Fluconazole Susceptibility Of Clinical Isolates Of Candida Species And Detection Of Candida Albicans ERG11 Mutations

Species distribution and Susceptibilities to fluconazole of clinical Isolates of Candida speciesObjective:This study was performed to establish the species distribution and site of isolation, and to investigate fluconazole susceptibility of Candida clinical strains from non-AIDS patients .Methods:A total of 2681 Candida spp. isolates were collected and these strains were differentiated by CHROMagar, Vitek2 and using polymerase chain reaction. Susceptibility testing was performed by a broth microdilution method according to the CLSI (formerly NCCLS) M27-A2 reference method for broth antifungal susceptibility testing of yeasts.Results:There were 1903 (71.0%) Candida albicans, 486 (18.1%) Candida glabrata, 142 (5.3%) Candida tropicalis, 99 (3.7%) Candida krusei and 51 (1.9%) Candida spp. The clinical strains were isolated from Sputum (1690/63%), Vagina (623/23.2%), Urine (169/6.3%), Feces (102/3.8%), Blood (44/1.6%), (10/0.4%) and Other sites (43/1.6%). Candida albicans constituted greater than 60% of the Candida spp. isolated from respiratory (76.1%) , genital (68.2%) , and digestive(63.7%) tract specimens, but accounted for only 47.3% and 31.8% of Candida spp. isolates obtained from bloodstream and urine cultures;C. glabrata accounted for 40.9% of all Candida spp. isolated from blood, for 40% from Ascites and for 34.9% from urine, but for less than 25% of isolates from other sites of infection;Candida tropicalis accounted for 12.4% of all urinary tract isolates but was less common(﹤7%)in other sites of infection. Overall, 87.4% of all Candida isolatestested were susceptible (S) to fluconazole,4.1% were susceptible dose-dependent(S-DD) and 8.4% were resistant(R) . The resistant rate of Candida albicans, Candida glabrata, Candida tropicalis and Candida krusei to fluconazole was 4.5% (86/1903), 6.4% (31/468), 7.0% (10/142) and 100% (99/99). The fluconazole-resistant rate of all the Candida isolats from vagina, feces, urine, blood, sptum, ascites and other sites was 13.5% (84), 9.8% (10), 8.3% (14), 6.8% (3), 6.6%(112), 0% (0) and 7.0% (3).Conclusions:1. This study indicates that Candida albicans remains the predominant reasons for the fungal infections, but Candida glabrata has emerged as the second most common cause among the clinical Candida isolates in a chinese tertiery-care hospital.2. C. albicans was the species most commonly isolated from respiratory, genital and digestive tract infections; however the Candida glabrata and Candida tropicalis were the predominant isolated species from bloodstream and urinary tract. Episodes due to non-albicans species of Candida appear to be increasing, non-albicans Candida were the leading causes of candidemia and urinary tract infection.3. Candida isolates have remained highly susceptible to fluconazole, resistance is still uncommon in bloodstream and respiratory tract infection, however Candida species from Vagina had higher rates(13.5%) of resistance to fluconazole.4. Notably, Fluconazole remains an effective drug against Candida infection in this hospital, Candida krusei and vaginitis must be taken into consideration when choosing antifungal agents for calculated therapy Contribution of mutations in the ERG11 gene to fluconazole resistance in Candida albicansObjective:To analyse the relationship between mutations in the ERG11 gene of Candida ablicans clinical isolates and fluconazole resistance.Methods:The ERG11 genes of 72 isolates of Candida albicans (7 susceptible, 16 susceptible dose-dependent and 49 resistant) were amplified and sequenced.Results:27 silent mutations and 14 missense mutations were detected in 72 sequenced isolates: (i) there were 4 missense mutations only in resistant isolates, including D225H, K342R, G450E and V488I; (ii) a mutation of G129A occurred only in a clinical isolate of fluconazole-SDD Candida albicans; (iii) and there were 7 missense mutations in both fluconazole-resistant and fluconazole-SDD isolates, including Y132H, A114S, Y257H, V437I, G465S, G448E and K128T.There were 7 multi-site missense mutation patterns in 65 fluconazole-resistant or fluconazole-SDD isolates: (i) D116E, K128T, Y132H and G465S appeared simultaneously in 5 isolates; (ii) A114S and Y257H appeared simultaneously in 14 isolates without any other missense or silent mutation; (iii) Y132H and G450E appeared simultaneously in 2 fluconazole-resistant isolates without any other missense or silent mutation; (iv) Y132H and G448E appeared simultaneously in 3 isolates without any other missense mutation; (v) D116E and V488I appeared simultaneously in 3 fluconazole-resistant isolates;(vi) D116E, E266D and V488I appeared simultaneously in 2 fluconazole-resistant isolates; and (ⅶ) D116E, E266D and V437I appeared simultaneously in 3 isolates.Another single missense mutation patterns in clinical isolates such as V437I and K342R were identified in fluconazole-resistant isolates.Conclusions:1. This study further indicates that both multi-site missense mutation patterns and single missense mutation patterns may contribute to fluconazole resistance or dose-dependence. However multiple mutations in ERG11 are required to confer decreased susceptibility to fluconazole.2. The multiple mutations of D116E, K128T, Y132H and G465S; D116E,E266D and V437I;D116E,E266D and V488I were firsted detected.