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

Anneta Naidoo,Pravi Moodley,Raveen Parboosing

doi:10.4102/ajlm.v5i1.269

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.

Highlights

Hepatitis C virus (HCV) infects 3% of the global population,[1] but there is a paucity of data on HCV prevalence in children, children in sub-Saharan Africa.[2]
The external quality control specimens with known HCV viral loads of 30 000 IU/mL, 6000 IU/mL and 1200 IU/mL were detected by the polymerase chain reaction (PCR), whereas external quality control specimens with an HCV viral load of 240 IU/mL were undetectable (Figure 2)
The in-house HCV quality control specimens with viral loads of 3 580 000 IU/mL, 358 000 IU/mL, 35 800 IU/mL and 3580 IU/mL were all detected by the HCV PCR (Figure 3)

Summary

Introduction

Hepatitis C virus (HCV) infects 3% of the global population,[1] but there is a paucity of data on HCV prevalence in children, children in sub-Saharan Africa.[2] HCV is usually transmitted to infants vertically, with a rate that varies from 3% to 7%, it may increase two- to five-fold with HIV co-infection.[3] The prevalence of HCV is higher amongst HIV-positive patients because of shared routes of transmission and common risk factors.[4] The estimated prevalence of HCV in sub-Saharan Africa was 3% amongst HIV-negative individuals in 2002, but prevalence increased to 7% amongst individuals with HIV co-infection by 2014.5 The prevalence of HCV/HIV co-infection in South Africa was 0.1% in 20026 compared to 3.5% amongst individuals infected only with HCV.[7] There are reasons to suspect that the prevalence of HCV may be high in KwaZulu-Natal, South Africa. There is a paucity of data on the prevalence of hepatitis C virus (HCV) in children, in sub-Saharan Africa. 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

Objectives

Methods

Results

Discussion

Conclusion