Real-time polymerase chain reaction optimised for hepatitis C virus detection in dried blood spots from HIV-exposed infants, KwaZulu-Natal, South Africa

Abstract

BackgroundThere is a paucity of data on the prevalence of hepatitis C virus (HCV) in children, particularly in sub-Saharan Africa. A major obstacle in resource-limited settings for polymerase chain reaction (PCR) testing is the necessity for specimen transportation and storage at low temperatures. There are numerous recent studies of using real-time HCV PCR for diagnosis and screening of plasma and serum, but few have looked at using dried blood spot (DBS) specimens.ObjectivesThe aim of this study was to optimise a real-time HCV PCR method to detect HCV RNA from infant DBS specimens for use as a tool for HCV surveillance in KwaZulu-Natal, South Africa.MethodThe LightCycler® 2.0 instrument was used for the HCV PCR using the LightCycler® RNA Master SYBR Green I kit. Template volume, primer concentration and primer annealing temperatures were optimised and the method was used on 179 DBS specimens from HIV-exposed infants in KwaZulu-Natal.ResultsPrimer concentrations adjusted to 0.25 µM and a template volume of 10 µL improved the PCR amplification. Primer annealing temperatures lowered from 65 °C to 58 °C resulted in higher quantities of amplified PCR product. The limit of detection of the optimised HCV PCR assay was between 1200 IU/mL and 3580 IU/mL of HCV RNA. HCV was not detected in any of the 179 DBS specimens.ConclusionThe optimised real-time HCV PCR on infant DBS specimens performed well, but HCV was not found in this surveillance study. HIV infection may have little impact on the vertical transmission of HCV in this region.

Simultaneous determination of HCV genotype and NS5B resistance associated substitutions using dried serum spots from São Paulo state, Brazil

“…More importantly, many studies have shown no significant variation in the assay performance between the DBS and direct sample methods [31,35,36]. Also, DBS been proven efficacious for screening high-risk patients such as intravenous drugs users [37,38] and require no cold chain for onward transportation from the sample collection site to the point of final analysis [39,40]. Although, to date there is no standard operating procedure for elution and recovery of HCV RNA from desiccated material [41], the World Health Organization (WHO) testing guidelines recommend the use of DBS specimens as an option for HCV testing and analysis, especially where there are no facilities for samples processing [42].…”