Elsevier

Journal of Clinical Virology

Volume 97, December 2017, Pages 18-21
Journal of Clinical Virology

Short communication
Hepatitis C Virus (HCV) RNA screening and sequencing using dry plasma spots

https://doi.org/10.1016/j.jcv.2017.10.012Get rights and content
Under a Creative Commons license
open access

Highlights

  • Pooled dried plasma spots (DPS) can be used for large scale HCV RNA screening.

  • HCV RNA prevalence found 0.6% in a large cohort of HIV positive patients from Ghana.

  • All HCV RNA positive subjects were infected with genotype 2 viruses.

  • DPS allowed reproducible HCV RNA detection and sequencing of HCV core and NS5B.

Abstract

Background

HCV RNA screening of large sample repositories provides data on HCV epidemic patterns that may help guide control policies. In resource-limited settings, shipment of frozen samples to molecular laboratory facilities and testing of individual samples may be prohibitively expensive.

Objective

Our aim was to detect and sequence HCV RNA in a large HIV-positive cohort from Kumasi, Ghana, using pooled and individual dried plasma spots (DPS) produced from samples stored at −80 °C.

Study design

In the validation phase, replicate DPS were prepared with six dilutions (500–10,000 IU/ml) of the 4th International Standard for HCV and tested in three independent experiments. In the testing phase, DPS prepared with plasma samples from 875 HIV-positive subjects were pooled for screening, followed by testing of individual DPS of positive pools. Input from individual DPS was two 6 mm punches; pools comprised two punches from each of five DPS. Genotypes were determined by Sanger sequencing of HCV core and NS5B.

Results

With the dilution series, sensitivity of HCV RNA detection was ≥2500 IU/ml. Replicate DPS gave intra-assay and inter-assay coefficients of variation ≤1.4%. With the stored samples, HCV RNA was detected in 5/175 DPS pools and in one DPS from each positive pool, yielding a HCV RNA prevalence of 5/875 (0.57%; 95% confidence interval 0.07-1.07%). The five samples were sequenced as HCV genotypes 2l and 2r.

Discussion

DPS allowed reproducible HCV RNA detection, and pooling effectively contained the cost and labour of screening a previously untested, low-prevalence cohort. DPS were also suitable for HCV sequencing.

Keywords

HCV RNA
Sequencing
Dried plasma spots
Sub-Saharan Africa
Epidemiology

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