外阴阴道念珠菌病患者菌群调查及其ERG5基因突变研究

目的?了解外阴阴道念珠菌病（VVC）患者白念珠菌分离及ERG5基因突变情况。方法?收集无锡市妇幼保健院2018年6—12月妇产科门诊VVC患者阴道分泌物500例，通过镜下观察菌丝及孢子选取可疑标本，经沙保弱琼脂平板增菌后接种科玛嘉显色平板，进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）鉴定；用真菌快速培养鉴定药敏试剂盒对白念珠菌进行药敏试验；PCR扩增部分白念珠菌ERG5基因并进行测序分析。结果?显色平板鉴定结果显示，VVC病原以白念珠菌最多（54.7%），其次为光滑念珠菌（22.5%）、热带念珠菌（16.5%）、克柔念珠菌（4.2%）和其他真菌（2.1%）；156株显色平板鉴定结果为白念珠菌的菌株中，经MALDI-TOF MS鉴定为白念珠菌共155株。155株白念珠菌对5-氟尿嘧啶均敏感，对各类唑类药物呈现不同水平耐药性；对7株（3株唑类药物耐药菌株，3株唑类药物敏感菌株和1株质控菌株）进行ERG5扩增测序，检出2个突变位点（1个同义突变位点G528A，1个错义突变位点G528C）。结论?外阴阴道念珠菌以白念珠菌为主，并对各类唑类药物表现出不同的耐药率，耐药菌株中ERG5基因在G528C位点突变可能与耐药有关。

Investigation of flora and mutation of ERG5 gene in vulvovaginal candidiasis Jin Lei,Weifeng Shi

Objective: To understand the incidence of candida vaginitis and the main types of Candida in this area,and explore the common antifungal antimicrobial sensitivity of C.albicans isolated from clinical. The ERG5 genes of C.albicans was amplified by PCR and sequencing and to investigate whether the mutation of ERG5 genes of C.albicans was associated with its drug resistance.Methods: During Jun to Dec 2018,vaginal secretions from gynecologic and obstetric outpatients were collected, and 285 strains of Candida were obtained by smear microscopy and CHROMagar chromogenic medium culture.Additionally, the identification and susceptibility tests of 156 C.albicans were detected by rapid fungal culture and drug sensitivity tests,as well as mass spectrometry. The ERG5 genes of C. albicans was amplified by PCR and sequencing for the analysis of mutation sites.Results: Of 285 fungi detected for the first time, C.albicans C.smooth, C.tropicalis, C.krusei and other fungi accounted for 156 (54.7%), 64(22.5%), 47(16.5%), 12(4.2%),and 6(2.1%),respectively. 156 strains of C.albicans were further identified by MALDI-TOF MS, indicating that One strain could mot be reliably identified(Score below 1.700) and the rest were C.albicans . strain Antimicrobial susceptibility tests in vitro showed that 155 strains of C.albicans were sensitive to 5-fluorouracil (5-FC), and the sensitivity to amphotericin (AMB) and nystatin (NYS) was 99.3% and 98.7%,respectively. However, the resistance rates of 155 strains against ketoconazole(KET),fluconazole(FIU),itraconazole(ITR),voriconazole(VOR),clotrimazole(CLO),miconazole(MIC), Econazole (ECO) were 43.59%, 62.82%, 61.54%, 50.64%, 50.64%, 51.92% and 51.28%, respectively. The ERG5 genes in 7 strains of C.albicans were chosen to be amplified by PCR and sequencing. Two mutation loci of ERG5 were found at the same site, one of which was synonymous mutation (G528A) and the other was missense mutation (G528C).Conclusions: (1)The proportion of C.albicans in VVC patients showed a downward trend, while non C.albicans gradually increased in our hospital.(2)When the traditional phenotypic identification is difficult to determine, it can be supplemented by mass spectrometry technique. (3)C.albicans is highly sensitive to 5-FC, AMB and NYS, but the resistance to azole drugs increased. There are cross-resistance and multi-drug resistance among C.albicans.(4)The mutation of ERG5 gene at 528 locus in C.albicans may be related to azole resistance.