Analysis of the Influence of Tween Concentration, Inoculum Size, Assay Medium, and Reading Time on Susceptibility Testing of Aspergillus spp.

Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo, Km 2. 28220 Majadahonda, Madrid, Spain.
Journal of Clinical Microbiology (Impact Factor: 3.99). 04/2005; 43(3):1251-5. DOI: 10.1128/JCM.43.3.1251-1255.2005
Source: PubMed


The influence of several test variables on susceptibility testing of Aspergillus spp. was assessed. A collection of 28 clinical isolates was tested against amphotericin B, itraconazole, voriconazole, and
terbinafine. Inoculum size (104 CFU/ml versus 105 CFU/ml) and glucose supplementation (0.2% versus 2%) did not have significant effects on antifungal susceptibility testing
results and higher inoculum size and glucose concentration did not falsely elevate MICs. In addition, antifungal susceptibility
testing procedure with an inoculum size of 105 CFU/ml distinctly differentiated amphotericin B or itraconazole-resistant Aspergillus strains in vivo from the susceptible ones. Time of incubation significantly affected the final values of MICs, showing major
increases (two to six twofold dilutions, P < 0.01 by analysis of variance) between MIC readings at 24 and 48 h, but no differences were observed between antifungal
susceptibility testing results obtained at 48 h and at 72 h. Significantly higher MICs were uniformly associated with higher
concentrations of Tween (P < 0.01), used as a dispersing agent in the preparation of inoculum suspensions. The geometric mean MICs showed increases
of between 1.5- and 10-fold when the Tween concentration varied from 0.1% (the geometric means for amphotericin B, itraconazole,
voriconazole, and terbinafine were 1.29, 0.69, 1.06, and 0.64 μg/ml, respectively) to 5% (the geometric means for amphotericin
B, itraconazole, voriconazole, and terbinafine were 1.97, 5.79, 1.60, and 4.66 μg/ml, respectively). The inhibitory effect
of Tween was clearly increased with inoculum sizes of 105 CFU/ml and was particularly dramatic for itraconazole, terbinafine, and Aspergillus terreus. The inoculum effect was not observed when the Tween concentration was below 0.5% (P > 0.01).

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