and 6w.36 In a global collective study of HCV patients treated with different Sofosbuvir-based regimens, including over 14,000 patients, a new GT 6 subtype closely related with 6 g and 6w was identified in Tainan.29 This further supported that GT 6 g might has existed in Southern Taiwan for centuries with new subtype evolved as these patients with GT 6 were significantly older than non-GT 6 patients. In conclusions, genotyping using Abbott GT II/PLUS and 50 UTR/core sequencing and verified the authenticity of GT 6 with E1 and NS5B sequences phylogenetic analysis, we found that GT 6 infection was not rare (18.3%) in Southern Taiwan. The low GT 6 prevalence of previous studies in Taiwan could be underestimated due to the limitation of genotyping methods and/or geographically varied genotype distribution. HCV GTs determination with working flow included GT II, PLUS assay and sequencing could be useful in areas with high rate of GT 1 no subtype or GT 6 prevalence. To clarify the time and routes of transmission and epidemiology of GT 6 HCV, especially subtype 6 g in Taiwan, more data are needed.

Financial disclosure The study was supported by grants from the Chimei Medical Center, Liouying, Taiwan to JJ Chen (CLFHR10722) and HD Tung (CLFHR10702).

Conflicts of interest All authors declare that they have no conflicts of interest.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jfma.2019.07.021.

References 1. Chen DS, Kuo GC, Sung JL. Hepatitis C virus infection in an area hyperendemic for hepatitis B and chronic liver disease: the Taiwan experience. J Infect Dis 1990;162:817e22. 2. Sun CA, Chen HC, Lu CF, You SL, Mau YC, Ho MS, et al. Transmission of hepatitis C virus in Taiwan: prevalence and risk factors based on a nationwide survey. J Med Virol 1999;59: 290e6. 3. Lu SN, Wang JH, Kuo YK, Kuo HL, Chen TM, Tung HD, et al. Predicting the prevalence of antibody to hepatitis C virus (HCV) in a community by the prevalence of elevated levels of alanine aminotransferase: a method to identify areas endemic for HCV. Am J Trop Med Hyg 2002;67:145e50. 4. Yu ML, Chuang WL, Chen SC, Dai CY, Hou C, Wang JH, et al. Changing prevalence of hepatitis C virus genotypes: molecular epidemiology and clinical implications in the hepatitis C virus hyperendemic areas and a tertiary referral center in Taiwan. J Med Virol 2001;65:58e65. 5. Chen CH, Sheu JC, Wang JT, Huang GT, Yang PM, Lee HS, et al. Genotypes of hepatitis C virus in chronic liver disease in Taiwan. J Med Virol 1994;44:234e6. 6. Wu JS, Lee HF, Hsiao HL, Lu HY, Chou WH, Lu CF, et al. Genogype distribution of hepatitis C virus infection in Taiwan. J Med Virol 1994;44:74e9.

Please cite this article as: Chen J-J et al., High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.07.021

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High GT 6 HCV prevalence in Southern Taiwan 7. Chan CY, Lee SD, Hwang SJ, Lu RH, Lu CL, Lo KJ. Quantitative branched DNA assay and genotyping for hepatitis C virus RNA in Chinese patients with acute and chronic hepatitis C. J Inf Dis 1995;171:443e6. 8. Kao JH, Chen PJ, Lai MY, Yang PM, Sheu JC, Wang TH, et al. Genotype of hepatitis C virus in Taiwan and the progression of liver disease. J Clin Gastroenterol 1995;21:233e7. 9. Yu ML, Chuang WL, Lu SN, Chen SC, Wang JH, Lin ZY, et al. The genotypes of hepatitis C virus in patients with chornic hepatitis C virus infection in southern Taiwan. Kaohsiung J Med Sci 1996; 12:605e12. 10. Wu CH, Lee MF, Kuo HS. Distribution of hepatitis C virus genotypes among blood donors in Taiwan. J Gastroenterol Hepatol 1997;12:625e8. 11. Lee PL, Wang JH, Tung HD, Lee CM, Lu SN. A higher than expected recovery rate from hepatitis C infection amongst adolescents: a community study in a hepatitis C-endemic township in Taiwan. Trans R Soc Trop Med Hyg 2004;98:367e72. 12. Lee CM, Lu SN, Hung CH, Tung WC, Wang JH, Tung HD, et al. Hepatitis C virus genotypes in southern Taiwan: prevalence and clinical implications. Trans R Soc Trop Med Hyg 2006;100: 767e74. 13. Cheng CH, Chen HL, Lin IT, Wu CH, Lee YK, Wong MW, et al. The genotype distribution of hepatitis C in southeastern Taiwan: clinical characteristics, racial difference, and therapeutic response. Kaohsiung J Med Sci 2015;31:597e602. 14. Lee YM, Lin HJ, Chen YJ, Lee CM, Wang SF, Chang KY, et al. Molecular epidemiology of HCV genotypes among injection drug users in Taiwan: full-length sequences of two new subtype 6w strains and a recombinant form_2b6w. J Med Virol 2010;82: 57e68. 15. Mallory AM, Lucic D, Sears MT, Cloherty GA, Hillyard DR. Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5’UTR, core and NS5B sequencing. J Clin Virol 2014 May;60(1):22e6. 16. Chevaliez S, Bouvier-Alias M, Brillet R, Pawlotsky JM. Hepatitis C virus (HCV) genotype 1 subtype identification in new HCV drug development and future clinical practice. PLoS One 2009; 4(12):e8209. https://doi.org/10.1371/journal.pone.0008209. Published 2009 Dec 8. 17. Mallory MA, Lucic D, Ebbert MT, Cloherty GA, Toolsie D, Hillyard DR. Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay with reference to core and NS5B sequencing. J Clin Virol 2017;90:26e31. 18. Mokhtari C, Ebel A, Reinhardt B, Merlin S, Proust S, RoqueAfonso AM. Characterization of samples identified as hepatitis C virus genotype 1 without subtype by abbott RealTime HCV genotype II assay using the new abbott HCV genotype plus RUO test [published correction appears in J Clin Microbiol. 2016 sep; 54(9):2402]. J Clin Microbiol 2016;54(2):296e9. https: //doi.org/10.1128/JCM.02264-15. 19. He C, Germer JJ, Ptacek ER, Bommersbach CE, Mitchell PS, Yao JDC. Utility of the abbott RealTime HCV genotype plus RUO assay used in combination with the abbott RealTime HCV genotype II assay. J Clin Virol 2018 FebeMar;99e100:97e100. https://doi.org/10.1016/j.jcv.2018.01.004. Epub 2018 Jan 12. 20. National Health Insurance Administration of Ministry of Health and Welfare. https://www.nhi.gov.tw/english/News_Content. aspx?nZ996D1B4B5DC48343& smsZF0EAFEB716DE7FFA&sZ 5B9044CF1188EE23. November 12 2018. 21. Bukh J, Purcell RH, Miller RH. Sequence analysis of the 5’ noncoding region of hepatitis C virus. Proc Natl Acad Sci USA 1992 Jun 1;89(11):4942e6.

7 22. Pohjanpelto P, Lappalainen M, Widell A, Asikainen K, Paunio M. Hepatitis C genotypes in Finland determined by RFLP. Clin Diagn Virol 1996 Oct;7(1):7e16. 23. Mellor J, Holmes EC, Jarvis LM, Yap PL, Simmonds P. Investigation of the pattern of hepatitis C virus sequence diversity in different geographical regions: implications for virus classification. The International HCV Collaborative Study Group. J Gen Virol 1995 Oct;76(Pt 10):2493e507. 24. Yang R, Cong X, Du S, Fei R, Rao H, Wei L. Performance comparison of the versant HCV genotype 2.0 assay (LiPA) and the abbott realtime HCV genotype II assay for detecting hepatitis C virus genotype 6. J Clin Microbiol 2014 Oct; 52(10):3685e92. 25. Murphy DG, Willems B, Desche ˆnes M, Hilzenrat N, Mousseau R, Sabbah S. Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5’ untranslated region sequences. J Clin Microbiol 2007 Apr;45(4):1102e12. 26. Cai Q, Zhao Z, Liu Y, Shao X, Gao Z. Comparison of three different HCV genotyping methods: core, NS5B sequence analysis and line probe assay. Int J Mol Med 2013 Feb;31(2): 347e52. 27. Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, et al. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology 2014;59: 318e27. 28. International Committee on Taxonomy of Viruses (ICTV). https://talk.ictvonline.org/ictv_wikis/flaviviridae/w/sg_flavi/ 634/table-1—confirmed-hcv-genotypes-subtypes-june-2017; 2018. Accessed November 12. 29. Hedskog C, Parhy B, Chang S, Zeuzem S, Moreno C, Shafran SD, et al. Identification of 19 novel hepatitis C virus subtypesfurther expanding HCV classification. Open Forum Infect Dis 2019;6(3):ofz076. https://doi.org/10.1093/ofid/ofz076. Published 2019 Feb 22. 30. Tokita H, Okamoto H, Tsuda F, Song P, Nakata S, Chosa T, et al. Hepatitis C virus variants from Vietnam are classifiable into the seventh, eighth, and ninth major genetic groups. Proc Natl Acad Sci USA 1994 Nov 8;91(23):11022e6. 31. Simmonds P, Bukh J, Combet C, Dele ´age G, Enomoto N, Feinstone S, et al. A Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005 Oct;42(4):962e73. 32. Pybus OG, Barnes E, Taggart R, Lemey P, Markov PV, Rasachak B, et al. Genetic history of hepatitis C virus in east Asia. J Virol 2009 Jan;83(2):1071e82. 33. Markov PV, van de Laar TJ, Thomas XV, Aronson SJ, Weegink CJ, van den Berk GE, et al. Colonial history and contemporary transmission shape the genetic diversity of hepatitis C virus genotype 2 in Amsterdam. J Virol 2012 Jul; 86(14):7677e87. 34. Markov PV, Pepin J, Frost E, Deslandes S, Labbe ´ AC, Pybus OG. Phylogeography and molecular epidemiology of hepatitis C virus genotype 2 in Africa. J Gen Virol 2009 Sep;90(Pt 9): 2086e96. 35. Thong VD, Akkarathamrongsin S, Poovorawan K, Tangkijvanich P, Poovorawan Y. Hepatitis C virus genotype 6: virology, epidemiology, genetic variation and clinical implication. World J Gastroenterol 2014 Mar 21;20(11):2927e40. 36. An Y, Wu T, Wang M, Lu L, Li C, Zhou Y, et al. Conservation in China of a novel group of HCV variants dating to six centuries ago. Virology 2014;464e465:21e5.

Please cite this article as: Chen J-J et al., High prevalence of genotype 6 hepatitis C virus infection in Southern Taiwan using Abbott genotype assays, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2019.07.021