浅部与深部感染来源白念珠菌氟康唑耐药株药物敏感性及耐药基因突变比较

【摘要】 目的 比较浅部、深部感染来源的氟康唑耐药白念珠菌体外对8种抗真菌药物的敏感性及耐药基因突变。方法 26株深部感染来源白念珠菌耐药株、33株浅部感染来源白念珠菌耐药株，参照CLSI酵母菌检测方案M27-A4测定上述菌株对氟康唑、伏立康唑、伊曲康唑、泊沙康唑、两性霉素B、氟胞嘧啶、特比萘芬、米卡芬净8种药物单独或联合的体外敏感性。提取所有耐药菌株DNA，通过PCR检测ERG3、ERG11、FUR1 3种耐药基因的突变情况。满足正态分布和方差齐性检验的定量资料两组间比较采用独立样本t检验，不满足者组间比较采用Mann-Whitney U检验，定性资料组间比较采用卡方检验。结果 氟康唑、伊曲康唑、伏立康唑、泊沙康唑、氟胞嘧啶的最小抑菌浓度（MIC）在浅部感染组和深部感染组间差异均有统计学意义（均P＜0.05），而两性霉素B、米卡芬净的MIC两组间差异无统计学意义（均P > 0.05）。96.6%的菌株特比萘芬MIC值＞64 μg/ml，无法进行组间对比。15株白念珠菌（7株深部感染来源、8株浅部感染来源）特比萘芬 + 唑类（氟康唑、伏立康唑、伊曲康唑、泊沙康唑）联合药敏试验均显示为协同效应，部分抑菌浓度（FIC）指数0.033～0.187；氟胞嘧啶 + 唑类、氟胞嘧啶 + 两性霉素B、两性霉素B + 氟康唑组合均无明显协同作用，FIC指数0.56～1.125。浅部感染来源白念珠菌ERG3基因突变包括错义突变V351A（33株，100%），深部感染来源白念珠菌包括错义突变V351A（13株，50%）、A353T（4株，15%）；浅部感染来源白念珠菌ERG11基因突变包括错义突变I437V（32株，97%）、Y132H（23株，70%）、T123I（16株，48%）、K128T（6株，18%）、D116E（5株，15%）、A114S（4株，12%）、E266D（2株，6%）、G448E（2株，6%）、G465S（2株，6%），深部感染来源白念珠菌包括I437V（23株，88%）、E266D（13株，50%）、E260G（5株，19%）、V488I（4株，15%）；浅部感染来源白念珠菌 FUR1基因突变包括错义突变R101C（11株，33%），深部感染来源白念珠菌未检出错义突变。结论 浅部与深部感染来源白念珠菌氟康唑耐药株对药物的敏感性及基因突变位点均有所差别。

Comparison of drug susceptibility of and drug resistance mutations in fluconazole-resistant Candida albicans strains from superficial and deep infections

【Abstract】 Objective To compare the in vitro susceptibility of fluconazole-resistant Candida albicans strains from superficial and deep infections to 8 antifungal drugs, and to compare drug resistance mutations in these strains. Methods According to the Clinical and Laboratory Standards Institute （CLSI）protocol M27-A4, 26 deep infection-derived and 33 superficial infection-derived drug-resistant Candida albicans strains were tested for in vitro susceptibility to 8 antifungal drugs （fluconazole, voriconazole, itraconazole, posaconazole, amphotericin B, fluorocytosine, terbinafine, and micafungin） alone or in combination. DNA was extracted from all drug-resistant strains, and mutations in 3 drug resistance genes, including ERG3, ERG11 and FUR1, were detected by PCR. Normally distributed measurement data with homogeneous variance were compared between two groups by using two-independent-sample t test, non-normally distributed measurement data with non-homogeneous variance were compared using Mann-Whitney U test, and enumeration data were compared using chi-square test. Results The minimum inhibitory concentrations （MICs） of fluconazole, itraconazole, voriconazole, posaconazole and fluorocytosine all significantly differed between the superficial infection group and deep infection group （all P < 0.05）, while there was no significant difference in the MIC of amphotericin B or micafungin between the two groups （both P > 0.05）. The MIC of terbinafine was ＞64 μg/ml in 96.6% of the above strains, so could not be compared between groups. As combination drug susceptibility testing revealed, the combination of terbinafine with azoles （fluconazole, voriconazole, itraconazole or posaconazole） showed synergistic inhibitory effects against 15 Candida albicans strains （7 strains from deep infections, 8 strains from superficial infections）, with fractional inhibitory concentration （FIC） indices being 0.033 to 0.187; no marked synergistic effect was observed in the combinations between fluorocytosine and azoles, between fluorocytosine and amphotericin B, or between amphotericin B and fluconazole, with the FIC indices being 0.56 to 1.125. The missense mutation V351A in the ERG3 gene was identified in all the 33 （100%） superficial infection-derived strains, as well as in 13 （50%） deep infection-derived strains, and the mutation A353T in the ERG3 gene was identified in 4 （15%） deep infection-derived strains; as for the ERG11 gene, missense mutations identified in the superficial infection-derived strains included I437V （32 strains, 97%）, Y132H （23 strains, 70%）, T123I （16 strains, 48%）, K128T （6 strains, 18%）, D116E （5 strains, 15%）, A114S （4 strains, 12%）, E266D （2 strains, 6%）, G448E （2 strains, 6%）, and G465S （2 strains, 6%）, while missense mutations identified in the deep infection-derived strains included I437V （23 strains, 88%）, E266D （13 strains, 50%）, E260G （5 strains, 19%）, and V488I （4 strains, 15%）; the missense mutation R101C in the FUR1 gene was identified in 11 （33%） superficial infection-derived strains, but not identified in deep infection-derived strains. Conclusion The drug susceptibility and drug resistance mutations differed to some extent between superficial infection- and deep infection-derived fluconazole-resistant Candida albicans strains.