Effect of media composition and in vitro activity of posaconazole, caspofungin and voriconazole against zygomycetes

Gil-Lamaignere, Cristina; Hess, Regina; Salvenmoser, Stefanie; Heyn, Kathrin; Kappe, Reinhard; Müller, F.

doi:10.1093/jac/dki140

Cited by 24 publications

(22 citation statements)

References 12 publications

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“…After the microtitration trays were defrosted, they were inoculated with a final concentration of 2.5 ϫ 10 4 sporangiospores/ml from each of the 14 isolates and incubated at 37°C. For each isolate, four different 96-well trays were used, corresponding to four different incubation periods: 6,8,12, and 24 h. For each time point, an additional tray was prepared, whose wells contained the above-mentioned drug concentrations at a final volume of 200 l of RPMI, but no fungal inoculum. These trays served to provide background OD measurements for the XTT and spectrophotometric readings.…”

Section: Methodsmentioning

confidence: 99%

“…At 6,8, and 12 h after inoculation, the corresponding trays were removed from the incubator, and 50 l of saline containing 0.5 mg/ml XTT with 125 M menadione was added to each well to obtain final concentrations of 100 g/ml XTT and 25 M menadione. The trays were further incubated for 2 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…For four of the zygomycete isolates (one each of R. oryzae, C. bertholletiae, M. circinelloides, and A. corymbifera), for the purpose of modeling, the relative OD values obtained with the XTT assay (6,8, and 12 h) or spectrophotometric reading at 405 nm (24 h) were analyzed by nonlinear regression analysis, using a four-parameter logistic model (a sigmoid curve with variable slope) known as the E max model. Deviation from the model was tested by the runs test, and goodness of fit was checked by the r 2 value.…”

Section: Methodsmentioning

confidence: 99%

“…Comparison of best-fit values for these parameters was performed with one-way analysis of variance, followed by Dunn's test for multiple comparisons. The results of comparisons of the EC 50 and bottom-of-the-curve values were used to employ appropriate cutoff levels for determination of MIC-2 and MIC-0, respectively, with the early XTT method (6,8, and 12 h). Statistical analysis was performed using the GraphPad (San Diego, Calif.) Prism software.…”

Section: Methodsmentioning

confidence: 99%

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Rapid Susceptibility Testing of Medically Important Zygomycetes by XTT Assay

et al. 2006

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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 Infections caused by Zygomycetes have emerged as an increasingly important cause of morbidity and mortality among severely immunocompromised patients, including those with hematological malignancies and hematopoietic stem cell transplant recipients. The prognosis of zygomycosis in these patients remains extremely poor, despite antifungal treatment (10,16,18,19). For years, amphotericin B has been the drug of choice for these devastating infections. Of the recently introduced second-generation triazoles, voriconazole is not active against the Zygomycetes, while posaconazole has demonstrated activity in vitro, in animal models, and in case reports (4-6, 8, 20-22). As zygomycosis may be rapidly fatal, timely determination of the susceptibilities of clinical isolates to these antifungal agents is important.[A colorimetric method, based on reduction of the tetrazolium salt 2,3-bis{2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT) by mitochondrial dehydrogenases, has been recently introduced for in vitro susceptibility testing of filamentous fungi. This method quantifies fungal growth by measuring fungal metabolism, using appropriate concentrations of XTT and an electron transfer agent, such as menadione (1,11,12). For Aspergillus species, MICs determined by the XTT method at 24 and 48 h were comparable with those obtained by the National Committee for Clinical Laboratory Standards (now known as the Clinical and Laboratory Standards Institute [CLSI]) method (12). The sensitivity of the XTT assay may be increased depending on the menadione concentration (11). High menadione concentrations could increase the rate of XTT conversion and theoretically allow the detection of metabolic activity associated with small, early fungal growth that is not yet detected visually or spectrophotometrically. In this case, it is likely that the XTT method might even be useful for early determination of MICs of antifungal agents. This hypothesis has been previously discussed (12) but has not been investigated.In preliminary studies using the XTT assay for zygomycete isolates, high metabolic activity was observed even in the presence of small fungal growth (assessed spectrophotometrically) at 12 or 24 h postinoculation and preceded increases in biomass (C. Antachopoulos, J. Meletiadis, E. Roilides, T. Sein, and T. J. Walsh, Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother., abstr. M-1003, 2005). The present study investigated whether significant metabolic activity with the XTT assay could be demonstrated for the Zygomycetes before hyphal growth is macroscopically or spectrophotometrically detected (at 6 or 8 h postinoculation) and whether changes in this early metabolic activity in the presence of antifungal agents could be used f...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Rapid Susceptibility Testing of Medically Important Zygomycetes by XTT Assay

et al. 2006

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“…Compared with itraconazole and isavuconazole on a mg:mg basis, posaconazole has enhanced in vitro activity with reported 90% minimum inhibitory concentrations (MIC90) ranging from 1 to ≥ 4 mg/mL. 20,21,57,58,65,66 In the largest and most diverse collection of clinical isolates published so far, that included 217 clinical isolates of 11 species, 64-100% of the isolates were reported to be susceptible using an arbitrary breakpoint of 0.5 mg/mL or below. Comparatively higher MIC values were found for Mucor circinelloides 20 (Table 5).…”

Section: Posaconazolementioning

confidence: 99%