流式细胞术快速检测假丝酵母菌药物敏感性方法的建立及应用

Establishment and application of a flow cytometry-based rapid drug susceptibility test for Candida species

目的：建立基于流式细胞分析技术快速检测假丝酵母菌临床菌株药物敏感性或耐药性的方法。方法以碘化丙锭（ PI）为荧光染料，采用白假丝酵母菌ATCC90029株确定流式药敏试验门设置及最佳实验条件。采用所建立的真菌流式药敏试验检测110株假丝酵母菌临床菌株对氟康唑和伏立康唑的敏感性或耐药性并经典的M27-A3常量稀释法进行比较。结果调整电压可将白假丝酵母菌ATCC90029株活菌与死菌在流式细胞仪SS/log（ FL3）门中分为边界清晰的两群细胞，不同比例死菌与活菌混合液与流式药敏试验检测值之间有高度相关性（r＝0．999）。流式药敏试验30 min内可出结果，其最适菌液浓度为1．0×106/ml、药物与真菌最适孵育时间为3 h、最佳染色方法及时间为脱氧胆酸钠预处理后PI染色5 min。流式药敏试验与M27-A3常量稀释法对110株假丝酵母菌临床菌株对氟康唑和伏立康唑敏感或耐药总符合率分别为98．2％和87．3％。结论较之经典的常量稀释法，流式药敏试验用于检测真菌对药物的敏感性或耐药性具有快速、准确、敏感等优点，具有临床应用前景。

Objective To establish a flow cytometry-based drug susceptibility test for the rapid de-tection of antifungal susceptibility or resistance of Candida isolates.Methods The gate selection and opti-mal experimental conditions of flow cytometry-based drug susceptibility test were determined by using Candi-da albicans strain ATCC90029 as the test strain and propidium iodide （ PI） as the fluorescent dye .The es-tablished flow cytometry-based drug susceptibility test was used to detect the susceptibility or resistance to fluconazole or voriconazole of 110 isolates belonging to Candida species, and the obtained results were com-pared with those by using typical M 27-A3 constant dilution method .Results The killed and viable Candida albicans ATCC90029 strains were clearly divided into two groups on the figure of SS /log （FL3） by regulating voltages.There was a high correlation between the results of susceptibility test and the proportions of killed and viable fungi in mixture （r=0.999）.The flow cytometry-based drug susceptibility test could provide the results within 30 min and its optimal concentration of fungal suspension , time of drug-fungus incubation , dyeing method and time were 1.0×106/ml, 3 h incubation and sodium deoxycholate-pretreated plus PI dye-ing for 5 min, respectively .The total coincident rates between the established test and the constant dilution method were 98.2%and 87.3%in the detection of drug susceptibility of 110 fungal isolates to fluconazole and voriconazole .Conclusion The flow cytometry-based drug susceptibility test shows advantages of rapidi-ty, accuracy and high sensitivity compared with the constant dilution method .It has a great potential for clin-ical application .