Rapid susceptibility testing of medically important zygomycetes by XTT assay

Abstract

The XTT colorimetric assay quantifies fungal growth by measuring fungal metabolism and has been used successfully for susceptibility testing of Aspergillus species after 24 and 48 h of incubation. In the present study using 14 clinical isolates of Zygomycetes (Rhizopus oryzae [5 isolates], Cunninghamella spp. [3 isolates], Mucor spp. [3 isolates], and Absidia corymbifera [3 isolates]), significant metabolic activity was demonstrated before visual or spectrophotometric detection of fungal growth by performing the XTT assay as early as 6 h after inoculation. Testing of susceptibility to amphotericin B, posaconazole, and voriconazole was subsequently performed using the XTT method (100 microg/ml XTT, 25 microM menadione) at 6, 8, or 12 h after inoculation and the CLSI (formerly NCCLS) M38-A method with visual and spectrophotometric MIC determinations at 24 h after inoculation. Concentration-effect curves obtained with the use of the E(max) model (a sigmoid curve with variable slope) were comparable between the early XTT and spectrophotometric readings at 24 h. Complete inhibition of early metabolic activity with the azoles was delayed in comparison to that with amphotericin B. Using appropriate cutoff levels, agreement was demonstrated between the early XTT and 24-h spectrophotometric or visual readings. In particular, for MIC-0 (the lowest drug concentration showing absence of visual growth) of amphotericin B, overall agreement levels were 90 to 93% for the 6-h XTT assay and 100% for the 8- and 12-h time points. For MIC-0 of posaconazole, agreement levels were 86% for the 6-h XTT and 93 to 100% for the 8- and 12-h time points. The overall agreement levels for MIC-0 and MIC-2 (the lowest drug concentration showing prominent reduction of growth compared with the control well) of voriconazole (compared with 24-h spectrophotometric readings) were 93 to 98% for the 8- and 12-h XTT assays. These results support the use of the XTT method for rapid MIC determination for Zygomycetes.

FIG. 1.

Metabolic activity of Zygomycetes assessed by the XTT method at 6 h postinoculation, using different concentrations of XTT (200, 100, and 50 μg/ml) and menadione (MEN) (25, 6.25, and 1.56 μM). The error bars indicate standard errors of the means.

FIG. 2.

Photomicrographs (×82) of adjacent wells with serially decreasing concentrations of voriconazole at completion of the 6-h XTT assay (total, 8 h after inoculation). Gradual increases in XTT conversion rates are manifested by color changes from yellow to orange and coincide with germination of M. circinelloides conidia and progression to the formation of hyphae. MIC-0s were 32, 16, and 16 μg/ml according to the 24-h visual and spectrophotometric and 6-h XTT readings, respectively. GC, growth control. Voriconazole was used for this example because it provided more gradual concentration-effect curves than those of other agents (see Results), thus allowing a better demonstration of the different stages of transition from nongerminated conidia to elongated hyphae.

FIG. 3.

Concentration-effect sigmoid curves based on 6-h XTT and 24-h spectrophotometric (SPEC) readings for R. oryzae. For posaconazole (A) and voriconazole (B), the bottom values of the curve for the 6-h XTT measurements were significantly higher (relative OD₄₅₀, ∼0.4 and ∼0.2, respectively) than those of 24-h spectrophotometric readings (relative OD₄₀₅, ∼0). For amphotericin B (C), the bottom values of both curves were similar (relative ODs, ∼0). The error bars indicate standard errors of the means.