Development of azole resistance in Aspergillus fumigatus during azole therapy associated with change in virulence

Abstract

Four sequential Aspergillus fumigatus isolates from a patient with chronic granulomatous disease (CGD) eventually failing azole-echinocandin combination therapy were investigated. The first two isolates (1 and 2) were susceptible to antifungal azoles, but increased itraconazole, voriconazole and posaconazole MICs were found for the last two isolates (3 and 4). Microsatellite typing showed that the 4 isolates were isogenic, suggesting that resistance had been acquired during azole treatment of the patient. An immunocompromised mouse model confirmed that the in vitro resistance corresponded with treatment failure. Mice challenged with the resistant isolate 4 failed to respond to posaconazole therapy, while those infected by susceptible isolate 2 responded. Posaconazole-anidulafungin combination therapy was effective in mice challenged with isolate 4. No mutations were found in the Cyp51A gene of the four isolates. However, expression experiments of the Cyp51A showed that the expression was increased in the resistant isolates, compared to the azole-susceptible isolates. The microscopic morphology of the four isolates was similar, but a clear alteration in radial growth and a significantly reduced growth rate of the resistant isolates on solid and in broth medium was observed compared to isolates 1 and 2 and to unrelated wild-type controls. In the mouse model the virulence of isolates 3 and 4 was reduced compared to the susceptible ones and to wild-type controls. For the first time, the acquisition of azole resistance despite azole-echinocandin combination therapy is described in a CGD patient and the resistance demonstrated to be directly associated with significant change of virulence.

Figure 1. In vivo susceptibility of sequential A. fumigatus isolates from a CGD patient failing azole therapy.

Susceptibility is shown as survival curve (a and d), as fungal CFU kidney burden (b and e) and as Aspergillus DNA load in kidney tissue. The early susceptible isolate (isolate no 2) is shown in fig. a, b and c and the late resistant isolate (isolate no 4) in fig. c, d and e. AND: anidulafungin, POS: posaconazole, AND+POS: combination therapy of anidulafungin and posaconazole, d4: day 4, d8: day 8, * P<0.05 compared to control.

Figure 2. CYP51A mRNA levels in the 4 sequential A. fumigatus isolates and in a control wild type isolate AZN 8196 (WT) and a tri-azole resistant control isolate with over-expression of the CYP51A gene due to the L98H mutation and promoter tandem repeat.

Figure 3. In vivo virulence of sequential isolates of A. fumigatus in immunosuppressed mice.

Survival of mice in groups of six challenged with each of the four sequential isolates (1–4) or a control isolate (NCPF2109), respectively, is shown. The survival data was combined per isolate, thus each survival curve displays the mortality of six mice challenged with the indicated isolate, two of which were challenged with a high inoculum (5x10⁴ CFU/ml), two with an intermediate inoculum (10⁴ CFU/ml) and two with a low inoculum (2×10³ CFU/ml).

Figure 4. Growth rate of the 4 sequential isolates expressed as radial growth rate on solid V8 agar (a) and as optical densities in fluent medium (b).

The laboratory strain NCP2109 is included as unrelated comparator. In the radial growth experiment (a) the growth rate is expressed in mean diameter (mm) and Kr (mm/hour) after 24–72 hours of two separate experiments performed in triplicate. In the kinetic evaluation of growth over time in liquid medium (b) growth is expressed as optical densities measured every 10 min.