Study On Antifungal Susceptibility Test Of Candida Albicans And Detection Of Point Mutations In Fluconazole-resistant Isolates

[Objective] Candida albicans were collected and identified from clinical specimens, to investigate the susceptibility of C. albicans isolates to fluconazole and itraconazole. Clinical C. albicans strains resistant to fluconazole were collected, and were detected point mutations of ERG11, TAC1, MRR1, and UPC2 gene, in order to better understand the relationship between point mutations of ERG11, TAC1, MRR1, and UPC2 and resistance to azoles.[Methods] C. albicans were collected from clinical specimens and were identified by germ tube formation test, CHROMagar medium, and chlamydospore formation test. M27-A2 Broth microdilution method was used to assess the susceptibility of clinical C. albicans to fluconazole and itraconazole. Totally,11 fluconazole-resistant strains were obtained. Extracted DNA by urea pyrolysis method, and acquired target ERG11 gene, three fragments of TAC1 gene including T225A, A736V, N972D, N977D and G980E amino acids substitutions, two fragments of MRR1 gene including P683S and G997V amino acids substitutions, and one fragment of UPC2 gene including A643T and G648D amino acids substitutions by PCR amplification. Then the target fragments were sequenced.[Results] Totally,506 clinical isolates of C. albicans were collected. Sensitivity rate of C. albicans to fluconazole is 99.21%(502/506), dose dependent sensitivity rate is 0.20%(1/506), and resistant rate is 0.59%(3/506); sensitivity rate of C. albicans to itraconazole is 43.28%(219/506), dose dependent sensitivity rate is 53.16%(269/506), and resistant rate is 3.56%(18/506). There was one isolate presenting cross-resistant to both fluconazole and itraconazole. For ERG 11 gene,9 of 11 fluconazole-resistant strains were detected missense mutations resulting in 8 kinds of amino acids substitutions (prevalence 81.82%), they were E266D(n=5), D116E(n=3), Y132H(n=2), G448E(n=2), Y132F(n=1), K143Q(n=1), T229A(n=1), G464S(n=1). Among them, K143Q was a new mutation which was not reported before. For UPC2 gene,2 of 11 fluconazole-resistant strains were detected 1 kind of amino acids substitutions (prevalence 18.18%). The mutation was G648D(n=2). For TAC1 and MRR1 gene, the mutations mentained above were not detected. [Conclusion] Some of the clinical C. albicans isolates do resist or cross-resist to antifungal azoles. The resistant rate of itraconazole is higher than fluconazole. However, most of C. albicans isolates are susceptible to fluconazole and itraconazole. Fluconazole and itraconazole still can be used to treat clinical C. albicans infections. Owing to the high susceptible-dose dependent rate of itraconazole, increasing the dosage maybe a considerable choice to improve the clinical therapeutic effect. There were close relation between mutations of ERG 11 and resistance to azoles, but the number of mutations or distribution patterns mutations have no obvious regular pattern. Further investigation should be carried out to make clear the relationship between mutations of TAC1, MRR1, UPC2 gene and resistance to fluconazole. Multiple molecular mechanisms lead to the resistance to azoles in C. albicans, so in the future further explore should be done to beter understand molecular mechanisms of resistance to azoles.