Antifungal susceptibility testing results for four triazole drugs and AMB at 48 h are shown in Table . All triazoles demonstrated comparable degrees of antifungal activity with MICs of ≤4 μg/ml (POS, 97.6%; ITR, 96.3%; VOR, 95.9%; RAV, 93.5%). The triazoles were not active against five isolates of A. calidoustus, for which MICs were ≥4 μg/ml. In addition, one (14.3%) of seven isolates of A. versicolor was resistant to the triazoles tested, except VOR. Among the A. fumigatus isolates, only one (0.6%) of 181 isolates was triazole resistant, with MICs of ≥4 μg/ml for ITR and VOR. The MIC for this isolate was 0.25 μg/ml for POS, and RAV was not tested.
In vitro susceptibilities of 274 Aspergillus isolates causing IA in transplant recipients
Low MICs to AMB were present for most isolates, with 93.3% of all isolates inhibited at an MIC of ≤1 μg/ml. The exception was A. terreus, for which 15 (68%) of 22 isolates tested had MICs of >1 μg/ml. One A. nidulans isolate had an MIC of 4 μg/ml, and two N. udagawae isolates had an MIC of 2 μg/ml.
Overall, resistant isolates were not uncommon in this collection, with resistance to triazoles in 10 (3.6%) or AMB in 18 (6.6%) of 274 isolates. When evaluating the relationship between MIC and overall mortality at 6 or 12 weeks in patients who received AMB or VOR, having either a triazole- or AMB-resistant isolate was not significantly associated with mortality by univariate analysis (Table ). In addition, among A. terreus isolates, no difference in outcomes by MIC was noted (Table ). With linear regression, as can be seen in Fig. , there was not a significant relationship between increasing VOR MIC and mortality at 6 weeks (R2 = 0.61; P = 0.065) or 12 weeks (R2 = 0.18; P = 0.40) among patients who received VOR therapy. In contrast, an increasing AMB MIC was associated with decreased mortality at 6 weeks (R2 = 0.70; P = 0.04); but not at 12 weeks (R2 = 0.39; P = 0.18) (Fig. ).
Relationship between MICs and all-cause mortality in patients who received VOR and AMB therapy
FIG. 1. All-cause mortality based on Aspergillus MIC among 115 patients who received VOR therapy. Regression lines for the association of MIC with mortality at 6 weeks (R2 = 0.61; P = 0.065) and 12 weeks (R2 = 0.18; P = 0.40) are (more ...)
FIG. 2. All-cause mortality based on Aspergillus MIC among 111 patients who received AMB therapy. Regression lines for the association of MIC with mortality at 6 weeks (R2 = 0.70; P = 0.04) and 12 weeks (R2 = 0.39; P = 0.18) are (more ...)
An additional analysis with use of an epidemiological cutoff for VOR was performed. The modal MIC for VOR among A. fumigatus isolates was 0.5 μg/ml, and the modal MIC ± 1 twofold dilution encompassed 96% of the strains for VOR (range, 0.25 to 1 μg/ml). Using a VOR cutoff of ≤1 μg/ml, the analysis did not show an association with all-cause mortality in patients with A. fumigatus infection (at 6 weeks, P = 0.28; at 12 weeks, P = 0.59).
This collection represents a large number of isolates from transplant patients with proven or probable IA collected from 19 U.S. institutions from 2001 to 2006. Overall, triazole agents showed good in vitro activity, with POS having the lowest MICs. Similar to recent reports, triazoles were active in over 95% of isolates (8
). It is important to note that triazole resistance was uncommon among A. fumigatus
isolates; only 1 of 181 isolates was resistant to two of four triazoles tested. This is in contrast to a recent report from medical centers in The Netherlands, in which ITR resistance occurred in 13 patients (12.8%) (23
). It is important to note that our isolates came from transplant patients only, a very different population than that described by Snelders and colleagues (23
). In addition, different susceptibility testing methods may be responsible for some variability. Finally, as Snelders and colleagues suggest, perhaps azole resistance among isolates from The Netherlands may be related to the use of azole fungicides. Our findings are similar to those seen in a recent U.S. survey, suggesting global geographic variation in susceptibility patterns (19
). The impact of azole fungicides on resistance among U.S. isolates needs further exploration.
In the present study, all A. calidoustus
isolates tested had MICs of >4 μg/ml for the triazoles. This newly described species, although uncommon, appears to be an emerging problem (1
). A recent report by Varga and colleagues (26
) evaluated a large number of clinical and environmental Aspergillus ustus
isolates, using phylogenetic analysis, and subsequently described A. calidoustus
as a new species. All 27 A. calidoustus
isolates were triazole resistant, with MICs of ≥8 μg/ml (26
). Our findings reaffirm the elevated triazole MICs seen in A. calidoustus
isolates. Of the five patients with A. calidoustus
, only one (20%) died at the 12-week outcome endpoint, suggesting a potentially lower pathogenicity of this organism. Additional isolates and outcome data are required to confirm this.
Our study also confirmed earlier observations of the resistance of A. terreus
to AMB (24
). A. terreus
is reported in a wide range of immunocompromised patients. Correlations between in vitro and in vivo resistance and diminished therapeutic response have been reported for A. terreus
infections with elevated MICs to AMB (10
). That our study did not demonstrate this correlation may be related to the relatively small number (n
= 13) of patients with A. terreus
who received AMB and had outcome data available.
Our study evaluated the relationship between in vitro MICs and all-cause mortality at 6 and 12 weeks and did not confirm that “resistant” isolates were significantly associated with increased mortality, using several univariate analytic techniques. In addition, with use of an epidemiologic cutoff for VOR of ≤1 μg/ml among A. fumigatus
isolates, no significant association was present (22
). However, we were not able to confirm non-wild-type organisms with sequence analysis, and only 3 of 74 patients with A. fumigatus
who received voriconazole therapy had isolates with MICs of >1 μg/ml.
Multiple factors contribute to patient outcomes, especially when considering all-cause mortality at 6 and 12 weeks, so the true impact of antifungal susceptibility is difficult to determine. We were not able to take into account length of therapy, dosages of antifungal agents, serum concentrations achieved, combination antifungal therapy, attributable mortality, and other factors that may impact outcomes in patients with IA. Most importantly, although this collection represents a large number of isolates, only 10% were either triazole or AMB resistant, resulting in a lack of power to determine differences. On the basis of these data, the authors do not recommend routine susceptibility testing of Aspergillus isolates, especially in areas of low frequency of resistance. However, in areas such as The Netherlands, routine testing may be more appropriate. If an isolate is discovered with an elevated MIC in a patient with aspergillosis, a thorough evaluation is needed to assess clinical response. Because of the myriad of factors that can impact patient outcomes, careful attention to antifungal therapy, drug levels, and patient factors is necessary, and a change in the antifungal regimen should be considered.
In summary, IA remains an important problem in transplant patients. Among transplant patients enrolled in this study, resistance to azoles was uncommon. Continued surveillance of Aspergillus susceptibility patterns and impact on patient outcomes is warranted.