Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system

Background & Aims: Currently available HDV PCR assays are characterized by considerable run-to-run and inter-laboratory variability. Hence, we established a quantitative reverse transcription real-time PCR (RT-qPCR) assay on the open channel of a fully automated PCR platform (cobas6800, Roch...

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Autores principales:	Lisa Sophie Pflüger, Dominik Nörz, Tassilo Volz, Katja Giersch, Annika Giese, Nora Goldmann, Dieter Glebe, Jan-Hendrik Bockmann, Susanne Pfefferle, Maura Dandri, Julian Schulze zur Wiesch, Marc Lütgehetmann
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	HDV, Hepatitis delta virus RT-qPCR, Real time reverse transcription polymerase chain reaction cobas6800 molecular diagnostics viral hepatitis quantification Diseases of the digestive system. Gastroenterology RC799-869
Acceso en línea:	https://doaj.org/article/f3d9aadcae3c49dab7c4fe5a8f856e86
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spelling	oai:doaj.org-article:f3d9aadcae3c49dab7c4fe5a8f856e862021-11-20T05:11:37ZClinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system2589-555910.1016/j.jhepr.2021.100356https://doaj.org/article/f3d9aadcae3c49dab7c4fe5a8f856e862021-12-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2589555921001324https://doaj.org/toc/2589-5559Background & Aims: Currently available HDV PCR assays are characterized by considerable run-to-run and inter-laboratory variability. Hence, we established a quantitative reverse transcription real-time PCR (RT-qPCR) assay on the open channel of a fully automated PCR platform (cobas6800, Roche) offering improved consistency and reliability. Methods: A primer/probe-set targeting a highly conserved region upstream of the HDV antigen was adapted for use on the cobas6800. The lower limit of detection (LLOD) was determined using a dilution panel of the HDV WHO standard (n = 21/dilution). Linearity and inclusivity were tested by preparing 10-fold dilution series of cell culture-derived virus (genotype [GT]1-8; n = 5/dilution). Patient samples containing a variety of bloodborne viral pathogens were tested to confirm exclusivity (n = 60). Results: The LLOD of the HDV utility-channel (HDV_UTC) assay was determined as 3.86 IU/ml (95% CI 2.95–5.05 IU/ml) with a linear range from 10–10ˆ8 IU/ml (GT1). Linear relationships were observed for all HDV GTs with slopes ranging from -3.481 to -4.134 cycles/log and R2 from 0.918 to 0.994. Inter-run and intra-run variability were 0.3 and 0.6 Ct (3xLLOD), respectively. No false-positive results were observed. To evaluate clinical performance, 110 serum samples of anti-HDV-Ab+ patients were analyzed using the HDV_UTC and CE-IVD RoboGene assays. 58/110 and 49/110 samples were concordant positive or negative, respectively (overall agreement 97.3%). Quantitative comparison demonstrated a strong correlation (R2 0.8733; 95% CI 0.8914–0.9609; p value <0.0001). Conclusion: The use of highly automated, sample-to-result solutions for molecular diagnostics holds many inherent benefits over manual workflows, including improved reliability, reproducibility and dynamic scaling of testing capacity. The assay we established showed excellent analytical and clinical performance, with inclusivity for all HDV GTs and a limit of quantification of 10 IU/ml, making it a sensitive new tool for HDV screening and viral load monitoring. Lay summary: The hepatitis delta virus (HDV) causes a severe form of inflammation in the liver. We developed a tool for molecular diagnostics, a polymerase chain reaction HDV assay that showed great performance. It can be used to improve diagnosis of HDV, as well as for monitoring treatment responses. The assay allows for quantification of the virus in the tested samples and is performed on a fully automated platform (cobas6800), which provides various benefits including less hands-on time and excellent comparability of test results.Lisa Sophie PflügerDominik NörzTassilo VolzKatja GierschAnnika GieseNora GoldmannDieter GlebeJan-Hendrik BockmannSusanne PfefferleMaura DandriJulian Schulze zur WieschMarc LütgehetmannElsevierarticleHDV, Hepatitis delta virusRT-qPCR, Real time reverse transcription polymerase chain reactioncobas6800molecular diagnosticsviral hepatitisquantificationDiseases of the digestive system. GastroenterologyRC799-869ENJHEP Reports, Vol 3, Iss 6, Pp 100356- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	HDV, Hepatitis delta virus RT-qPCR, Real time reverse transcription polymerase chain reaction cobas6800 molecular diagnostics viral hepatitis quantification Diseases of the digestive system. Gastroenterology RC799-869
spellingShingle	HDV, Hepatitis delta virus RT-qPCR, Real time reverse transcription polymerase chain reaction cobas6800 molecular diagnostics viral hepatitis quantification Diseases of the digestive system. Gastroenterology RC799-869 Lisa Sophie Pflüger Dominik Nörz Tassilo Volz Katja Giersch Annika Giese Nora Goldmann Dieter Glebe Jan-Hendrik Bockmann Susanne Pfefferle Maura Dandri Julian Schulze zur Wiesch Marc Lütgehetmann Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system
description	Background & Aims: Currently available HDV PCR assays are characterized by considerable run-to-run and inter-laboratory variability. Hence, we established a quantitative reverse transcription real-time PCR (RT-qPCR) assay on the open channel of a fully automated PCR platform (cobas6800, Roche) offering improved consistency and reliability. Methods: A primer/probe-set targeting a highly conserved region upstream of the HDV antigen was adapted for use on the cobas6800. The lower limit of detection (LLOD) was determined using a dilution panel of the HDV WHO standard (n = 21/dilution). Linearity and inclusivity were tested by preparing 10-fold dilution series of cell culture-derived virus (genotype [GT]1-8; n = 5/dilution). Patient samples containing a variety of bloodborne viral pathogens were tested to confirm exclusivity (n = 60). Results: The LLOD of the HDV utility-channel (HDV_UTC) assay was determined as 3.86 IU/ml (95% CI 2.95–5.05 IU/ml) with a linear range from 10–10ˆ8 IU/ml (GT1). Linear relationships were observed for all HDV GTs with slopes ranging from -3.481 to -4.134 cycles/log and R2 from 0.918 to 0.994. Inter-run and intra-run variability were 0.3 and 0.6 Ct (3xLLOD), respectively. No false-positive results were observed. To evaluate clinical performance, 110 serum samples of anti-HDV-Ab+ patients were analyzed using the HDV_UTC and CE-IVD RoboGene assays. 58/110 and 49/110 samples were concordant positive or negative, respectively (overall agreement 97.3%). Quantitative comparison demonstrated a strong correlation (R2 0.8733; 95% CI 0.8914–0.9609; p value <0.0001). Conclusion: The use of highly automated, sample-to-result solutions for molecular diagnostics holds many inherent benefits over manual workflows, including improved reliability, reproducibility and dynamic scaling of testing capacity. The assay we established showed excellent analytical and clinical performance, with inclusivity for all HDV GTs and a limit of quantification of 10 IU/ml, making it a sensitive new tool for HDV screening and viral load monitoring. Lay summary: The hepatitis delta virus (HDV) causes a severe form of inflammation in the liver. We developed a tool for molecular diagnostics, a polymerase chain reaction HDV assay that showed great performance. It can be used to improve diagnosis of HDV, as well as for monitoring treatment responses. The assay allows for quantification of the virus in the tested samples and is performed on a fully automated platform (cobas6800), which provides various benefits including less hands-on time and excellent comparability of test results.
format	article
author	Lisa Sophie Pflüger Dominik Nörz Tassilo Volz Katja Giersch Annika Giese Nora Goldmann Dieter Glebe Jan-Hendrik Bockmann Susanne Pfefferle Maura Dandri Julian Schulze zur Wiesch Marc Lütgehetmann
author_facet	Lisa Sophie Pflüger Dominik Nörz Tassilo Volz Katja Giersch Annika Giese Nora Goldmann Dieter Glebe Jan-Hendrik Bockmann Susanne Pfefferle Maura Dandri Julian Schulze zur Wiesch Marc Lütgehetmann
author_sort	Lisa Sophie Pflüger
title	Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system
title_short	Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system
title_full	Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system
title_fullStr	Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system
title_full_unstemmed	Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system
title_sort	clinical establishment of a laboratory developed quantitative hdv pcr assay on the cobas6800 high-throughput system
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/f3d9aadcae3c49dab7c4fe5a8f856e86
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Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system

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