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

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.