Molecular markers for direct testing of antifungal resistance in Candida

Candida albicans is an opportunistic pathogen that resides as a commensal colonising the mouth, gastrointestinal tract, moist areas of the skin and genital areas. It is kept under control by local and systemic immune defence mechanisms, by microbial control, and by restricting its access to deeper tissues by epithelial barriers. If this control is lost Candida becomes an opportunistic pathogen and cause superficial and invasive infections including candidaemia with high mortality rate. There are five antifungal drug classes used to treat Candida infections but the emergence of resistance is an increasing problem in patient management and treatment outcomes. Methods used to diagnose invasive infection usually rely on culture based methods with low sensitivity and long turn-around times. Therefore, more rapid and accurate molecular tools are needed to guide the choice of appropriate antifungal drugs and improve patient outcomes. This thesis addressed these issues by focusing on the epidemiology of Candida infections at a UK tertiary referral hospital, and next-generation sequencing (NGS) and pyrosequencing as two molecular tools for rapid detection of polymorphisms associated with host adaptation and drug resistance in C. albicans isolates. A Candida database for a 12- year period, obtained from Wythenshawe Hospital, was analysed. The main finding was the reduction of candidaemia cases with no emergence of echinocandin resistance. Then, genome sequences obtained from NGS of 14 isolates from autoimmune polyendocrinopathy candidosis ectodermal dystrophy (APECED) were analysed and variants associated with APECED were identified. These included single nucleotide polymorphisms (SNPs) in cell wall and DNA repair genes that could enhance their adaptation in APECED patients. Seven APECED isolates were pyrosequenced for 27 SNPs found in the multi-drug resistance regulator MRR1 gene and the results were comparable to the results obtained from NGS for the same isolates. Pyrosequencing of three (L171P, V341E and L592F) of these 27 SNPs was used to screen 100 isolates with variable azole susceptibility from the Mycology Reference Centre Manchester depository. This method was shown to be accurate and rapid to detect polymorphisms in C. albicans.