Diagnostic Microbiology and Infectious Disease 82 (2015) 201–202
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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Virology
Potential risk of misclassification HCV 2k/1b strains as HCV 2a/2c using VERSANT HCV Genotype 2.0 assay Steven De Keukeleire ⁎, Patrick Descheemaeker, Marijke Reynders Department of Laboratory Medicine, AZ St-Jan Bruges, Bruges, Belgium
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Article history: Received 19 March 2015 Received in revised form 31 March 2015 Accepted 1 April 2015 Available online 7 April 2015 Keywords: Hepatitis C virus HCV genotyping and subtyping Recombinant forms HCV 2k/1b
a b s t r a c t The performance of a hepatitis C virus (HCV) NS5B sequencing method described by Murphy et al. (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;45(4):1102–12.) was compared with the VERSANT HCV Genotype 2.0 assay. The sequencing strategy led to detection of HCV recombinant genotype 2k/1b, previously identified as genotype 2a/2c, which reveals the importance of exact HCV genotyping and subtyping. © 2015 Elsevier Inc. All rights reserved.
Hepatitis C virus (HCV) is a major cause of cirrhosis and liver failure worldwide. The genomes of HCV variants display considerable sequence divergence and have been classified into seven genotypes and several subtypes, showing a different geographic distribution worldwide (Smith et al., 2014). The most accurate method for HCV genotyping is sequence analysis of an appropriate coding region, which is divergent enough to allow the discrimination of types and subtypes. The 5′ untranslated region (5′UTR) is the region of choice for qualitative and quantitative HCV RNA detection due to its high level of conservation and is consequently of limited use in genotype discrimination. The aims of this study were to compare the performance of the HCV NS5B sequencing method (Murphy et al., 2007) with the VERSANT HCV Genotype 2.0 assay, a second-generation line probe assay (LiPA; Siemens, Medical Solutions Diagnostics, Tarrytown, NY) based on the 5′UTR amplicon and to evaluate the potential consequences it has on patient management. HCV genotyping and subtyping remain essential criteria when considering the choice of antiviral treatment and treatment duration protocols, as applicable for genotype 2k/1b, which might have to be treated differently from 2a2c. During a 14-month period (January 2012 to March 2013), genotyping of HCV was performed on 77 HCV-positive isolates using the VERSANT HCV Genotype 2.0 assay (LiPA), which resulted in following genotype distribution: genotype 1a (n = 16; 21%), genotype 1b (n = 22; 28%), genotype 2a/2c (n = 2; 3%), genotype 3a (n = 19; 25%), genotype 4 (n = 7; 9%), genotype 4a/4c/4d (n = 4; 5%), and genotype 5a (n = 7; 9%) (Fig. 1). Among these isolates, 38 were selected for sequencing analysis. As shown in Table 1, 38 clinical isolates were selected for NS5B sequence analysis. An overall correlation of 92% (35/38) was observed ⁎ Corresponding author. Tel.: +32-50-45-26-44; fax: +32-50-45-26-19. E-mail address:
[email protected] (S. De Keukeleire). http://dx.doi.org/10.1016/j.diagmicrobio.2015.04.001 0732-8893/© 2015 Elsevier Inc. All rights reserved.
between the LiPA assay and the sequencing method. One out of 16 cases of subtype 1a was misclassified as genotype 1b by LiPA, and 2 LiPA types 2a/2c isolates were classified as HCV 2k/1b by sequencing of the NS5B region. This finding of LiPA misclassifying 1a subtype is in agreement with other studies, which recognized that the low heterogeneity of 5′UTR could contribute to the lack of discrimination of HCV subtypes, especially for genotype 1 (Avó et al., 2013). The LiPA assay does not seem to provide clear differentiation between subtypes of genotype 2 (Avó et al., 2013; Cantaloube et al., 2006; Ramière et al., 2014). Subsequently, routine sequencing testing identified 2 more patients infected by a 2k/1b during
Distribution of HCV genotypes AZ Sint–Jan Hospital Bruges-Ostend HCV genotype 4 9%
HCV genotype 5a 9%
HCV genotype 4a/4c/4d 5% HCV genotype 2a/2c 3%
HCV genotype 3a 25% HCV genotype 1a 21% HCV genotype 1b 28%
Fig. 1. Distribution of HCV genotypes in AZ Sint-Jan Hospital Bruges-Ostend.
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Table 1 Comparison of VERSANT and “in house” sequencing for 77 clinical HCV isolates. No. of isolates Genotype by VERSANT LiPA
Available
Selected for sequence analysis
Correlation (%) between VERSANT LipA and NS5B sequencing
Discrepancies
HCV genotype 1a HCV genotype 1b HCV genotype 3a HCV genotype 4 HCV genotype 5a HCV genotype 4a/4c/4d HCV genotype 2a2c
16 22 19 7 7 4 2
10 10 10 3 2 1 2
90 100 100 100 100 100 0
One isolate identified as HCV genotype 1b
77
38
35/38 (92%)
the period March 2013 to December 2014. This brings the total number of HCV 2k/1b–infected patients identified in our hospital to 3.3% (4/121). The reclassification of HCV subtype 1b into HCV subtype 1a has no implication on the choice of antiviral therapy but can influence the duration of treatment. Combination (triple) therapy of peginterferon (pegINF) α-2a or α-2b plus ribavirin (RBV) with a direct acting antiviral agent (DAA), boceprevir or telaprevir, is indicated for patients infected with HCV genotype 1 (Ghany et al., 2011). However, development of HCV resistance to protease inhibitors may be an issue. Only a single mutation is required to induce resistance in HCV genotype 1a, whereas 2 mutations are required in genotype 1b (Sarrazin and Zeuzem, 2010). The recombinant genotype 2k/1b was first described in St Petersburg in 2002 in intravenous (IV) drug users (Kalinina et al., 2002). Since its discovery, the genotype 2k/1b has been detected in many countries, including Ireland, Uzbekistan, Cyprus, France, the Netherlands, and Russia (Demetriou et al., 2009; Kalinina et al., 2002; Karchava et al., 2015; Kurbanov et al., 2008a, 2008b; Moreau et al., 2006; Morel et al., 2010; Raghwani et al., 2012). In all these cases, the patients originated from Russia or Georgia. The recombinant genotype 2k/1b has a 5′ genome region that is most closely related to subtype 2k and a 3′ genome region that is most closely related to the global epidemic subtype 1b (Raghwani et al., 2012). The sequence strategy led to the detection of the HCV recombinant genotype 2k/1b in 4 patients, initially erroneously identified as genotype 2a/2c by the VERSANT method. From clinical point of view, particular attention should be paid to patients from the Caucasus region (Ramière et al., 2014). Based on our observations, the number of patients infected with the recombinant 2k/1b HCV strain may be underestimated when using the VERSANT LiPA technique. Due to the limited information on susceptibility of the recombinant 2k/1b HCV genotype to antiviral treatment, no recommendation exists on duration or optimal therapy (Karchava et al., 2015). Maybe an association with DAA could be beneficial. In our study, for the 2 patients receiving antiviral treatment, a therapeutic continuation of 48 weeks instead of the expected 24-week protocol was needed to obtain a sustained viral response (SVR). Literature shows discrepant results regarding pegINF treatment and SVR. Kurbanov et al. (2008a, 2008b) showed higher susceptibility of the 2k/1b genotype to pegINF/RBV treatment in contrast to Morel et al. (2010). Currently, a new DAA, sofosbuvir, shows pangenotypic HCV activity in chronic HCV patients with high rates of SVR when given with RBV (Koff, 2014). However, in Belgium, sofosbuvir is only reimbursed for chronic HCV patients with cirrhosis, and in many Eastern European countries, the access to this agent is extremely limited for HCV patients (Karchava et al., 2015). Our results support the importance of correct identification of infecting HCV genotype among HCV patients for treatment selection and/or SVR prediction (Morel et al., 2010; Quer et al., 2015). Despite the fact that the number of patients in the present study is small, the incidence of these 2k/1b HCV strains is very likely to be underestimated, and they may have spread all over Western Europe. Further studies
Subtype 4a Both isolates identified as HCV genotype 2k/1b
will be necessary to determine the incidence of this strain in Belgium and other European regions, in particular among IV drug users. Acknowledgments We would like to thank all technicians of the laboratory of molecular virology of AZ St-Jan Bruges for their work and dedication. References Avó AP, Agua-Doce I, Andrade A, Pádua E. Hepatitis C virus subtyping based on sequencing of the C/E1 and NS5B genomic regions in comparison to a commercially available line probe assay. J Med Virol 2013;85(5):815–22. Cantaloube JF, Laperche S, Gallian P, Bouchardeau F, de Lamballerie X, de Micco P. Analysis of the 5′ noncoding region versus the NS5b region in genotyping hepatitis C virus isolates from blood donors in France. J Clin Microbiol 2006;44(6):2051–6. Demetriou VL, van de Vijver DA, Cyprus HCV Network, Kostrikis LG. Molecular epidemiology of hepatitis C infection in Cyprus: evidence of polyphyletic infection. J Med Virol 2009; 81(2):238–48. Ghany MG, Nelson DR, Strader DB, Thomas DL, Seeff LB. An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 Practice Guideline by the American Association for the Study of Liver Diseases. Hepatology 2011;54(4): 1433–44. Kalinina O, Norder H, Mukomolov S, Magnius LO. A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg. J Virol 2002;76(8):4034–43. Karchava M, Waldenström J, Parker M, Hallack R, Sharvadze L, Gatserelia L, et al. High incidence of the hepatitis C virus recombinant 2k/1b in Georgia: recommendations for testing and treatment. Hepatol Res 2015. Koff RS. Review article: the efficacy and safety of sofosbuvir, a novel, oral nucleotide NS5B polymerase inhibitor, in the treatment of chronic hepatitis C virus infection. Aliment Pharmacol Ther 2014;39(5):478–87. Kurbanov F, Tanaka Y, Avazova D, Khan A, Sugauchi F, Kan N, et al. Detection of hepatitis C virus natural recombinant RF1_2k/1b strain among intravenous drug users in Uzbekistan. Hepatol Res 2008a;38(5):457–64. Kurbanov F, Tanaka Y, Chub E, Maruyama I, Azlarova A, Kamitsukasa H, et al. Molecular epidemiology and interferon susceptibility of the natural recombinant hepatitis C virus strain RF1_2k/1b. J Infect Dis 2008b;198(10):1448–56. Moreau I, Hegarty S, Levis J, Sheehy P, Crosbie O, Kenny-Walsh E, et al. Serendipitous identification of natural intergenotypic recombinants of hepatitis C in Ireland. Virol J 2006;3:95. Morel V, Descamps V, François C, Fournier C, Brochot E, Capron D, et al. Emergence of a genomic variant of the recombinant 2k/1b strain during a mixed Hepatitis C infection: a case report. J Clin Virol 2010;47(4):383–6. Murphy DG, Willems B, Deschênes 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; 45(4):1102–12. Quer J, Gregori J, Rodriguez-Frias F, Buti M, Madejon A, Perez-del-Pulgar S, et al. Highresolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods. J Clin Microbiol 2015;53(1):219–26. Raghwani J, Thomas XV, Koekkoek SM, Schinkel J, Molenkamp R, van de Laar TJ, et al. Origin and evolution of the unique hepatitis C virus circulating recombinant from 2k/1b. J Virol 2012;86(4):2212–20. Ramière C, Tremeaux P, Caporossi A, Trabaud MA, Lebossé F, Bailly F, et al. Recent evidence of underestimated circulation of hepatitis C virus intergenotypic recombinant strain RF2k/1b in the Rhône-Alpes region, France, January to August 2014. Euro Surveill 2014;19. Sarrazin C, Zeuzem S. Resistance to direct antiviral agents in patients with hepatitis C virus infection. Gastroenterology 2010;138(2):447–62. 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(1):318–27.
