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Detecting and defining immunity to human cytomegalovirus (HCMV) in health; combining QuantiFERON®-CMV and flow cytometry.

Willis, S. L., 2023. Detecting and defining immunity to human cytomegalovirus (HCMV) in health; combining QuantiFERON®-CMV and flow cytometry. Masters Thesis (Masters). Bournemouth University.

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WILLIS, Sophie Louise_M.Res._2023.pdf
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Human cytomegalovirus (CMV) is an archaic and ubiquitous member of the beta-herpesvirus family (Herpesviridae), infecting approx. 83% of adults worldwide. Once infection has resolved, CMV enters a life-long latent state, with potential to reactivate. Infection is usually asymptomatic in healthy individuals, while severe disease can occur in the immunosuppressed. Following allogeneic haematopoietic stem cell transplant (allo-HSCT), reactivation of CMV can cause severe morbidity and mortality. CMV DNA monitoring by polymerase chain reaction (PCR) and timely administration of pre-emptive antiviral treatment in the case of reactivation are vital to avert such outcomes. T-cell mediated immunity is key in controlling CMV infection and assays designed to measure it would improve the management of CMV infection in transplant patients; however, they are lacking in standard clinical practice. This study aimed to evaluate the performance of QuantiFERON-CMV, a commercial assay that detects CMV T cell mediated immune responses by detecting IFNy produced by CD8+ T cells. To confirm results from the QuantiFERON-CMV assay and to provide additional information on CMV-specific T cell frequency and phenotype, blood samples were assessed in parallel by flow cytometry. Finally, serology (detection of CMV IgG) was used to identify if any volunteers had been previously exposed to CMV. We hypothesised that only volunteers with a positive CMV IgG result would have detectable CMV-specific T cells by QuantiFERON-CMV and/or flow cytometry. This is a preliminary project to a clinical study which aims to evaluate the QuantiFERON-CMV assay in monitoring CMV immune reconstitution in allo-HSCT recipients. Preliminary investigations sought to validate the flow cytometry approach using a human T cell line and then primary human T cells. Together, these assays confirmed that flow cytometry can be used to identify activated T cells accumulating intracellular IFNy, and that T cell subsets could also be distinguished by the gating strategy employed. Blood samples from eleven healthy volunteers were tested for the frequency/phenotype of CMV-specific T cells by QuantiFERON-CMV and flow cytometry and for the presence and titre of anti-CMV IgG. Six of the eleven healthy volunteers were CMV IgG positive and five were CMV IgG negative. The flow cytometry assay detected a CD8+ IFNy+ T cell response in four of the six CMV IgG positive volunteers. Of these four volunteers 0.1-3.3% of the total CD8+ T cell population was specific to CMV. Phenotypic analysis showed that the majority of CMV- specific CD8+ T cells were of the TEMRA subset. Overall, the QuantiFERON-CMV levels positively correlated with CMV IgG antibody titres, and QuantiFERON-CMV results also correlated with the frequency of CMV-specific CD8+ T cells detected by flow cytometry. We also conclude that the QuantiFERON-CMV assay is more sensitive than flow cytometry. These data support the evaluation of QuantiFERON- CMV in the allo-HSCT setting.

Item Type:Thesis (Masters)
Additional Information:If you feel that this work infringes your copyright please contact the BURO Manager.
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Group:Faculty of Science & Technology
ID Code:39280
Deposited By: Symplectic RT2
Deposited On:18 Dec 2023 12:25
Last Modified:19 Jan 2024 09:31


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