Elsevier

Journal of Clinical Virology

Volume 82, September 2016, Pages 139-144
Journal of Clinical Virology

Evaluation of dried blood spot samples for hepatitis C virus detection and quantification

https://doi.org/10.1016/j.jcv.2016.07.009Get rights and content

Highlights

  • Optimization of qualitative and quantitative methods for HCV RNA in serum and DBS.

  • In house qPCR for HCV in serum showed good correlation to commercial assay.

  • Detection limit of qPCR for HCV in DBS is 58.5 copies/ml.

  • Our method enabled HCV genotyping in DBS samples.

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

Hepatitis C virus
Dried blood spot (DBS)
Quantitative methods
Genotyping
Optimization

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