DEVELOPING FAST AND EFFICIENT MOLECULAR DIAGNOSTIC TOOLS FOR HUMAN INVASIVE CANDIDIASIS

Infection with yeasts of the genus Candida can cause invasive human candidiasis in hospitalised immunosuppressed patients. Mortality rates are high due to the lack of specific clinical signs of infection and of reliable diagnostic tools. Early diagnosis could facilitate implementation of accurate treatment and save lives but currently used diagnostic methods based on microbial culture and taxonomic identification are time- consuming, cumbersome and non-specific. To address this problem, this research explores the potential of two different biomarker detection platforms as simple nucleic acid biosensors for the diagnosis of invasive candidiasis. These include Multiplex Probe Amplification (MPA) and Multi-component Nucleic Acid Enzymes (MNAzymes). In all cases assay specificity is conferred by diagnostic probes and amplification primers designed from the internal transcribed spacer (ITS) and D1/D2 domains of the ribosomal DNA locus. A single-tube MPA assay was developed which relies on multiple-biomarker probe pairs with unique melting temperature profiles. Specific probe hydrolysis during amplification leads to measurable change(s) in post- amplification melting profiles compared with the negative control. Nine Candida species associated with invasive candidiasis were targeted by the MPA-Candida assay, including antifungal-resistant C. auris, C. glabrata and C. krusei. A unique internal control certifies assay functionality and avoids false-negative results. The MPA-Candida assay can also be coupled with an independent pan-fungal assay, exhibiting detection limits of 10-100 genomic copies. In vitro characterisation of the assay and its evaluation against a standardised Candida panel and a commercially available Candida diagnostic kit are reported. The MPA-Candida assay was further optimised to incorporate a pan-fungal biomarker for a single-tube reaction in either two or three fluorescence detection channels. Finally, the engineering and characterisation of an isothermal MNAzyme assay for Candida diagnosis was also investigated. Here, homology-dependent self-assembly of a hydrolytic MNAzyme is driven by a synthetic single-stranded oligonucleotide complementary to the target biomarker. Catalytic activity is monitored by hydrolysis of dual-labelled substrate probe, which is detectable in a qPCR instrument. In conclusion, the development of effective diagnostic tools for invasive candidiasis could save thousands of lives and find clinical, epidemiological and outbreak control applications.