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目的对新疆首次临床分离的5株新生隐球菌进行分子鉴定、RAPD-PCR基因分型及体外药物敏感性研究。方法5株新生隐球菌分子鉴定采用核糖体DNA大亚基(LSU rDNA)D1/D2基因区域分子鉴定。分子分型采用引物SEQ-6结合RAPD-PCR法扩增5株新疆临床分离新生隐球菌菌株及5株由上海长征医院提供分离自上海新生隐球菌菌株,根据扩增产物带型进行基因型判定。采用临床实验室标准化协会(CLSI)的酵母微量液基稀释法(M27-A3)测定5株新疆临床分离新生隐球菌菌株对6种抗真菌药(伏立康唑、伊曲康唑、两性霉素B、特比萘芬、氟康唑、5-氟胞嘧啶)的体外敏感性。结果 5株新疆临床分离隐球菌菌株经过D1/D2区域序列分析在基因Bank中比对后鉴定为新生隐球菌。5株新疆临床分离新生隐球菌和5株上海新生隐球菌菌株经RAPD-PCR法扩增,带型显示分为A、B、C、D 4个基因型,其中新疆5株菌株A型1株、其余4株均为B型,上海菌株B型为1株,C型3株、D型1株,5株新疆临床分离新生隐球菌株对伏立康唑、伊曲康唑、两性霉素B、特比萘芬的MIC值(μg/mL)范围依次为:0.062 5~0.25、0.25~1、0.125~0.5、1~2,对氟康唑、5-氟胞嘧啶MIC值较高,MIC值范围依次为:4~16、8~32。结论新疆临床分离5株隐球菌株采用D1/D2基因序列鉴定为新生隐球菌。RAPD-PCR分型显示新疆临床分离5株新生隐球菌株以B型基因型为主。A型和B型对伏立康唑、两性霉素B敏感,对伊曲康唑、特比萘芬剂量依赖型敏感,对氟康唑、5-氟胞嘧啶耐药。
OBJECTIVE: To identify the first clinically isolated Cryptococcus neoformans from Xinjiang and analyze the genotypes and in vitro drug sensitivity of RAPD-PCR. Methods Five strains of Cryptococcus neoformans were identified by molecular identification of D1 / D2 gene of LSU rDNA. Molecular Typing Five strains of Cryptococcus neoformans clinically isolated from Xinjiang and five strains of Cryptococcus neoformans isolated from Shanghai were provided by primer SEQ-6 combined with RAPD-PCR and genotypes were determined according to the genotype of the amplified product . Five strains of clinical isolates of Cryptococcus neoformans from Xinjiang clinically determined by CLSI (M27-A3) were tested against 6 antifungal agents (voriconazole, itraconazole, amphotericin B, Terbinafine, fluconazole, 5-fluorocytosine) in vitro. Results Five clinical isolates of Cryptococcus neoformans from Xinjiang were identified as Cryptococcus neoformans by the sequence analysis of D1 / D2 region in Bank. Five clinical isolates of Cryptococcus neoformans and five strains of C. neoformans from Xinjiang were amplified by RAPD-PCR, and the genotypes were divided into four genotypes, A, B, C, and D, of which five isolates belonged to type A , The remaining four were type B, Shanghai strain B type 1 strain, C type 3 strain, D type 1 strain, 5 strains of clinical isolates of Cryptococcus neoformans Xinjiang voriconazole, itraconazole, amphotericin B, special MIC values (μg / mL) for flunarfin ranged from 0.062 5 to 0.25,0.25 to 1, from 0.125 to 0.5 and from 1 to 2, respectively. MIC values for fluconazole and 5- Followed by: 4 ~ 16,8 ~ 32. Conclusion The clinical isolates of five Cryptococcus from Xinjiang were identified as Cryptococcus neoformans using the D1 / D2 gene sequence. RAPD-PCR typing showed that clinical isolates of 5 strains of Cryptococcus neoformans in Xinjiang belonged to type B genotype. A and B were sensitive to voriconazole and amphotericin B, sensitive to itraconazole and terbinafine, and resistant to fluconazole and 5-fluorocytosine.