The in vitro susceptibility of 45 Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus isolates against caspofungin (CAS) was assessed by the CLSI reference method with ...spectrophotometric reading and by a colorimetric method that employed the dye MTT. Perfect agreement was found between the minimal effective concentrations (MECs) and the MIC-2 values (50% growth reduction) obtained by the MTT method. The agreement found between the MICs obtained by the CLSI and the MTT method was dependent on the MIC endpoint used, the antifungal agent tested, and the species investigated.
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 Esubscript 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.
Objectives: The in vitro effects of caspofungin combined with voriconazole and amphotericin B were tested in triplicate experiments against nine clinical isolates of Aspergillus fumigatus, ...Aspergillus flavus and Aspergillus terreus. Methods: The isolates were tested against a range of concentrations of voriconazole (0.015–1.0 mg/L), caspofungin (0.125–256 mg/L) and five concentrations of amphotericin B (0.1–0.5 mg/L) with a microdilution chequerboard method based on the CLSI M38-A reference method and the results were analysed with the fractional inhibitory concentration (FIC) index. The effect of individual drugs on the FIC index of each of the double combinations was also evaluated. Results: The triple combination of voriconazole, caspofungin and amphotericin B against all Aspergillus spp. was synergistic (FIC index 0.49–0.57) at low median concentrations of amphotericin B (0.10–0.22 mg/L) and voriconazole (0.07–0.15 mg/L) over a wide range of caspofungin concentrations (4.32–17.28 mg/L). Antagonistic interactions (FIC index 1.65–2.15) were found at higher median concentrations of amphotericin B (0.3–0.5 mg/L) and voriconazole (0.23–0.68 mg/L) over a similarly wide range of caspofungin concentrations (1.47–32 mg/L). Conclusions: These concentration-dependent interactions may have important clinical implications, which require further evaluation in animal models of invasive aspergillosis.
Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of ...deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from Candida albicans in vitro and in vivo. We developed a C. albicans-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from C. albicans alone, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis. The analytical qPCR assay was highly specific and sensitive (10 fg). Cells of C. albicans incubated in Hanks balanced salt solution (±10% bovine serum albumin BSA) or RPMI (±10% BSA) showed a significant release of DNA at T equal to 24 h compared to T equal to 0 h (P </= 0.01). C. albicans incubated with H-MNCs exhibited a greater release of DNA than C. albicans cells alone over 24 h (P = 0.0001). Rabbits with disseminated candidiasis showed a steady increase of detectable DNA levels in plasma as disease progressed. Plasma cultures showed minimal growth of C. albicans, demonstrating that DNA extracted from plasma reflected fungal cell-free DNA. In summary, these studies of the kinetics of DNA release by C. albicans collectively demonstrate that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, that phagocytic cells may play an active role in increasing this release over time, and that plasma is a suitable blood fraction for the detection of C. albicans DNA.
Summary
The antifungal activities of miconazole, terbinafine, itraconazole, UR 9825, voriconazole and amphotericin B against 11 clinical isolates of Exophiala spp. were tested by the broth ...microdilution method. All drugs were very active against Exophiala spp.. The 90% minimal inhibitory concentration (MIC90) ranged from 0.125 to 1 μg ml−1. Terbinafine was the most active drug against Exophiala spinifera, Exophiala dermatitidis and Exophiala castellanii and seems to be a promising agent in the treatment of infections caused by these fungi.
An assay system in which polymerase chain reaction (PCR) amplification of the ITS-1 region of ribosomal DNA (rDNA) is combined with a reverse-hybridization line probe assay (LiPA) was used for the ...identification of six Candidaspecies and four Aspergillusspecies in pure cultures of clinical isolates, as well as in bronchoalveolar lavage (BAL) fluid samples from 42 patients with various underlying diseases. The results were compared with the results obtained with conventional routine identification methods as well as with a commercial enzyme-linked immunosorbent assay (ELISA) galactomannan detection assay and an Aspergillus-specific PCR. No discrepancies between the PCR-LiPA system and routine methods were found for pure cultures of Candidaand Aspergillusspecies except in the case of Aspergillus versicolor. In BAL fluid samples in which Candidaspecies were cultured, the PCR-LiPA system identified more species than did the routine methods. When routine analyses of patient samples were supplemented by adding data obtained by repurifying and re-identifying cultures and by taking isolates obtained from other body sites into account, the results agreed with PCR-LiPA system results in 81% of the cases (34 42). Most of the remaining discrepancies (6 8) involved cases in which such supplementary data were not available. In BAL fluid samples from which A. fumigatus was cultured, the agreement between the PCR-LiPA system and the routine methods was low. Only 2 of 11 BAL samples shown to contain A. fumigatusin ELISA and genus-specific PCR assays were positive in PCR-LiPA system. The PCR-LiPA system enables the simultaneous detection and identification of different fungal species present in pure or mixed populations within 6 h in a single assay. Optimization is required, however, before it is useful as a diagnostic tool in the clinical microbiology laboratory.
Objectives To develop and evaluate a new method for rapid susceptibility testing of Aspergillus spp. based on early metabolic signalling of high-inoculum biomass. Methods Susceptibility to ...amphotericin B and voriconazole was studied in 39 clinical isolates of Aspergillus spp. (16 Aspergillus fumigatus, 11 Aspergillus flavus, 12 Aspergillus terreus). At 6 or 8 h after inoculation for A. fumigatus and A. flavus, and at 8 or 12 h after inoculation for A. terreus, 100 µg/mL of the tetrazolium salt XTT and 25 µM menadione were added and absorbance measured at 450 nm after 2 h of incubation at 37°C. Inocula used were 106 conidia/mL for A. fumigatus and A. terreus and 105 conidia/mL for A. flavus, as lower inocula exhibited very low metabolic activity at these time points. Data were analysed with the sigmoid Emax model and compared with visual (lowest drug concentration showing no growth) and spectrophotometric MIC determination at 48 h (CLSI M38-A method). Results The Emax model described well the concentration–effect relationship for early metabolic activity and 48 h fungal biomass (median r2: 0.97 and 0.93, respectively). Use of the model allowed characterization and quantification of species- and drug-related differences in pharmacological inhibition of early metabolic activity as well as calculation of appropriate cutoff levels for MIC determination with the XTT assay. Using these cutoff levels, for A. fumigatus and A. flavus at both time points (6 and 8 h) and for A. terreus at 12 h, the agreement (± one dilution) of the XTT assay with the CLSI method was 91–100% and its reproducibility was 97–100%. Conclusions This newly developed high-inoculum-based method provides rapid and reproducible MIC determinations for Aspergillus spp.
Non-parametric and parametric approaches of two competing zero-interaction theories - the Loewe additivity and the Bliss independence - were evaluated for analyzing the in vitro interactions of ...various antifungal drugs. Fifty-one data sets, derived from three drug combinations, tested in triplicate against 17 clinical yeast and mold isolates with a two-dimensional checkerboard microdilution technique, were selected to span from strong synergy to strong antagonism. These were analyzed with the standard FIC index model and modern concentration-effect response surface models: the fully parametric model developed by Greco et al. and the 3-D analysis developed by Prichard et al. The FIC index model is subjective, sensitive to experimental errors and resulted in approximated results and variable conclusions depending on the MIC endpoints determined and interpretation endpoints used. By using the MIC-2 endpoint (lowest drug concentration showing 50% of growth) for calculating the FIC indices, problems due to trailing phenomena were reduced and weak interactions could be detected; higher levels of reproducibility and agreement with the other models were achieved using the MIC-0 and MIC-1 (lowest drug concentration showing 10 and 25% of growth, respectively). High reproducibility was achieved in interpreting the FIC indices when the cutoffs of 0.25 and 4 (for single experiments) and the cutoff of 1 (for replicates) were used for defining the limits of additivity/indifference. Although the fully parametric Greco model did not describe precisely the entire response surface of all antifungal drug interactions, it was able to differentiate synergistic from non-synergistic interactions with a non-unit, reproducible, concentration-independent interaction parameter, including its uncertainty, without requiring replication. The Bliss independence based models resulted in mosaics of synergistic and antagonistic combinations, raising questions about the concentration-dependent nature of antifungal drug interaction. The sum of all statistically significant interactions were used as a summary interaction parameter for the entire response surface, concluding synergy or antagonism when it was positive or negative, respectively. The cutoffs of 100% and 200% were used to distinguish weak and moderate interactions, respectively in 12-16×8-12 checkerboard formats. Semi-parametric approaches need particular care as experimental errors are not eliminated from the entire response surface.
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