Diagnosis of Hepatitis C Using Dried Blood Spots

UoM administered thesis: Phd

  • Authors:
  • Benjamin Brown

Abstract

Hepatitis C virus (HCV) is a major worldwide health issue, infecting approximately 71.1 million people worldwide, with 143 000 thought to be chronically infected in England. The vast majority of these (92.1%) infections are found in injecting drug users. A major breakthrough in screening has been achieved in the past few years with the introduction of increased screening for HCV, specifically targeting the injecting drug user population. Dried blood spots were shown to be a suitable sample type to use in these circumstances, overcoming previous issues caused by poor venous access and limited availability of sufficiently trained phlebotomists and increasing testing in this population. Despite the improvements brought about through the use of this sample type several issues still remain including: - A large population of injecting drug users are still unaware of their hepatitis C infection. - Testing from dried blood spots uses commercial CE marked HCV serological assays, but these assays are not CE marked for use with dried blood spots. - Optimisation of commercial assays has increased the sensitivity achievable from dried blood spots to a high level (97-100%) this does not match that available from serum samples. Only an assay specifically designed for dried blood spot samples will be able to increase the sensitivity past what is currently achievable. - Reliable identification of acute HCV infection is not currently possible. The HCV Core, NS3, NS4, NS5a and NS5b proteins were produced using flashBAC™ ultra baculovirus expression system. Low protein yield in NS4, NS5a and NS5b protein production was overcome with the use of a baculovirus transfer vector incorporating an anti-apoptotic vankyrin expression cassette. Proteins were purified using immobilized metal affinity chromatography. Solubility issues in the NS4, NS5a and NS5b proteins were overcome using urea or guanidine based solubilisation. Purified proteins were optimised in individual ELISAs, and their performance assessed for the detection of anti-HCV in both serum and dried blood spot samples. Results from the individual ELISAs were combined to give a multi analyte array type profile. Performance for detection of anti-HCV in serum samples was high, with a sensitivity of 91.6% while maintaining a specificity of 100%. Performance with DBS samples was higher, giving a sensitivity and specificity of 100%. This matched the performance of the commercial Roche Elecsys® anti-HCV assay and improved on the performance of the commercial Ortho anti-HCV assay for screening of anti-HCV from DBS samples, while removing the need for anti-HCV confirmation testing in the majority (93.5%) of samples.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
  • Pamela Vallely (Supervisor)
  • Paul Klapper (Supervisor)
  • Raymond Borrow (Supervisor)
Award date31 Dec 2020