RESUMO
BACKGROUND: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. METHODS: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. RESULTS: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5' UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. CONCLUSIONS: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.
Assuntos
Técnicas de Genotipagem/métodos , Hepacivirus/genética , Hepatite C/diagnóstico , Técnicas de Sonda Molecular , Kit de Reagentes para Diagnóstico , Análise de Sequência de RNA/métodos , Regiões 5' não Traduzidas , Sequência de Bases , Comércio , Genômica/métodos , Genótipo , Técnicas de Genotipagem/economia , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Técnicas de Sonda Molecular/economia , Filogenia , RNA Viral/análise , RNA Viral/isolamento & purificação , Kit de Reagentes para Diagnóstico/economia , Estudos Retrospectivos , Análise de Sequência de RNA/economia , Centros de Atenção TerciáriaRESUMO
INTRODUCTION: We aim to report on results and challenges of different methods used for hepatitis C (HCV) genotyping in a Cambodian HCV/HIV coinfection project. METHODS: Samples of 106 patients were available. HCV genotyping was initially (63 samples) done by the LightPower Taqman real-time PCR method (Viet A Corp.) and quality controlled using the Versant 2.0 line probe assay (Siemens Healthcare). Next, following interim quality control results, all 106 samples were (re)genotyped with Versant 2.0, complemented with 5'UTR/core sequencing for uninterpretable/incomplete Versant results. RESULTS: Using Versant, 103 (97.2%) of the 106 HCV-coinfected patients had an interpretable genotype result: 1b (50.5%), 6 non-a/non-b (30.1%), 1a (6.8%), 6a or b (4.9%), 2 (3.9%), 1 (2.9%) and 3 (1.0%). For 16 samples that were interpreted as genotype 1 or 1b per Versant's current instructions, it could not be excluded that it concerned a genotype 6 infection as the core region line patterns on the Versant test strip were unavailable, inconclusive or atypical. Upon sequencing, seven of these were genotyped as 1b and nine as genotype 6. Combining Versant and sequencing results, a definitive genotype was assigned in 104 patients: 1b (44.2%), 6 non-a/non-b (39.4%), 1a (6.7%), 6a or b (4.8%), 2 (3.8%) and 3 (1.0%). Genotyping by LightPower and Versant was discordant for 23 (of 63) samples. The LightPower assay misclassified all genotype 6 non-a/non-b samples as genotype 1, which indicates that this assay is only using 5'UTR information. CONCLUSIONS: HCV genotype 1b and genotype 6 non-a/non-b were most common. With Versant 2.0 (using 5'UTR and core information), genotype classification (1 or 6) remained inconclusive in 15% of samples. The locally available method (LightPower assay) failed to identify genotype 6 non-a/non-b, which highlights that methods using 5'UTR information only should not be used in Cambodia. Regional/national guidelines should be explicit about this. TRIAL REGISTRATION: This study was performed as part of a larger cross-sectional study on the burden of hepatitis C coinfection in HIV patients in Cambodia (Clinical.trials.gov: HCV-Epi NCT02361541).