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Comparison of Sanger sequencing for hepatitis C virus genotyping with a commercial line probe assay in a tertiary hospital.
BMC Infect Dis. 2019 Aug 22; 19(1):738.BI

Abstract

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.

Authors+Show Affiliations

Microbiology Department, Cliniques universitaires St Luc, Université catholique de Louvain, Brussels, Belgium. sylviegoletti@hotmail.com. Pôle de Microbiologie médicale, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels, Belgium. sylviegoletti@hotmail.com.Pôle de Microbiologie médicale, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels, Belgium.Pôle de Microbiologie médicale, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels, Belgium.AIDS Reference Laboratory, Clinical Chemistry, Microbiology and Immunology Department, Ghent University, Ghent, Belgium.Microbiology Department, Cliniques universitaires St Luc, Université catholique de Louvain, Brussels, Belgium. Pôle de Microbiologie médicale, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels, Belgium.Microbiology Department, Cliniques universitaires St Luc, Université catholique de Louvain, Brussels, Belgium. Pôle de Microbiologie médicale, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels, Belgium.HepatoGastroenterology Department, Cliniques universitaires St Luc, Université catholique de Louvain, Brussels, Belgium.HepatoGastroenterology Department, Cliniques universitaires St Luc, Université catholique de Louvain, Brussels, Belgium.Microbiology Department, Cliniques universitaires St Luc, Université catholique de Louvain, Brussels, Belgium. Pôle de Microbiologie médicale, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels, Belgium.

Pub Type(s)

Comparative Study
Evaluation Study
Journal Article

Language

eng

PubMed ID

31438880

Citation

Goletti, Sylvie, et al. "Comparison of Sanger Sequencing for Hepatitis C Virus Genotyping With a Commercial Line Probe Assay in a Tertiary Hospital." BMC Infectious Diseases, vol. 19, no. 1, 2019, p. 738.
Goletti S, Zuyten S, Goeminne L, et al. Comparison of Sanger sequencing for hepatitis C virus genotyping with a commercial line probe assay in a tertiary hospital. BMC Infect Dis. 2019;19(1):738.
Goletti, S., Zuyten, S., Goeminne, L., Verhofstede, C., Rodriguez-Villalobos, H., Bodeus, M., Stärkel, P., Horsmans, Y., & Kabamba-Mukadi, B. (2019). Comparison of Sanger sequencing for hepatitis C virus genotyping with a commercial line probe assay in a tertiary hospital. BMC Infectious Diseases, 19(1), 738. https://doi.org/10.1186/s12879-019-4386-4
Goletti S, et al. Comparison of Sanger Sequencing for Hepatitis C Virus Genotyping With a Commercial Line Probe Assay in a Tertiary Hospital. BMC Infect Dis. 2019 Aug 22;19(1):738. PubMed PMID: 31438880.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Comparison of Sanger sequencing for hepatitis C virus genotyping with a commercial line probe assay in a tertiary hospital. AU - Goletti,Sylvie, AU - Zuyten,Siméon, AU - Goeminne,Léonie, AU - Verhofstede,Chris, AU - Rodriguez-Villalobos,Hector, AU - Bodeus,Monique, AU - Stärkel,Peter, AU - Horsmans,Yves, AU - Kabamba-Mukadi,Benoît, Y1 - 2019/08/22/ PY - 2018/09/03/received PY - 2019/08/15/accepted PY - 2019/8/24/entrez PY - 2019/8/24/pubmed PY - 2019/10/28/medline KW - Genotyping KW - Hepatitis C virus KW - Sequencing SP - 738 EP - 738 JF - BMC infectious diseases JO - BMC Infect Dis VL - 19 IS - 1 N2 - 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. SN - 1471-2334 UR - https://www.unboundmedicine.com/medline/citation/31438880/Comparison_of_Sanger_sequencing_for_hepatitis_C_virus_genotyping_with_a_commercial_line_probe_assay_in_a_tertiary_hospital_ DB - PRIME DP - Unbound Medicine ER -