Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/16502
Title: Evaluation of dried blood spot samples for hepatitis C virus detection and quantification
Authors: Marques, Brunna Lemos Crespo
Espírito-Santo, Márcia Paschoal do
Marques, Vanessa Alves
Miguel, Juliana Custódio
Silva, Elisangela Ferreira da
Nogueira, Cristiane Alves Villela
Lewis-Ximenez, Lia Laura
Lampe, Elisabeth
Villar, Livia Melo
Affilliation: Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Hospital Universitário Clementino Fraga Filho. Divisão de Hepatologia. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil.
Abstract: Abstract BACKGROUND: Dried blood spots (DBS) could be an excellent alternative for HCV diagnosis, since it is less invasive and can be stored and transported without refrigeration. OBJECTIVES: The aim of this study was to optimize quantitative and qualitative methods for HCV detection in DBS. STUDY DESIGN: DBS and serum samples were collected from 99 subjects (59 anti-HCV/HCV RNA positive and 40 seronegative samples). Seven extraction methods and different PCR parameters were evaluated in DBS samples in the quantitative RT-PCR (qRT-PCR) developed to amplify the 5' noncoding region of HCV. A qualitative PCR for amplification of NS5B region of HCV was also valued and the nested-PCR sequenced. RESULTS: The qRT-PCR showed good correlation to commercial assay for HCV viral measurement in serum. To quantify HCV RNA in DBS, it was necessary to increase reverse transcriptase and cDNA concentration. HCV RNA quantification in DBS demonstrated sensitivity of 65.9%, 100% of specificity and kappa statistic of 0.65. The median viral load of DBS samples was 5.38 log10 copies/ml (minimum value=1.76 and maximum value=10.48 log10 copies/ml). HCV RNA was detected in NS5B regions and nucleotide sequences obtained in 43 serum and 11 DBS samples. The presence of the same subtype was observed in paired serum and DBS samples. CONCLUSIONS: In this study, it was possible to demonstrate that, despite the low sensitivity, the optimized protocol was able to determine the viral load, as well as, the infecting HCV genotype, validating the usefulness of DBS for viral load determination and molecular epidemiology studies of HCV.
Keywords: Dried blood spot (DBS)
Genotyping
Hepatitis C virus
Optimization
Quantitative methods
keywords: Ponto de sangue seco
Genotipagem
Vírus da Hepatite C
Otimização
Métodos quantitativos
Issue Date: 2016
Publisher: Elsevier
Citation: MARQUES, Brunna Lemos Crespo; et al. Evaluation of dried blood spot samples for hepatitis C virus detection and quantification. Journal of Clinical Virology, v.82, p.139-144, 2016.
DOI: 10.1016/j.jcv.2016.07.009
ISSN: 1386-6532
Copyright: restricted access
Appears in Collections:IOC - Artigos de Periódicos

Files in This Item:
File Description SizeFormat 
Brunna_marques_etal_IOC_2016.pdf719.31 kBAdobe PDF    Request a copy



FacebookTwitterDeliciousLinkedInGoogle BookmarksBibTex Format mendeley Endnote DiggMySpace

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.