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

Hepatitis C Virus (HCV) RNA screening and sequencing using dry plasma spots.
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Geretti AM, King S, Adjei-Asante K, Appiah LT, Owusu DO, Sarfo FS, Chadwick D, Phillips RO, Beloukas A,


Geretti AM, King S, Adjei-Asante K, Appiah LT, Owusu DO, Sarfo FS, Chadwick D, Phillips RO, Beloukas A, (click to view)

Geretti AM, King S, Adjei-Asante K, Appiah LT, Owusu DO, Sarfo FS, Chadwick D, Phillips RO, Beloukas A,

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Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 2017 10 2397() 18-21 pii 10.1016/j.jcv.2017.10.012

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 6mm 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.

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