背景:可以通过使用含唑的平板常规进行烟曲霉分离株中的唑耐药性筛选(E.Def10.2方法),这需要在接种物调整之前过滤分生孢子悬浮液。
目的:我们评估了省略分生孢子悬浮液的过滤步骤是否会对E.Def10.2的性能产生负面影响。患者/方法烟曲霉严格分离株(n=92),根据EUCAST微量稀释E.Def9.4方法分类为对唑敏感或对唑耐药,被研究过。唑抗性分离株具有野生型cyp51A基因序列(n=3)或TR34-L98H(n=26),G54R(n=5),TR46-Y121F-T289A(n=1),F46Y-M172V-N248T-D255E-E427K(n=1),F165L(n=1),或G448S(n=1)cyp51A基因替换。根据EUCASTE.Def10.2程序制备内部含唑的琼脂平板。通过加入蒸馏水(Tween200.1%)获得分生孢子悬浮液。随后,在接种物调整至0.5McFarland之前,将悬浮液过滤或不过滤。使用微量稀释作为黄金标准,协议,灵敏度,并评估用两种接种物接种的琼脂平板的特异性。
结果:使用未过滤或过滤的分生孢子悬浮液的琼脂筛选方法的协议对伊曲康唑(100%)很高,伏立康唑(100%),泊沙康唑(97.8%)。当使用未过滤的悬浮液时,排除耐药性的程序的敏感性(100%)和特异性(98.2%)也很高。隔离物包含TR34-L98H,G54R,用改进的方法检测到TR46-Y121F-T289A取代。
结论:未过滤的分生孢子悬浮液在筛选烟曲霉时不会对E.Def10.2方法的性能产生负面影响。
BACKGROUND: Azole resistance screening in A. fumigatus isolates can be routinely carried out by using azole-containing plates (E.Def 10.2 method), that requires filtering conidial suspensions prior inoculum adjustment.
OBJECTIVE: We evaluated whether skipping the filtration step of conidial suspensions negatively influences the performance of the E.Def 10.2. Patients/Methods A. fumigatus sensu stricto isolates (n=92), classified as azole-susceptible or azole-resistant according to the EUCAST microdilution E.Def 9.4 method, were studied. Azole-resistant isolates had either wild type cyp51A gene sequence (n = 3) or the TR34 -L98H (n = 26), G54R (n = 5), TR46 -Y121F-T289A (n = 1), F46Y-M172V-N248T-D255E-E427K (n = 1), F165L (n=1), or G448S (n=1) cyp51A gene substitutions. In-house azole-containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions were obtained by adding distilled water (Tween 20 0.1%). Subsequently, the suspensions were either filtered or left unfiltered prior to inoculum adjustment to 0.5 McFarland. Using microdilution as the gold standard, agreement, sensitivity, and specificity of the agar plates inoculated with two inoculums were assessed.
RESULTS: Agreements for the agar screening method with either unfiltered or filtered conidial suspensions were high for itraconazole (100%), voriconazole (100%), and posaconazole (97.8%). Sensitivity (100%) and specificity (98.2%) of the procedure to rule in or out resistance when unfiltered suspensions were used were also high. Isolates harbouring the TR34 -L98H, G54R, and TR46 -Y121F-T289A substitutions were detected with the modified method.
CONCLUSIONS: Unfiltered conidial suspensions does not negatively influence the performance of the E.Def 10.2 method when screening for A. fumigatus sensu stricto.