Saudi Cultural Missions Theses & Dissertations
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Item Restricted Quantitative Detection of Hepatitis C Virus RNA in Dried Blood Spots(The University of Manchester, 2024-05-16) Ismaeel, Loui; Klapper, PaulAn 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 0Item Restricted Pathological and Immunological Changes in Host Cells in Response to Leishmania mexicana Infection(Nottingham Trent University, UK, 2019-04) Alhajri, Salah Mahrous; Selman, AliAbstract Introduction: Leishmaniasis is a group of parasitic diseases caused by obligate intracellular protozoa of the genus Leishmania, with more than 20 pathogenic species. Leishmania infects approximately 12 million people annually in 98 countries. The deaths associated with this disease ranges between 20,000 - 30,000 per year (WHO, 2018). Therefore, the need for treatments or vaccines get more urgent. Macrophages are the ultimate host cell for the Leishmania parasite where it survives and multiplies. Though, a lot is known on how the Leishmania parasite survives and multiplies inside macrophages, there are still aspects related to pathophysiological and immunological responses to their infection that need further investigation to aid in the development of new vaccines or drugs for this disease. In this study, a virulent and avirulent L.mexicana model was developed to examine their interaction with bone marrow derived macrophages (BMDM) from susceptible (Balb/c) and resistant (C57) mice in vitro. Methods: Virulent L.mexicana parasite MNYC/BZ/62/M379 (P1) was maintained by subcutaneous inoculation of Balb/c mice. Avirulent L.mexicana passage twenty (P20) was produced by sub culturing of L.mexicana passage one (P1) twenty times in vitro. The effects of 20 continuous passages on virulence-associated gene (LPG1, LPG2, A2, CHAT1, CPB2, CPB2.8, CPC, GP63, LACK, and MAPK9) were investigated using qPCR. The expression of LPG and PS was also investigated using flow cytometry and immunofluorescence analysis. Growth characteristics and morphology of avirulent (P20) and virulent (P1) L.mexicana parasites grown in two media (Schneider's Drosophila and RPMI1640) in vitro were investigated under different culture conditions (temperature, and oxygen) using light immunofluorescence microscopy, EM and AFM. Differentiation into amastigotes under several conditions was investigated by estimation of the number of amastigotes. The infectivity of the parasites at each passage was also assessed by hemocytometry and Alamar blue assay. Survival of parasites inside macrophages was assessed visually by labelling the parasites with CFSE stain and the ability to form PV in BMDM from C57 and Balb/c mice. qPCR was used assess the expression pro-inflammatory cytokine expression (TNF- α, IL-6, IL-1β and TGF-β) and ELISA was used for estimation of TNF-α in the culture supernatants. Annexin V stain and flow cytometry analysis was used to assess apoptosis of infected cells. qPCR was used to assess the expression of genes associated with apoptosis (Bax, BCL 2, Caspase 1, Caspase 8, Caspase 9 and PD 1). The effect of supernatants derived from cultures of infected BMDM on the P1 and P20 promastigotes growth and virulence genes regulation was also investigated by qPCR. v Results: Twenty passages of L.mexicana in vitro caused significant changes in parasite morphology, ability to differentiate into amastigotes and downregulation of all tested virulence associated genes. Expression of LPG decreased, and PS increased on the surface of L.mexicana promastigotes. P20 infected both Balb/c and C57 BMDM but failed to survive inside BMDM both mice strains. P1 survived and inhibited apoptosis accompanied by significant downregulation of Caspase 8 by qPCR. Both P1 and P20 induced the release of TNF-α as confirmed by qPCR and ELISA. P1 promastigotes incubated in conditioned media derived from Balb/c BMDM infected with P1, enhanced their growth accompanied by upregulation of LPG1, CHT1, CPB2 and CPB2.8. While, incubation in conditioned media derived from C57 BMDM infected with P1 inhibited their growth and caused downregulation of LPG1, LPG2 CPB2, CPB2.8, CHT1 and A2. Conclusion: Culturing of L.mexicana in vitro for 20 passages has produced significant changes in their ability to differentiate from promastigotes into amastigotes, the ability to survive in macrophages and regulation of apoptosis associated genes. Supernatants produced by BMDM infected with P1 enhanced the growth rate of P1 promastigotes, when derived from Balb/c and inhibited their growth when derived from C57 cells. Understanding differences between P1 and P20 and their interaction with mammalian host may help in identifying the virulence factors of P1 L.mexicana which may aid in development of vaccines or drugs against this disease.12 0