Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Marañón, Universidad Complutense de Madrid, Madrid, Spain.
Antimicrob Agents Chemother. 2012 Jan;56(1):174-8. doi: 10.1128/AAC.00301-11. Epub 2011 Oct 17.
Secondary resistance to azoles in Aspergillus fumigatus isolates from patients taking long-term itraconazole therapy has been described. We studied the acquisition of secondary azole resistance in 20 A. fumigatus isolates with no mutations at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene. Adjusted conidium inocula (3 × 10(7) CFU/ml) of each isolate were prepared and progressively or directly exposed to increasing itraconazole concentrations, ranging from 0.5 μg/ml to 16 μg/ml. Itraconazole, voriconazole, and posaconazole MICs were determined using the CLSI M38-A2 procedure before (MIC(initial)) and after (MIC(final)) exposure to itraconazole. In both procedures, the MIC(final) was significantly higher than the MIC(initial). However, after progressive exposure to itraconazole, the MICs of the three azoles were higher than after direct exposure. No mutations were found at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene of isolates growing at the highest concentration of itraconazole. More concentrated conidium inocula (2 × 10(9) CFU/ml) plated in itraconazole at 4 μg/ml revealed the presence of heteroresistant populations in two initially wild-type isolates. These isolates became resistant to itraconazole and posaconazole only after use of the concentrated inoculum. These heteroresistant isolates harbored a mutation at codon G54, and the MICs of itraconazole and posaconazole were >16 μg/ml. In all procedures, A. fumigatus short tandem repeat (STRAf) typing was used to demonstrate that the genotype did not change before or after exposure to itraconazole.
已有报道称，长期接受伊曲康唑治疗的烟曲霉患者分离株中出现了唑类药物继发耐药。我们研究了 20 株烟曲霉分离株在 cyp51A 基因的 54、98、138、220、432 或 448 密码子处没有突变的情况下获得唑类药物继发耐药的情况。每个分离株的调整后的分生孢子接种物（3×10(7)CFU/ml）均进行了制备，并分别或直接暴露于逐渐增加的伊曲康唑浓度（0.5 μg/ml 至 16 μg/ml）中。使用 CLSI M38-A2 程序，在（初始 MIC）和暴露于伊曲康唑后（最终 MIC），分别测定了伊曲康唑、伏立康唑和泊沙康唑的 MIC。在这两种程序中，最终 MIC 均明显高于初始 MIC。但是，在逐渐暴露于伊曲康唑后，三种唑类药物的 MIC 均高于直接暴露。在生长于伊曲康唑最高浓度的分离株的 cyp51A 基因的 54、98、138、220、432 或 448 密码子处没有发现突变。在伊曲康唑 4 μg/ml 中接种更浓缩的分生孢子接种物（2×10(9)CFU/ml），揭示了最初为野生型的两个分离株存在异质耐药群体。仅在用浓缩接种物处理后，这些分离株才对伊曲康唑和泊沙康唑产生耐药性。这些异质耐药分离株携带 G54 密码子的突变，伊曲康唑和泊沙康唑的 MIC 值均＞16 μg/ml。在所有程序中，均使用烟曲霉短串联重复（STRAf）分型来证明在暴露于伊曲康唑之前或之后基因型没有改变。
