Unit of Mycology, Statens Serum Institut, Copenhagen, Denmark.
Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark.
J Antimicrob Chemother. 2017 Dec 1;72(12):3325-3333. doi: 10.1093/jac/dkx319.
Azole-resistant Aspergillus fumigatus is emerging worldwide. Reference susceptibility testing methods are technically demanding and no validated commercial susceptibility tests for moulds currently exist. In this multicentre study a 4-well azole-containing screening agar method was evaluated using clinically relevant isolates.
Forty WT and 39 cyp51A mutant A. fumigatus [G54 (n = 10), M220 (n = 10), TR34/L98H (n = 9) and TR46/Y121F/T289A (n = 10)] were tested individually and as simulated mixed samples (sampling 4 WT and 1 mutant colonies). EUCAST MICs were determined following E.Def 9.3. In-house and commercial 4-well plates containing agars supplemented with 4 mg/L itraconazole, 1 mg/L voriconazole, 0.5 mg/L posaconazole and no antifungal, respectively, were evaluated. Growth was scored (0-3) by two independent observers in three laboratories. Inter-plate, inter-observer, essential and categorical agreement, sensitivity and specificity were calculated.
CYP51A genotype and antifungal compound-specific MICs and growth patterns were documented. The inter-observer agreement was excellent with 86%-99% identical scores (range 80%-100%) for both plates. The qualitative agreement (no growth versus growth) was excellent (median 95%-100%, range 87%-100%, overall). The overall sensitivity and specificity for the 4-well plate (no growth versus growth) was 99% (range 97%-100%) and 99% (95%-100%), respectively. The sensitivity for simulated WT/mutant specimens was 94% (range 83%-100%) for the WT-TR34/L98H combination, but 100% for the WT/G54W combination. The performance remained unchanged using only itraconazole- and voriconazole-containing agars, but was lower for the other combinations.
Implementation of the 4-well screening plate in routine laboratories will allow easy and reliable detection of the most common azole-resistant A. fumigatus.
唑类耐药烟曲霉在全球范围内不断出现。参考药敏检测方法技术要求高，目前尚无经过验证的商业化霉菌药敏检测方法。本研究采用多中心方法，使用临床相关分离株对 4 孔含唑类药物筛选琼脂法进行评估。
40 株野生型（WT）和 39 株 Cyp51A 突变型烟曲霉[G54（n=10）、M220（n=10）、TR34/L98H（n=9）和 TR46/Y121F/T289A（n=10）]单独检测，并模拟混合样本（分别取 4 个 WT 和 1 个突变菌落）进行检测。根据 E.Def 9.3 确定 EUCAST MIC 值。分别使用含有 4mg/L 伊曲康唑、1mg/L 伏立康唑、0.5mg/L 泊沙康唑和无抗真菌药物的 4 孔琼脂平板进行检测。由 3 个实验室的 2 名独立观察者对平板进行评分（0-3 分）。计算平板间、观察者间、定性和分类一致性、敏感性和特异性。
记录了 Cyp51A 基因型和抗真菌化合物特异性 MIC 值和生长模式。两种平板的观察者间一致性极好，评分完全一致（范围 80%-100%，得分 86%-99%）。平板定性（无生长与生长）的一致性极好（中位数 95%-100%，范围 87%-100%，总体）。4 孔平板（无生长与生长）的总敏感性和特异性分别为 99%（范围 97%-100%）和 99%（95%-100%）。WT/TR34/L98H 组合的模拟 WT/突变标本的敏感性为 94%（范围 83%-100%），但 WT/G54W 组合的敏感性为 100%。仅使用伊曲康唑和伏立康唑含琼脂时，性能保持不变，但其他组合的性能较低。
在常规实验室中实施 4 孔筛选平板将能够轻松可靠地检测最常见的唑类耐药烟曲霉。
