Development of biomarkers for the identification of pathogenic Candida species
Year: 2012
Type: dissertation
Abstract: In the last decades the incidence of fungal infections has increased exponentially, due to the development of more aggressive therapeutic techniques, which increase the number of immunocompromised and in risk individuals. The Candida species are the most common etiological agents isolated from opportunistic fungal infections in these patients and Candida albicans appears to be the most common species isolated. However, infections caused by nonalbicans species, such as C. parapsilosis, C. glabrata, C. tropicalis and C. krusei, are increasing alarmingly. It is thought that the susceptibility to antifungal drugs varies according to species, thus, the rapid and correct identification of infecting species are crucial. Microsatellite sequences have been largely used as molecular targets to differentiate and characterize strains. However, no studies have been performed using microsatellite DNA for Candida species identification. Therefore, the main objectives of this work were the evaluation of the potential of microsatellite markers for species differentiation and for identification of specific C. albicans lineages. After an intensive search for described microsatellite markers for the main Candida species, several markers were selected for C. albicans, C, glabrata, C. parapsilosis, C. tropicalis and C. krusei. These markers were combined into a multiplex strategy and this new developed system tested in 81 strains from 10 different species. All tested loci amplified correctly in single and multiplex conditions, except for the C. tropicalis selected locus that was unable to amplify in multiplex. After removal of the C. tropicalis primers, this system showed 100% specificity and 100% sensibility. To test if the microsatellites were able to identify specific C. albicans lineages two microsatellite markers were used, CAI and CAVIII. These markers are located in two repeat-containing ORFs, CAI is located in the terminal-3’ of RLM1 gene and CAVIII in the terminal-3’ of SAP8 gene. CAI microsatellite has been previously described and CAVIII was described for the first time in this study. CAVIII demonstrated to be highly specific for C. albicans strains and presented a discriminatory power of 0.72. The two microsatellite markers were tested in 144 unrelated C. albicans strains isolated from different body locations, allowing the statistical differentiation of strains from oral cavity, vulvovaginal infections and from extra-mucosal (respiratory tract and urine) infections. The number of extra-mucosal strains was increased to 224 and statistical analysis based in CAI genotypes, demonstrated significant differences between genotypes of strains isolated from superficial (oral and vagina) and invasive infections (respiratory tract, urine and blood). The results obtained allowed to conclude that the microsatellite loci analysis can be used to differentiate the most common Candida species, being an alternative in clinical diagnosis. Moreover, it was also possible observe that analysis of repeat containing ORFs, such as RLM1 and SAP8 is able to differentiate lineages of C. albicans.
Author: Joana Carvalho-Pereira
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W85018998