Molecular typing and phylogenetic analysis of Candida albicans isolates from different patient populations : a thesis presented in partial fulfilment of the requirements for the degree of PhD in Molecular Biology and Genetics at Massey University, Palmerston North, New Zealand

Abstract

An important question in understanding the epidemiology of Candida albicans is to determine whether certain strains, e.g. HSP strains (highly successful strains), more prevalent in Candidosis patients than in healthy individuals, replace commensal strains under certain conditions and whether the replacing strains may cause Candidosis. This question was investigated in experiments which monitored the genetic diversity of commensal C. albicans isolates obtained from individuals before and after they were exposed to conditions that may predispose them to the development of Candidosis. The distinctiveness of C.albicans strains isolated from individuals was analysed using the phylogenetic method of split decomposition. The method was found to provide a good representation of the phylogenetic information in strain replacement Ca3 data. Our study highlighted difficulties in monitoring of strain replacement with Ca3 methodology. An indication for strain replacement was observed in one patient at low risk to acquire Candidosis. However, the observations that cancer patients, who were at a high risk of developing Candidosis, were colonised with diverse strains and that healthy individuals could be colonised with different commensal C.albicans strains within one body location, cautioned against overinterpretation of this finding. These results demonstrated the need for extensive sampling of larger numbers of isolates from different body locations when evaluating replacement hypotheses. In investigating potential sources of C.albicans infections we successfully isolated this fungus from the hospital environment of high risk patients, demonstrating the potential of the hospital environments as a source for infection causing strains. In characterising strains, a nonradioactive fingerprinting protocol was developed for a more convenient use of Ca3 fingerprinting. Until now the existence of the HSP group has been based entirely on Ca3 fingerprinting data. To test for the existence of this group we have analysed amplified fragment length polymorphisms (AFLP) of 36 C.albicans isolates from different geographical regions. Phylogenetic reconstruction from both data forms (Ca3 and AFLP data) were highly congruent and suggest a worldwide distribution of HSP strains. Study of the tree building properties of AFLP and Ca3 data using Quartet Puzzling and a tree comparison metric showed that AFLP data were more treelike than the Ca3 data. However, whilst both AFLP and Ca3 methods provided high resolution data to identify strains and substrains of C.albicans, the need for population based studies to test for strain replacement makes the use of either method limited. For this reason, both nuclear ITS and AFLP derived PCR markers were investigated for their potential use in such studies. In particular, one AFLP derived PCR marker that was partially characterised appears very promising for future strain replacement studies. It is likely to provide a simple diagnostic test for rapid identification of HSP strains.