Besides seven major hepatitis C virus (HCV) genotypes (GT), a number of intergenotypic recombinant strains have been described. These so-called chimeras combine genetic characteristics of different HCV genotypes.
KEYWORDS: 2k/1b, hepatitis C virus, assay, chimera, recombinant
ABSTRACT
Besides seven major hepatitis C virus (HCV) genotypes (GT), a number of intergenotypic recombinant strains have been described. These so-called chimeras combine genetic characteristics of different HCV genotypes. However, correct genotype classification is important, as choice and duration of direct-acting antiviral (DAA) treatment is mainly based on the viral genotype. Therefore, misclassification of chimeras might lead to suboptimal treatment of patients infected with these strains. For example, 2k/1b chimeras are typically described as HCV genotype 2 strains by commercially available hybridization assays, but real-time PCR-based tests recognizing another HCV region might be more suitable for correct chimera detection. In this study, the analytic capacity of the hybridization-assay Versant HCV Genotype 2.0 (LiPA 2.0) and the real-time PCR-based-assays cobas HCV GT and Abbott RealTime HCV Genotype II were tested in a selected cohort of 230 patients infected with HCV genotype 1 (n = 53) and 2 (n = 177) and 48 patients infected with HCV 2/1 chimeric strains. While the Versant HCV Genotype 2.0 (LiPA 2.0) assay failed to identify chimeras in all of the patients (48/48, 100%), cobas HCV GT and Abbott HCV Genotype II assays identified chimeras correctly in 90% (43/48) and 65% (31/48) of the cases, respectively. In conclusion, while the hybridization-based Versant HCV Genotype 2.0 (LiPA 2.0) assay seems to be unsuitable for detection of HCV 2/1 chimeras, use of the real-time PCR-based assays cobas HCV GT and Abbott RealTime HCV Genotype II led to a higher rate of chimera detection.
INTRODUCTION
Chronic infection with hepatitis C virus (HCV) can lead to the development of liver cirrhosis and hepatocellular carcinoma. According to a recent WHO hepatitis C fact sheet (https://www.who.int/en/news-room/fact-sheets/detail/hepatitis-c), there are approximately 71 million people worldwide who are chronically infected with HCV, and approximately 399,000 people die every year due to HCV-related morbidity. HCV variants are characterized by a high genetic diversity due to the high viral turnover rate and lack of a proofreading activity of the viral RNA-dependent RNA polymerase. At least seven genotypes (GT) and 67 subtypes with up to 30% nucleotide divergence have been described (1). In addition, several intergenotypic recombinant HCV strains, so-called chimeras, have been found across the globe. These chimeras combine genetic characteristics of different HCV genotypes. The HCV variant 2k/1b, which was first described from a patient in St. Petersburg, Russia, has been infrequently but consistently detected among patients originating from the former Soviet Union (2–4). This chimera consists of a sequence fragment originating from an HCV genotype 2k strain (coding for the core region [core]ore, E1, E2, p7, and NS2) and a fragment originating from an HCV genotype 1b strain (coding for nonstructural protein [NS] 3, NS4A/B, and NS5A/B). Its recombination site was found at nucleotides −232 to −219 in the carboxyterminal part of NS2 (4). Phylogenetic analyses have revealed that 2k/1b chimeras are extremely closely related, indicating a single recombination event in the past (4).
2k/1b chimeras are typically described as HCV genotype 2 strains by commercially available hybridization assays, which use the 5′ untranslated region (UTR) for genotype determination and include the HCV core for subtype discrimination (5). In contrast to hybridization assays, real-time PCR-based tests, which use the 5′-UTR for genotype determination and nonstructural protein (NS) 5B for subtype discrimination, may be suitable for correct chimera detection (4). In addition to HCV 2k/1b chimeras, other chimeras with united fragments of other HCV genotype 1 and 2 subtypes were also described, but with a much lower prevalence (4, 6–8).
Since the choice of direct-acting antiviral (DAA) treatment is based on a number of factors, including the viral genotype, misclassification of HCV genotypes may potentially lead to insufficient treatment of patients infected with these chimeric strains. For example, treatment of patients infected with 2k/1b chimeras leads to poor sustained virologic response (SVR) rates of <10% when treated with sofosbuvir (SOF)-ribavirin (RBV) for 12 weeks, like patients infected with genotype 2, as the antiviral targets for SOF within the replication HCV machinery are encoded in the genotype 1b parts of the chimeras (4, 9). In contrast, SVR rates are high in this population when treated with DAA regimens approved for HCV-genotype 1, including ledipasvir (LDV) and SOF (4).
In this study, we analyzed the analytic capacity of the widely used reverse hybridization-based genotyping assay Versant HCV Genotype 2.0 (LiPA 2.0) and the real-time PCR-based assays cobas HCV GT and Abbott RealTime HCV Genotype II in a large cohort of patients infected with HCV genotype 1b, genotype 2, or HCV 2/1 chimeras. Population-based sequencing of HCV core, the NS2/NS3 junction, and NS5B was used as the reference method, as previously described (4).
MATERIALS AND METHODS
Study population.
Serum samples of 278 patients infected with either HCV genotype 1 (subtype 1b) or genotype 2 were collected at study sites in Germany, Israel, and Italy for routine HCV resistance testing and genotype reevaluation at the University Hospital Frankfurt, Germany, as described elsewhere (4, 10). The present study aimed to investigate the proper discrimination of the most frequent HCV intergenotype recombinant/chimera 2k/1b from different HCV genotype 2 subtypes and HCV subtype 1b isolates.
The retrospective and anonymized analysis of leftover serum samples was approved by the local ethics committee (Ethikkomission der J. W. Goethe-Universität Frankfurt), and the study was performed in accordance with provisions of the Declaration of Helsinki and Good Clinical Practice guidelines.
Amplification and population-based sequencing of core, NS5B, and NS2/NS3 junction.
HCV core, the NS5B polymerase gene, and the NS2/NS3 junction were amplified by nested PCR and further analyzed by population-based sequencing. HCV RNA extraction, amplification, and sequencing were performed as described previously (4, 11). Sequencing of core and NS5B determination were performed for genotype and subtype determination in all samples. Sequencing of the NS2/NS3 junction was performed if core and NS5B sequencing revealed evidence for a recombinant chimera strain. The primers used were described recently (4, 11). All sequences were proofread and then subtype analyses were performed using Megablast bioinformatics software (12). Moreover, sequences were manually aligned against their respective prototype sequences (con1, subtype 1b; H77, subtype 1a; HC-J6, subtype 2a; GenBank accession number D10988, subtype 2b; VAT96, subtype 2k; and GenBank accession number JX227952, subtype 2k/1b) using BioEdit version 7.2.5 (4).
HCV genotyping with Versant HCV Genotype 2.0 (LiPA 2.0), cobas HCV GT, and Abbott RealTime HCV Genotype II assays.
HCV-positive serum samples of 278 patients were genotyped using the Versant HCV Genotype 2.0 (LiPA 2.0; Siemens Healthineers, Erlangen, Germany) reverse hybridization-based genotyping assay according to the instructions of the assay protocol, and with the cobas HCV genotyping test for use on the cobas 4800 system (Roche Molecular Diagnostics, Pleasanton, CA, USA), a real-time PCR-based genotyping assay for use on the cobas 4800 platform, according to the instructions in the manual. Due to lack of sufficient leftover volumes of several samples of the genotype 1b and genotype 2 control cohort, only serum samples with known chimeras (n = 48) were additionally genotyped using the Abbott RealTime HCV Genotype II assay (Abbott Molecular Inc., Des Plaines, IL, USA) on the m2000rt platform according to the assay manual. Detectable genotypes/subtypes of each assay, as well as the corresponding detection principles, are summarized in Table 1.
TABLE 1.
Characteristics of different genotyping assays
| Characteristic | Genotyping assay |
||
|---|---|---|---|
| Versant HCV Genotype 2.0 (LiPA 2.0) | cobas HCV GT | Abbott RealTime HCV Genotype II | |
| No. of detectable genotypes | 1–6 | 1–6 | 1–6 |
| Detectable subtypes | 1a, 1b, 2b, 3a, 4b, 6a, 6 (c-l) | 1a, 1b | 1a, 1b |
| Principle of the assay | Reverse hybridization technology based on sequences from 5′-UTR for genotypes 1 to 6 and core region for genotype 1a, 1b, and 6c-l | Real-time PCR targeting 5′-UTR, core and NS5B | Real-time PCR targeting 5′-UTR for GT 1–6 identification and NS5B for GT1a and GT1b subtyping |
| Chimera classification | By different genotype detection in 5′-UTR compared to core | By different genotype detection in 5′-UTR compared to core and to NS5B | By different genotype detection in 5′-UTR compared to NS5B |
RESULTS
Sequencing of core and NS5B were performed for genotype and subtype determination in all 278 samples. Sequencing of the NS2/NS3 junction was performed if core and NS5B sequencing revealed evidence for a recombinant chimera strain. Sequencing analysis revealed the following subtypes and chimeras: 1b (n = 53), 2a (n = 49), 2b (n = 75), 2c (n = 38), 2d (n = 1), 2f (n = 3), 2i (n = 4), 2k (n = 6), 2 m (n = 1), 2a/1b (n = 1), 2b/1a (n = 1), and 2k/1b (n = 46). Data about HCV viral load was available for 222/278 patients. The mean viral load was log 5.83 ± 0.98 IU/ml, including 5 samples (one genotype 2a/1b, two genotype 2b, and two genotype 2k/1b) with very low viral loads of <500 IU/ml (minimal viral load, 228 IU/ml).
Performance of Versant HCV Genotype 2.0 (LiPA2.0).
The sequenced serum samples from 278 patients infected with HCV genotype 1, genotype 2, or chimeras were genotyped using the Versant HCV Genotype 2.0 (LiPA 2.0). Known chimeras (HCV 2a/1b, 2b/1a, and 2k/1b) were not captured by Versant HCV Genotype 2.0 (LiPA 2.0) (Fig. 1, Table 2). Most of the chimeras (HCV 2k/1b, n = 46; HCV 2a/1b, n = 1) were classified as HCV genotype 2a/c (n = 37) or HCV genotype 2 (n = 10). Samples classified as genotype 2b by the Versant HCV Genotype 2.0 (LiPA 2.0) assay harbored only one HCV 2b/1a chimera according to sequence analysis. Analysis of the primary data revealed no evidence for a specific hybridization pattern by Versant HCV Genotype 2.0 (LiPA 2.0), which indicated the presence of HCV 2/1 recombinants (data not shown).
FIG 1.
Percentage of chimeras identified by the hybridization-based assay Versant HCV Genotype 2.0 and the real-time PCR-based assays cobas HCV GT and Abbott RealTime HCV Genotype II.
TABLE 2.
Genotype determination by different assays in comparison to population-based sequencingc
| Genotype | Population based sequencing | Assay |
||
|---|---|---|---|---|
| Versant HCV genotype 2.0 (LiPA 2.0) | cobas HCV GT | Abbott realtime HCV Genotype II | ||
| All samples (n) | 278 | 278 | 275a | 48b |
| 1 (n) | 53 | 53 | 53 | |
| 1a | 0 | 0 | 0 | |
| 1b | 53 | 53 | 51 | 11 |
| 2 (n) | 177 | 225 | 179 | 4 |
| 2a | 49 | |||
| 2b | 75 | 73 | ||
| 2c | 38 | |||
| 2d | 1 | |||
| 2f | 3 | |||
| 2i | 4 | |||
| 2k | 6 | |||
| 2m | 1 | |||
| HCV indeterminate (n) | 2 | |||
| 2/1 chimera (n) | 48 | 0 | 43 | 31 |
| 2/1a | 1 | 0 | 1 | 0 |
| 2b/1a | 1 | |||
| 2/1b | 47 | 0 | 42 | 29 |
| 2a/1b | 1 | |||
| 2k/1b | 46 | |||
| Other | 0 | 2 | ||
| Identification of chimeras (total) (n [%]) | 0 (0) | 43 (90) | 31 (65) | |
Three measurements were invalid.
Only assay results for chimeras detected by population-based sequencing were compared to results from population-based sequencing.
In the 230 nonchimeric samples, all samples detected as genotype 1b and most of the samples detected as genotype 2b (71/75) were consistent with our population-based sequencing (Fig. 2b). In almost half of the cases (106/230), the subtype was specified by sequencing (Fig. 2a). Specification of the subtype appeared only in genotype 2 samples, as the Versant HCV Genotype 2.0 (LiPA 2.0) assay is not designed for precise subtyping of most genotype 2 subtypes, except genotype 2b (Fig. 2b).
FIG 2.
(a) Overview of genotype and subtype determination by the hybridization-based assay Versant HCV Genotype 2.0 and the real-time PCR-based assay cobas HCV GT in comparison to that by population-based sequencing. If genotype or subtype were consistent with sequencing results, calls were termed consistent; otherwise, the subtype was refined by the sequencing analysis. (b and c) Detailed subtype determination by the hybridization-based assay Versant HCV Genotype 2.0 (b) and by the real-time PCR-based-assay cobas HCV GT (c) in comparison to that by population-based sequencing. If subtype was consistent with sequencing results, calls were termed consistent; otherwise, the subtype was refined by the sequencing analysis.
Performance of cobas HCV GT.
All of the 278 serum samples were also genotyped using the cobas HCV GT assay. In 3 patients, the measurement was invalid, and the HCV genotype could therefore only be determined in 275 samples. Tested chimeras were correctly identified in 90% (43/48) and not identified in 10% (5/48 overall; one HCV 2a/1b and four HCV 2k/1b variants) of the clinical samples (Fig. 1, Table 2). Among the five samples not identified as chimeras, all five samples were classified as HCV genotype 2 by the cobas assay. Two of these samples had very low viremia (240 and 390 IU/ml, respectively, data not shown). All samples not identified as chimeras by the cobas HCV GT assay were also not correctly identified as chimeras by the Abbott RealTime HCV Genotype II assay.
In the nonchimeric samples the subtype was specified in the majority (175/227) of the samples by our sequencing, as no HCV genotype 2 subtype discrimination is available when using the cobas HCV GT assay (Fig. 2a). Overall, 2 HCV genotype 1 and 173 HCV genotype 2 samples were specified with regard to the HCV subtype by population-based sequencing of the core and NS5B regions (Fig. 2c).
Performance of Abbott RealTime HCV Genotype II assay.
Due to limited serum resources, only 48 samples with known HCV GT 2/1 chimeras were also analyzed by the Abbott RealTime HCV Genotype II assay. Overall, chimeras were correctly identified in 65% (31/48) and not identified in 35% (17/48) of the samples (Fig. 1 and Table 2). Among samples classified as chimeras, the majority (29/31) were correctly classified as GT 2/1b, one was classified as GT 1a/1b/2, and one was classified as GT 1b/2/4. Among the 17 samples not identified as chimeras, two were classified as HCV indeterminate, four as genotype 2 (GT2), and eleven as GT1b. Of the four samples classified as GT2, three had very low viremia (228 IU/ml, 240 IU/ml, and 390 IU/ml), below the official limit of detection (LOD) of the assay (LOD for GT2, 250 IU/ml; LOD for GT1b, 500 IU/ml [13]).
DISCUSSION
The worldwide prevalence of intergenotypic recombinant HCV chimeras is low. However, significant variations may exist between different geographical regions and countries. In particular, the prevalence of the so-called St. Petersburg variant (HCV intergenotype recombinant 2k/1b) is unknown in many regions and may have been underestimated in some countries (5). For example, in Georgia, which was one of the first countries to implement a national strategy for HCV elimination, a very high prevalence (76%) of HCV 2k/1b strains was found among patients originally genotyped as HCV genotype 2 (14). This is particularly noteworthy, as genotype misclassification may lead to suboptimal treatment of patients infected with these recombinant strains, particularly where no pangenotypic DAA regimens are available or affordable and choice of treatment is still based on the viral genotype (4, 15). In the present study, we therefore aimed to analyze the analytic capacity of different commercial genotyping assays with regard to detection of the therapeutically relevant genotype 1 in 2/1 chimera. In our study, 278 serum samples of patients with HCV genotype 1b, genotype 2, or HCV chimeras, characterized by population-based sequencing, were reanalyzed with two different widely used commercially available genotyping assays, Versant HCV Genotype 2.0 (LiPA 2.0) and the real-time PCR-based assay cobas HCV GT. In addition, all samples with chimeras detected by population-based sequencing were reanalyzed using another widely used commercially available genotyping assay, Abbott RealTime HCV Genotype II.
We found a high determination rate when using the Versant HCV Genotype 2.0 (LiPA 2.0) assay for samples representing HCV genotypes 1 (subtype 1b) and 2. However, this assay could not correctly identify any of the tested HCV GT 2/1 chimeras. This result was expected, as the Versant HCV Genotype 2.0 (LiPA 2.0) uses the 5′-UTR for genotype determination and includes the core region for subtype discrimination (16). Importantly, these regions are located in the HCV genotype 2-originated fragments of the chimeras. However, analysis of hybridization patterns did not reveal any specific pattern among the chimeras that might be suitable to indicate the presence or absence of HCV GT 2/1 recombinants. Most chimeras were misclassified as HCV genotypes 2a/c or 2. On the other hand, no 2k/1b chimeras were detected by population sequencing in samples classified as HCV genotype 2b by the Versant HCV Genotype 2.0 (LiPA 2.0) assay. This is in accordance with another study in which HCV 2k/1b chimeras were misclassified as HCV genotype 2a/c by the Versant HCV Genotype 2.0 (LiPA 2.0) (5).
In contrast to hybridization assays, real-time PCR-based tests, which use the 5′-UTR for genotype determination and NS5B for subtype discrimination are expected to be more suitable for recombinant detection, as the NS5B region should be correctly classified as having a genotype 1 origin (4). Indeed, the real-time based assay cobas HCV GT correctly classified the vast majority (90%) of analyzed chimeras. However, 10% of analyzed samples were not identified as intergenotypic recombinant strains. Here, all were classified as HCV genotype 2 isolates, which harbor the risk of undertreatment with sofosbuvir plus ribavirin if no pangenotypic DAA regimens are available or affordable. Interestingly, two of these samples had very low viremia, with a viral load of <500 IU/ml, but above the LOD of the cobas HCV GT (17). This might indicate that the risk for nondetection of chimeras might be higher in low-viremic samples. In addition, all samples not identified as chimeras by the cobas HCV GT were also not detected as chimeras by the Abbott RealTime HCV Genotype II assay.
Altogether, the rate of undetected intergenotypic recombinant strains was higher for the Abbott RealTime HCV Genotype II assay. Here, 65% of chimera samples were correctly identified, while 35% of the samples were classified as genotype 1b, 2 or “indeterminate.” Again, three of the undetected chimeras had low viremia, with viral loads below the official LOD of the tests, indicating that the risk for nondetection of chimeras might be higher in low-viremia samples. Interestingly, the assay classified the chimeras with low viral loads as genotype 2, which indicates a higher sensitivity of the assay for the GT2 fragment in samples with low viral loads. Supporting this, the LOD of the Abbott RealTime HCV Genotype II assay is lower for genotype 2 (250 IU/ml) than for genotype 1b (500 IU/ml) (13). However, in the majority of cases with a nondetected HCV 2k/1b chimera, the assigned HCV genotype by the Abbott RealTime HCV Genotype II assay was HCV genotype 1b (12/18 cases), which would lead to an HCV genotype 1b-based DAA therapy with high SVR rates and no risk of undertreatment (4).
The underlying mechanism for the observed differences in sensitivity of chimera detection for the two real-time PCR-based tests with a higher rate of correct detection for the cobas HCV GT is not completely clear. However, the cobas HCV GT uses three targets in the 5-UTR, core, and NS5B for genotyping and subtyping, whereas the Abbott RealTime HCV Genotype II uses two targets in the 5′-UTR and NS5B (see Fig. 3). Using an additional target in core might lead to a higher sensitivity to identify the genotype 2 part of the chimeras in the case of the cobas HCV GT in comparison to that of the Abbott RealTime HCV Genotype II. Indeed, most of the chimeras not detected by the Abbott RealTime HCV Genotype II were classified as genotype 1b, missing the genotype 2 fragment of the chimeras. In addition, the official LODs of the cobas HCV GT for genotype 2 and 1b are lower than the LODs of the Abbott RealTime HCV Genotype II, which might also indicate a higher sensitivity of cobas HCV GT for these genotypes.
FIG 3.
Schematic representation of viral recombination in HCV 2k/1b and targets for genotype typing/subtyping by the assays Versant HCV Genotype 2.0 (LiPA 2.0), cobas HCV GT, and Abbott RealTime HCV Genotype II. UTR, untranslated region; E, envelope protein; NS, nonstructural protein; gt, genotype.
Taken together, our HCV genotype panel of clinical samples represents one of the largest sample sets of HCV recombinants analyzed to date. Based on our findings, the two tested commercially available real-time PCR-based assays seem to be more suitable for HCV GT 2/1 chimera identification than the tested hybridization-based assay. However, even with the real-time PCR-based assays, relevant percentages (10 to 35%) of samples were not identified as chimeras in this study, with a higher chimera detection rate for the cobas HCV GT. Therefore, correct identification of HCV GT 2/1 chimeras by commercially available genotyping assays remains challenging. From a clinical perspective, however, this issue may be solved by enabling worldwide access to pangenotypic DAA regimens, particularly in former Soviet countries where the St. Petersburg HCV variant is most prevalent.
ACKNOWLEDGMENTS
This study was funded, in part, by Siemens Healthineers (Erlangen, Germany). The genotyping kits were provided by Siemens Healthineers, Roche Diagnostics (Pleasanton, CA, USA) and Abbott GmbH & Co. KG (Wiesbaden, Germany). This study was also supported by a DZIF (Deutsches Zentrum für Infektionsforschung) grant entitled “HCV Treatment Optimization” to Christoph Sarrazin (TTU 05.809).
REFERENCES
- 1.Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, Simmonds P. 2014. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology 59:318–327. doi: 10.1002/hep.26744. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Hedskog C, Doehle B, Chodavarapu K, Gontcharova V, Crespo Garcia J, De Knegt R, Drenth JP, McHutchison JG, Brainard D, Stamm LM, Miller MD, Svarovskaia E, Mo H. 2015. Characterization of hepatitis C virus intergenotypic recombinant strains and associated virological response to sofosbuvir/ribavirin. Hepatology 61:471–480. doi: 10.1002/hep.27361. [DOI] [PubMed] [Google Scholar]
- 3.Kalinina O, Norder H, Mukomolov S, Magnius LO. 2002. A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg. J Virol 76:4034–4043. doi: 10.1128/JVI.76.8.4034-4043.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Susser S, Dietz J, Schlevogt B, Zuckerman E, Barak M, Piazzolla V, Howe A, Hinrichsen H, Passmann S, Daniel R, Cornberg M, Mangia A, Zeuzem S, Sarrazin C. 2017. Origin, prevalence and response to therapy of hepatitis C virus genotype 2k/1b chimeras. J Hepatol 67:680–686. doi: 10.1016/j.jhep.2017.05.027. [DOI] [PubMed] [Google Scholar]
- 5.De Keukeleire S, Descheemaeker P, Reynders M. 2015. Potential risk of misclassification HCV 2k/1b strains as HCV 2a/2c using VERSANT HCV Genotype 2.0 assay. Diagn Microbiol Infect Dis 82:201–202. doi: 10.1016/j.diagmicrobio.2015.04.001. [DOI] [PubMed] [Google Scholar]
- 6.Bhattacharya D, Accola MA, Ansari IH, Striker R, Rehrauer WM. 2011. Naturally occurring genotype 2b/1a hepatitis C virus in the United States. Virol J 8:458. doi: 10.1186/1743-422X-8-458. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Yokoyama K, Takahashi M, Nishizawa T, Nagashima S, Jirintai S, Yotsumoto S, Okamoto H, Momoi MY. 2011. Identification and characterization of a natural inter-genotypic (2b/1b) recombinant hepatitis C virus in Japan. Arch Virol 156:1591–1601. doi: 10.1007/s00705-011-1038-4. [DOI] [PubMed] [Google Scholar]
- 8.Kageyama S, Agdamag DM, Alesna ET, Leano PS, Heredia AM, Abellanosa-Tac-An IP, Jereza LD, Tanimoto T, Yamamura J, Ichimura H. 2006. A natural inter-genotypic (2b/1b) recombinant of hepatitis C virus in the Philippines. J Med Virol 78:1423–1428. doi: 10.1002/jmv.20714. [DOI] [PubMed] [Google Scholar]
- 9.Zimmermann T, Jansen PL, Sarrazin C, Vollmar J, Zeuzem S. 2018. Z Gastroenterol 56:e53–e115. doi: 10.1055/a-0598-5242. [DOI] [PubMed] [Google Scholar]
- 10.Dietz J, Susser S, Vermehren J, Peiffer K-H, Grammatikos G, Berger A, Ferenci P, Buti M, Müllhaupt B, Hunyady B, Hinrichsen H, Mauss S, Petersen J, Buggisch P, Felten G, Hüppe D, Knecht G, Lutz T, Schott E, Berg C, Spengler U, von Hahn T, Berg T, Zeuzem S, Sarrazin C. 2018. Patterns of resistance-associated substitutions in patients with chronic HCV infection following treatment with direct-acting antivirals. Gastroenterology 154:976–988.e4. doi: 10.1053/j.gastro.2017.11.007. [DOI] [PubMed] [Google Scholar]
- 11.Dietz J, Susser S, Berkowski C, Perner D, Zeuzem S, Sarrazin C. 2015. Consideration of viral resistance for optimization of direct antiviral therapy of hepatitis C virus genotype 1-infected patients. PLoS One 10:e0134395. doi: 10.1371/journal.pone.0134395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. 2008. NCBI BLAST: a better web interface. Nucleic Acids Res 36:W5–W9. doi: 10.1093/nar/gkn201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Abbott Molecular, Inc. 2013. Abbott RealTime HCV Genotype II package insert. Abbott Molecular, Inc, Des Plaines, IL. [Google Scholar]
- 14.Karchava M, Waldenström J, Parker M, Hallack R, Sharvadze L, Gatserelia L, Chkhartishvili N, Dvali N, Dzigua L, Dolmazashvili E, Norder H, Tsertsvadze T. 2015. High incidence of the hepatitis C virus recombinant 2k/1b in Georgia: recommendations for testing and treatment. Hepatol Res 45:1292–1298. doi: 10.1111/hepr.12505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Stelzl E, Haas B, Bauer B, Zhang S, Fiss EH, Hillman G, Hamilton AT, Mehta R, Heil ML, Marins EG, Santner BI, Kessler HH. 2017. First identification of a recombinant form of hepatitis C virus in Austrian patients by full-genome next generation sequencing. PLoS One 12:e0181273. doi: 10.1371/journal.pone.0181273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Verbeeck J, Stanley MJ, Shieh J, Celis L, Huyck E, Wollants E, Morimoto J, Farrior A, Sablon E, Jankowski-Hennig M, Schaper C, Johnson P, Van Ranst M, Van Brussel M. 2008. Evaluation of Versant hepatitis C virus genotype assay (LiPA) 2.0. J Clin Microbiol 46:1901–1906. doi: 10.1128/JCM.02390-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Roche Molecular Systems, Inc. 2018. Cobas HCV GT package insert. Roche Molecular Systems, Inc., Pleasanton, CA. [Google Scholar]