Browsing by Author "Ismaeel, Loui"

Now showing 1 - 1 of 1

Restricted
Quantitative Detection of Hepatitis C Virus RNA in Dried Blood Spots
(The University of Manchester, 2024-05-16) Ismaeel, Loui; Klapper, Paul
An estimated fifty-eight million people have chronic hepatitis C virus (HCV) infection, with about 1.5 million new infections occurring each year. A sizeable proportion of these new infections occur among injecting drug users. Dried blood spots (DBS) have revolutionised HCV diagnosis in this difficult to reach population and have further advantages when used in healthcare resource-poor regions of the world. Diagnosis of infection may be made serologically but confirmation of chronic infection necessitates the detection of HCV RNA and response to therapy must be monitored by quantitation of HCV RNA in blood (‘viral load’). A current limitation of the use of DBS is that quantitation of HCV RNA is not possible from standard DBS samples as test blood volume cannot be accurately measured. In the present work a volumetric microfluidic device (HemaXis™ DB 10) was evaluated for preparation of DBS samples to allow quantitation of HCV RNA from the DBS. The HemaXis device was used in conjunction with a custom-made Perkin Elmer 226 paper collection card. Cross contamination between samples was avoided using 10mm diameter perforations to allow individual processing of samples. Extraction of RNA used a modified Qiagen extraction kit and the internally controlled (MS2) HCV RNA quantitative PCR had a limit of detection in DBS specimens of 8.46 IU/mL comparing well with the limit of detection of the reference assay (Cobas® AmpliPrep TaqMan® HCV Test, v2.0; Roche Diagnostics) in EDTA plasma which is 8.5 IU/mL when using a sample size of 500µL. Previously tested HCV RNA positive (n = 125) and HCV RNA and HCV antibody negative (n = 138) serum and plasma samples were used to prepare mock DBS samples by mixing with packed cells from an HCV RNA and HCV antibody negative donor blood. The HemaXis device was used to deliver 10µL samples for each spot. QPCR of mock DBS samples prepared from the 125 HCV RNA positive whole blood samples gave 104 positive HCV RNA results from the DBS. This gave a qualitative test sensitivity of 85.6% and specificity of 100%. A Bland-Altman plot of the differences between the two tests showed that on average the DBS system determines a log 3.37 (2,344 IU/mL) lower value than testing of plasma. The reduction was independent of viral load. Review of the results of internal control testing suggested many samples had evidence of inhibition suggestive of sample deterioration between the time of initial testing of whole blood specimens and the preparation of the mock DBS specimens. Contemporaneous testing of DBS and plasma would be needed to confirm this suggestion. The DBS prepared using the HemaXis were also evaluated for use in genotyping and sequencing of the NS5B and core regions of HCV. Determination of genotype with discrimination of subtypes and of mixed infections was shown to be possible from the DBS samples. The consistent (albeit lower) viral load determination made when using HemaXis prepared DBS shows that this technology has promise as a method for quantitation of HCV RNA load from DBS. Together with the proven ability to genotype HCV using the same sample, the utility of DBS in the diagnosis and management of HCV infection will be further extended.
17 0