In Vitro Activity of Amphotericin B Against Aspergillus terreus Isolates from Different Countries and Regions

Invasive aspergillosis is the most common infection caused by filamentous fungi which is observed in immunocompromised patients. Amphotericin B deoxycholate was considered the “gold standard” for therapy of invasive aspergillosis for more than three decades. However, the recently published American and European guidelines recommend intravenous or oral voriconazole as primary treatment and lipid-based amphotericin B as an alternative 1, 2. Aspergillus fumigatus is the Aspergillus species most commonly associated with invasive aspergillosis, A. flavus is second most common, and A. terreus accounts for a minority of cases. However, an increasing frequency of this last species has been reported in some institutions, where it was isolated from up to 12.5% of the cases 3,4. In addition, the A. terreus resistance to amphotericin B observed in vitro 5,6 and in vivo 7,8 represents a serious concern. The aim of the present study was to investigate the in vitro susceptibility to amphotericin B of A. terreus patient isolates from the USA and Europe. A total of 145 A. terreus clinical isolates, mainly from respiratory tract and nails, were analysed. The isolates were identified on the basis of morphologic characters. Ninety-seven were collected in Europe, that is 61 from Italy (Igiene Università Milano, IUM collection) and 37 from Belgium and France (IHEM collection at Scientific Institute of Public Health, Bruxelles, Belgium). A total of 47 were collected in USA, and among these 24 were from the West Coast and 23 from the northeastern section of the country. The in vitro susceptibility to amphotericin B was investigated by the broth microdilution method, according to the Clinical Laboratory Standards Institute (formerly the National Committee Clinical Laboratory Standards) reference method for filamentous fungi 9. Tests were performed in the Department of Public Health-Microbiology-Virology, University of Milan, Italy. The A. terreus conidia were harvested after the isolates were subcultured on potato dextrose agar at 35°C for 3-5 days and suspended in normal saline containing 0.025% Tween 20. The conidial suspensions were counted with a hemocytometer and the inocula adjusted to a final concentration of 4 x 103 to 5 x 104 conidia/ml. Amphotericin B (Sigma, Milano, Italy) was dissolved in dimethyl sulfoxide and then diluted twofold to final concentrations ranging from 0.03 to 16 mg/L 9. The dilutions in the microtiter plates were prepared and used for testing within 24 hours. Testing was performed in RPMI 1640 without sodium bicarbonate (Sigma), buffered to pH 7.0 with 0.165 M MOPS (Sigma) supplemented with 0.03% Lglutamine (Sigma). Reference strains Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as quality control isolates for susceptibility testing. Petromyces alliaceus IUM 81-713 was included in each testing session as an amphotericin B-resistant control. Tests were performed in duplicate. Isolates showing different results in the two runs were tested again and the MIC most frequently obtained was utilized. The minimal inhibitory concentration (MIC, mg/L) endpoint was the lowest drug concentration showing no visible growth after 48 hours of incubation at 35°C. Bartlett’s test was used to test the homogeneity of variances across geographic groups and Kruskal-Wallis test to compare the groups showing statistically significant differences. Table 1 shows the MIC profiles of the isolates by geographic origin. Amphotericin B at a concentration ≤1 mg/L was able to inhibit the growth of 97% (59 out of 61) of the isolates from Italy, and 84% of the isolates from Belgium and France. The American isolates, 96% of which from the West Coast but only 61% from the Northeast, were inhibited by this amphotericin B concentration. In this last group four isolates were clearly resistant, being inhibited only by a concentration of 4 and 16 mg/L. These results show a different pattern of susceptibility to amphotericin B of A. terreus isolates from difREPRINT