Development of novel molecular assays for the detection of cryptococcus spp. and pneumocystis jiroveci
Cardiff Metropolitan University
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Invasive fungal diseases such as Cryptococcosis and Pneumocystis Pneumonia are significant diseases of immunocompromised individuals and are often considered AIDS defining illnesses. The prevalence of these diseases is relatively low but proportionally many cases are undiagnosed or at least not identified soon enough to prevent disease progression. Proven cases of cryptococcosis and PCP are dependent on conventional culture methods, confirmatory commercial kits based on serology and histopathology. Clinicians are relying more and more on non-invasive, non-culture diagnostic techniques and PCR technology provides this capacity. This project utilised current PCR methodology and associated techniques to help produce molecular assays capable of detecting Cryptococcus and Pneumocystis jiroveci in clinical samples. Extraction methods were trialled and evaluated for the types of clinical samples most frequently received for the identification of the two organisms. Stored anonymised clinical samples and simulated samples were used throughout the project. Samples were simulated using commercially available products and stored anonymised clinical specimens. They were then spiked with Cryptococcus neoformans isolates and placed on an automated DNA extraction and purification system. The resulting samples consisted of eluted DNA exracts for a range of cryptococcal isolates. PCR primers and probes for the ITS2 region between 23S and 18S section of rRNA common to all fungal species were designed by performing multiple sequence alignments on a range of fungal taxa. Efficiency of primers was determined by amplifying DNA extracted form samples; both clinical and simulated. PCR was optimized and performed using block based thermocyclers and post amplification analysis was done by using gel electrophoresis of amplified products. Probe specificity was evaluated by amplifying extracted DNA on a Real-Time PCR platform. Using this system, amplification was seen only when probes specific to the target organism were used. The project was successful in developing functional diagnostic assays for Cryptoccocus spp. and Pneumocystis jiroveci that were quantitative as well as being qualitative. This not only makes the assays suitable for diagnosis of cryptococcosis and PCP but also as a means of monitoring disease progression and success of antifungal therapy.
MSc Biomedical Science
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