The analysis of marker CDC 3 showed that all homozygous strains, including those from the patient, were plotted in one group except for the CNM- CL 7020 strain (Figure 1A). Due to the unexpected result for CNM-CL7020, the PCR product was sequenced (6x sequence coverage) and a 3 bp insertion at 67 pb from the forward primer was found. Heterozygous

strains click here were distributed in four groups according to their fragment length. The heterozygous strains CNM-CL 7694 and ATCC 64550 were plotted together although one of the alleles were different (Table 3). When we performed EF 3 fragments analysis by HRM, six different groups were plotted one of them contained strains from the patient while the control population was distributed into five groups according to its fragment size or whether they were homozygous or heterozygous (Figure 1B). Finally, HRM analysis of the HIS3 marker showed six different groups. Strains from the patient were grouped together again. Strains in the control population were grouped based on their fragment size pattern (Figure 1C). Discrimination power for CDC 3 marker was 0.53, for EF 3 it was 0.62 and for the HIS 3 marker it was 0.68. The combination of the three markers provided a DP AZD1480 ic50 value of 0.77 (Table 4). Discussion Typing methods have been described as useful tools for the differentiation

between strains isolated only once and those able to cause recurrent infections. Several methods have been developed to analyze microevolution and structure of C. albicans species. Although MLST (MultiLocus Sequence Typing) has been chosen as the most discriminatory technique [5, 32], several articles have recently pointed towards the suitability of MLP [14–16, 29]. In this study, nine isolates from a case of recurrent urinary infection were genotyped using microsatellites and a new HRM analysis method. Antifungal susceptibility testing revealed that strains from the patient

were susceptible and resistant in vitro to fluconazole in a random way. Microvariation between colonies due to exposure of C. albicans to azole antifungal agents has been widely described [10, 16] and the need to perform intercolony assays has also been reported [25, 33, 34]. We performed an inter-colony test modified from Schoofs et al. [25] and we were able to prove the coexistence of colonies resistant and susceptible to azoles in a high number of the strains oxyclozanide tested. The number of azole-resistant colonies was variable depending on azole concentration. A genotyping method based on HRM analysis was developed taking into account previous works showing that if the number of genotypes is higher than seven, the curve definition is not the best possible [35]. Based on that premise, for each marker we selected seven strains with different genotype, previously analysed by capillary electrophoresis. C. albicans microsatellites (CDC3, EF3 and HIS3) were amplified using LightCycler® 480 ResoLight as intercalating dye.