JAC Advance Access originally published online on December 21, 2006
Journal of Antimicrobial Chemotherapy 2007 59(2):230-237; doi:10.1093/jac/dkl488
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Use of high inoculum for early metabolic signalling and rapid susceptibility testing of Aspergillus species
1 Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA 2 Third Department of Pediatrics, Aristotle University, Hippokration Hospital, Thessaloniki, Greece
* Corresponding author. Tel: +1-301-402-0023; Fax: +1-301-480-2308; E-mail: walsht{at}mail.nih.gov
Received 14 July 2006; returned 29 September 2006; revised 3 November 2006; accepted 6 November 2006
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Abstract |
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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.
Keywords: XTT , metabolic activity , Aspergillus spp
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Introduction |
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Invasive aspergillosis constitutes a major cause of morbidity and mortality among immunocompromised patients.1,2 Although Aspergillus fumigatus is most commonly isolated from clinical specimens, non-fumigatus Aspergillus species are increasingly reported. Some of these species, such as Aspergillus terreus, exhibit reduced susceptibility to amphotericin B.2–5 Voriconazole resistance among Aspergillus spp. is still uncommon; however, the wide use of this newer triazole as primary treatment, prophylaxis, or pre-emptive therapy may be associated with increased resistance among clinical strains in the future. Recently, reduced susceptibility of clinical isolates of Aspergillus spp. to voriconazole has been demonstrated6–10 and correlation with clinical failure has been reported.10 Novel mechanisms that confer resistance of A. fumigatus isolates to all azole agents have been described; these mechanisms include amino acid substitutions at methionine-220, leucine-98 or glycine-138 in the cyp51A gene.7,10,11
Antifungal susceptibility testing of Aspergillus isolates may be an important guide to therapy.12 However, following the M38-A broth microdilution method approved by the Clinical and Laboratory Standards Institute (CLSI), MIC determination for Aspergillus species requires 48 h of incubation.13,14 Given the need for prompt treatment of invasive aspergillosis with appropriate antifungal agents, several methods for rapid susceptibility testing have been developed, including a radiometric assay measuring the inhibition of 14CO2 production,15 a flow cytometry assay of conidial viability,16 a fluorescence-based microplate assay,17 a method measuring glucose utilization,18 and a turbidometric method assessing the lag phases of fungal growth curves in the presence of antifungal agents.19 Some of these methods may require expensive equipment or potentially hazardous substances. The glucose utilization and fluorescence-based microplate assays generated MICs in 16 h for Aspergillus spp.,17,18 while the turbidometric method allowed determination of antifungal drug resistance after 8.8–11.4 h for A. fumigatus and 6.7–8.5 h for Aspergillus flavus.19
Using the XTT assay, we previously demonstrated that increases in metabolic activity precede increases in biomass for Zygomycetes and that, using higher menadione concentrations, significant metabolic activity could be detected even in the absence of hyphal growth macroscopically.20,21 We subsequently showed for Zygomycetes that MIC values in agreement with the CLSI M38-A method could be obtained with the XTT assay as early as 8 h after inoculation, using the inoculum suggested by the CLSI M38-A method.21
However, a new methodological strategy for development of a rapid susceptibility assay is necessary for Aspergillus spp., which do not exhibit the rapid growth and high metabolic activity of Zygomycetes. We therefore considered the development of a high-inoculum-based assay in order to elicit an early XTT signal from a large biomass of Aspergillus spp. As the use of high-inoculum biomass for early metabolic signalling has not been well studied for filamentous fungi, we first initiated a series of experiments to investigate the optimal conditions for detection of early metabolic activity of Aspergillus spp. using this approach. We subsequently applied these conditions for rapid testing of susceptibility to amphotericin B and voriconazole in a large collection of A. fumigatus, A. flavus and A. terreus isolates. Using mathematical modelling we generated appropriate cutoff levels for endpoint determination of MIC values of these species with the XTT methodology and compared these values with those determined with the CLSI method.
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Materials and methods |
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Isolates
A collection of 39 clinical isolates of Aspergillus spp. was used, including 16 isolates of A. fumigatus, 11 of A. flavus and 12 of A. terreus. Conidia were harvested after isolates were subcultured on potato dextrose agar at 35°C for 5–7 days, and were suspended in normal saline containing 0.025% Tween 20. Conidial suspensions were counted with a haematocytometer and diluted into RPMI medium (see below) at the desired concentrations. Inoculum sizes were verified by quantitative colony counts on Sabouraud dextrose agar plates. Reference strains Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were used as quality controls.
The assay was performed in RPMI 1640 medium with L-glutamine but without bicarbonate, buffered to pH 7.0 with 0.165 M 3-N-morpholinopropanesulphonic acid (Cambrex Bio Science Walkersville, Inc. MD, USA).
The tetrazolium salt XTT [2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide] (Sigma-Aldrich, St Louis, MO, USA) was dissolved in normal saline at concentrations of 0.5, 1 or 2 mg/mL. Menadione (Sigma-Aldrich) was initially dissolved in absolute ethanol at a concentration of 10 mg/mL and subsequently added to the above XTT solutions at concentrations of 0.125, 0.5, 1.25 or 2 mM for each solution. In this way a total of 12 solutions with different XTT/menadione concentrations were prepared.
Assessment of early metabolic activity by XTT assay
These preliminary studies investigated the metabolic activity at early phases of growth for Aspergillus species, by performing the XTT assay at different time points after inoculation using various concentrations of XTT/menadione and inocula. Briefly, inocula of 2.5 x 104, 105 or 106 conidia/mL of six Aspergillus isolates (two each of A. fumigatus, A. flavus and A. terreus) were incubated in RPMI 1640 medium at 37°C in flat-bottom 96-well microtitration plates (Costar 3596, Corning Inc., Corning, NY, USA) at a volume of 200 µL/well. After 6, 8, 10 or 12 h of incubation, 50 µL of one of the above XTT/menadione solutions was added to each well, as previously described.21,22 All 12 solutions were used, resulting in final concentrations of 100, 200 or 400 µg/mL XTT, each combined with 25, 100, 250 or 400 µM menadione. The microtitration plates were further incubated at 37°C for 2 h in order to allow conversion of XTT into its formazan derivative and subsequently shaken for 1–2 min (Wallac Plate Shake 1296-004, Wallac OY, Turku, Finland) until complete dissolution of formazan derivatives. Absorbance was then measured at 450 nm with a microtitration plate spectrophotometric reader (Elx808, Bio-Tek Instruments, Winooski VT, USA). For each well, XTT conversion was calculated after subtraction of the background absorbance, which was the absorbance of a simultaneously incubated well with 200 µL of RPMI and 50 µL of the same XTT/menadione solution but no inoculum. Four replicates were used for each species, time point and XTT/menadione concentration.
Antifungal susceptibility testing
Susceptibility to amphotericin B and voriconazole was determined for all 39 Aspergillus isolates using both the rapid XTT and the CLSI M38-A methods.13,14 Clinical preparations of amphotericin B (Bristol-Myers Squibb, Princeton, NJ, USA) and voriconazole (Pfizer Inc., New York, NY, USA) were dissolved in sterile distilled water. Serial dilutions of these agents in RPMI 1640 were initially prepared in 96-well microtitration plates, as suggested by the CLSI M38-A method, in order to yield concentrations of 0.015–16 mg/L for amphotericin B and 0.008–8 mg/L for voriconazole at a final volume of 200 µL after inoculation. Trays were maintained at –70°C until the day of testing.
(i) CLSI method.
After the microtitration trays were thawed, they were inoculated with a final concentration of 2.5 x 104 conidia/mL from each of the 39 isolates and incubated at 37°C. In every tray, a row of wells containing the same drug concentrations as other rows and RPMI 1640 medium up to a total volume of 200 µL, without fungal inoculum, served to provide background measurements for calculation of fungal biomass. After 48 h of incubation fungal growth was assessed visually and spectrophotometrically. For visual assessment, a concave mirror was used and MIC was determined as the lowest drug concentration showing absence of visual growth. For spectrophotometric assessment, fungal biomass was measured as optical density (OD) at 405 nm, as previously described.21,22 The percentage fungal biomass of each well in relation to the drug-free control was calculated after subtraction of corresponding background ODs as:
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10% was determined as MIC. (ii) Rapid XTT assay. The conditions of rapid XTT assay (inocula, time points, concentrations of XTT and menadione) employed for rapid MIC determination of Aspergillus spp. were based on the results of the preliminary studies of early metabolic activity (see the Results section). In particular, for A. fumigatus the final inoculum used was 106 conidia/mL and the XTT assay was performed at 6 and 8 h after inoculation. For A. flavus, the inoculum used was 105 conidia/mL and the XTT assay was also performed at 6 and 8 h after inoculation. Finally, for A. terreus 106 conidia/mL were used and the XTT assay was performed at 8 and 12 h after inoculation. The final concentrations of XTT and menadione used were 100 µg/mL and 25 µM, respectively.
The microtitration trays were inoculated with the pre-specified inoculum and incubated at 37°C for 6, 8 or 12 h, as described above for different Aspergillus species. At the above time points, 50 µL of saline containing 0.5 mg/mL XTT with 0.125 mM menadione were added to each well in order to obtain final concentrations of 100 µg/mL XTT and 25 µM menadione, and the trays were further incubated for 2 h at 37°C. After shaking of the trays, absorbance (A) was measured at 450 nm and the percentage metabolic activity for each well and drug concentration in relation to the drug-free control was calculated as:
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For all 39 Aspergillus isolates, the % metabolic activity (6, 8 or 12 h XTT assay) and % biomass (48 h) were analysed by non-linear regression analysis, using a four parameter logistic model (sigmoid curve with variable slope) known as the Emax model and described by the equation: y = bottom + (top – bottom)/ (1 + 10(LogEC50–x) x slope), where x is the log10 of drug concentration, y is the corresponding % metabolic activity or biomass, and the four parameters are the top and bottom plateaus of the sigmoid curve, the EC50, which is the drug concentration producing 50% of the Emax (maximum metabolic activity or biomass), and the slope, which describes the steepness of the curve. Deviation from the model was tested by the runs test and goodness of fit was checked by the r2 value. In order to compare the relative position of the concentration–effect curves of early metabolic activity versus those of 48 h biomass on the x-axis, for each isolate the ratio: EC50 of 48 h biomass measurements/EC50 of metabolic activity readings with 6, 8 or 12 h XTT assay was calculated and expressed as log2; in this way any ‘shifting’ of the curves of metabolic activity compared with those of fungal biomass was expressed as number of 2-fold drug dilutions.
In order to estimate the early metabolic activity corresponding to the CLSI MIC, using the above equation we calculated the EC10 for every concentration–effect curve corresponding to spectrophotometric measurements of fungal biomass at 48 h, which was the drug concentration producing 10% of the maximum biomass, as follows: EC10 = 10Log[a/(1 – a)]/slope + Log EC50, where a = 0.10. Subsequently, using the best-fit values obtained for the four parameters of the model from the concentration–effect curves of early metabolic activity, we calculated the metabolic activity that the above drug concentration (EC10) demonstrated at early time points (6, 8 or 12 h) directly from the Emax model equation, by replacing x with the log10 of this drug concentration (EC10).
As no cutoff levels have been defined for MIC determination for Aspergillus spp. based on early metabolic activity, for a given species, drug and time point, a range of values of % metabolic activity were evaluated as cutoff levels for MIC determination with the rapid XTT method. This range included values that fulfilled the following conditions: (i) exceeding the upper range of bottom values of the concentration–effect curves of early metabolic activity; (ii) approximating the median value of % metabolic activity (at the given time point) of the above defined as EC10 drug concentration (provided that this was equal to, or higher than, the upper range of bottom values). Condition (i) was considered a prerequisite, as significant metabolic activity at early time points was often detected at drug concentrations equal to or greater than the CLSI-determined MIC (see the Results section); the bottom values of the XTT curves in fact represent this activity. Consequently, the cutoff levels were set higher than the detected metabolic activity at inhibitory drug concentrations, taking into account the inter-strain and experimental variation. Cutoff values fulfilling conditions (i) and (ii) were subsequently evaluated on the raw data and the cutoff giving the best agreement (see below) between the two methods was reported. Model building was performed using GraphPad Prism Software (4.0b, San Diego, CA, USA).
Agreement between CLSI and rapid XTT methods
The MIC values for all 39 Aspergillus isolates obtained with the early XTT assays using the suggested cutoff levels were compared with corresponding values from visual and spectrophotometric (405 nm) readings at 48 h. The percentage of relative (± one dilution) agreement between the MICs obtained with different methods and time points for all isolates of each species and for each antifungal agent was reported.
Reproducibility of the rapid XTT method
The median MIC of those obtained with the three experiments using the rapid XTT method was calculated for each isolate, time point and antifungal agent. The reproducibility was subsequently defined as the percentage of MICs (obtained for each species, time point and antifungal agent) that were within one 2-fold dilution from the median MIC.21,22
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Assessment of early metabolic activity by XTT assay
The use of 2.5 x 104 conidia/mL and 100 µg/mL XTT with 25 µM menadione resulted in very low XTT conversion rates (A450 < 0.1) when the XTT assay was performed at 6, 8 or 10 h after inoculation for A. fumigatus, at 6 h for A. flavus and at 6, 8, 10 or 12 h after inoculation for A. terreus. Increasing the XTT concentration to 200 or 400 µg/mL had no significant influence on the obtained metabolic activity; whereas inhibitory effects were observed with menadione concentrations 
100 µM (Figure 1). This inhibitory effect of high menadione concentrations on XTT conversion was observed to be independent of the XTT concentration that was used. Notable inter-species differences in early metabolic activity were also observed, with higher XTT conversion rates detected for A. flavus and lower for A. terreus (Figure 1).
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When 105 conidia/mL were studied, robust metabolic activity (A450 > 0.1) was detected with the XTT assay performed as early as 6 h after inoculation for A. flavus but not for A. fumigatus or A. terreus. For these two species, 106 conidia/mL were required to obtain similar conversion rates (A450 > 0.1) with the 6 h XTT assay. The absence of significant effect of XTT concentration on XTT conversion at this early time point and the inhibitory effect of higher menadione concentrations also were observed using 105 or 106 conidia/mL. For A. flavus and A. fumigatus, examination of microtitration plates under the inverted microscope at the end of the 2 h incubation period following addition of the XTT/menadione solution (i.e. 8 h, in total, after inoculation) demonstrated that XTT conversion, manifested by colour change, increased with progression from germination to formation of hyphae. For the slowly germinating A. terreus isolates, increased metabolic activity was observed even in the presence of swollen conidia, not yet germinated.
Antifungal susceptibility testing
For all 39 isolates the viable inoculum, based on cfu counts, ranged between 0.20 and 2.04 (median: 0.73) times the target inoculum. Using 106 conidia/mL for A. fumigatus and 105 conidia/mL for A. flavus allowed rapid determination of MICs with the XTT assay performed as early as 6 h for all isolates of these two species; for A. fumigatus the median absorbance with the 6 h XTT assay was 0.74 (range: 0.33–1.11), while for A. flavus the median absorbance was 0.40 (range: 0.21–0.74). For A. terreus, however, despite the use of 106 conidia/mL, very low XTT conversion (A450 < 0.1) was observed for 7 of 12 isolates with the 6 h XTT assay, and for 4 of 12 isolates with the 8 h XTT assay. Consequently, for A. terreus, the rapid MIC determination with XTT methodology was evaluated using the additional 12 h time point, at which significant metabolic activity was detected for all isolates. Data analysis and comparison with the CLSI method for A. terreus was performed only for the 8 and 12 h XTT assays, including 8 and 12 isolates, respectively.
For all isolates tested, the Emax model described well the concentration–effect relationship of early metabolic activity and fungal biomass, with r2 ranging between 0.89 and 0.99 (median: 0.97) for the XTT measurements (6, 8, 12 h) and between 0.82 and 0.99 (median: 0.93) for the 48 h spectrophotometric readings. No statistical deviations from the model were found with the runs test. However, some differences were observed between the early metabolic activity and 48 h biomass concentration–effect curves regarding the bottom, slope and EC50 values, as depicted in Figure 2 and Table 1. These differences appeared to be species- and drug-related, while there were also trends observed among different time points. For A. fumigatus (both drugs) and A. terreus (amphotericin B) the concentration–effect curves of early metabolic activity were shifted to the left compared with those of 48 h biomass (Figure 2). This was also manifested by higher ratios of the corresponding EC50 values, which exceeded one 2-fold drug dilution (Table 1).
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The bottom values of early XTT curves for voriconazole (all species) and amphotericin B (A. flavus, 6 h assay) were higher than those of 48 h biomass (Figure 2). The median (range) of these bottom values as presented in Table 1 suggests that for voriconazole (all species) and for amphotericin B [in the case of A. flavus (6 h XTT) and A. terreus (8 h XTT)] significant metabolic activity (up to 25% of control for amphotericin B and up to 45% for voriconazole) could be detected at the early time points, even in the presence of drug concentrations
MIC. For each species, drug and time point, the range of values of early metabolic activity that were evaluated as cutoff levels for MIC determination included values fulfilling conditions (i) and (ii) described in the Materials and methods section. For all species and drugs, the bottom values and the metabolic activity corresponding to EC10 tended to decrease at later time points (i.e. 8 versus 6 h, 12 versus 8 h, Table 1), as did the corresponding cutoff levels (Table 2).
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For A. fumigatus (both drugs) and A. terreus (amphotericin B), in order to adjust for the shifting of curves of early metabolic activity to the left of those of 48 h fungal biomass (see above), a one-dilution higher MIC value than the one determined with the cutoff levels was used for comparing the rapid XTT method with the CLSI method.
Table 2 summarizes the optimal conditions (XTT/menadione concentrations, inocula, time points, cutoff levels) giving the best agreement with CLSI method and the need, or not, for adjustment of obtained MICs, for rapid susceptibility testing of A. fumigatus, A. flavus and A. terreus to amphotericin B and voriconazole with the XTT assay.
Agreement between CLSI and rapid XTT methods
Using the conditions, cutoff values and adjustment summarized in Table 2, MIC values obtained for Aspergillus spp. with the rapid XTT assay were comparable to those determined with the CLSI method. Differences in median MIC values obtained with the two methods did not exceed one dilution (Table 3). The relative agreement between the XTT and CLSI methods ranged from 90 to 100% for all species, agents and time points (Table 4) except the 8 h XTT assay for A. terreus, for which agreement was 81% for MIC of amphotericin B (when compared with visual determination at 48 h) and 80% for MIC of voriconazole (compared with spectrophotometric determination at 48 h). When discrepancies were observed between the rapid XTT and CLSI methods, differences among the MICs did not exceed two dilutions in 84.7% and three dilutions in 95.5% of discordant pairs. These discrepancies were not consistently observed in repeated experiments.
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Reproducibility of the rapid XTT method
The reproducibility of the XTT method ranged between 97 and 100% for all species, drugs and time points with the exception of the 8 h XTT assay for A. terreus, for which reproducibility was 92% (standard error: 6%) and 93% (standard error: 5%) for MIC determination of amphotericin B and voriconazole, respectively.
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Discussion |
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In this study we developed and evaluated a new assay for rapid susceptibility testing of Aspergillus spp., which is based on the use of high-inoculum biomass in order to elicit a robust early metabolic signal from this slowly growing mould. This assay provides reproducible determination of MIC values that are in agreement with the CLSI M38-A method as early as 8 h after inoculation for A. fumigatus and A. flavus. We chose amphotericin B and voriconazole to study this rapid susceptibility method, as these compounds are likely to be administered as primary therapy in seriously ill patients with invasive aspergillosis.1
In previous studies with Zygomycetes, an increase in menadione concentration up to 25 µM was sufficient for eliciting an early metabolic signal with the XTT assay, using inocula suggested by the CLSI M38-A method (0.4 x 104–5 x 104 sporangiospores/mL).20,21 When the same conditions and inocula were studied for Aspergillus spp., however, very low conversion rates were observed at the early time points. Increasing the XTT concentration used in the assay did not significantly affect XTT conversion at these early time points, in agreement with studies in Zygomycetes.20,21 The use of menadione concentrations greater than 25 µM was also evaluated in an attempt to increase early XTT conversion.20,21,23 Interestingly, a decrease in XTT conversion was observed using 250 or 400 µM menadione for all species, and even 100 µM for A. terreus. This inhibition probably represented a toxic effect of high doses of menadione on fungal cells. Such a toxic effect of menadione has been reported for mammalian cell lines and the yeast Saccharomyces cerevisiae, and may be mediated through impairment of oxidative phosphorylation and generation of reactive oxygen intermediates.24–27
The use of high-inoculum biomass for early metabolic signalling has been evaluated previously for rapid susceptibility testing in bacteria,28 but not in filamentous fungi. In our study the use of 105–106 conidia/mL yielded robust metabolic activity at early time points for Aspergillus spp., which allowed subsequent determination of MICs. The sigmoid Emax model described well this metabolic activity in the presence of increasing concentrations of amphotericin B and voriconazole. Notable differences in the obtained concentration–effect curves were, however, observed among species, antifungal agents or even time points, which may represent differential pharmacological effects of the two drugs that become manifested at early phases of fungal growth. For example, the shifting of XTT curves to the left compared with those of 48 h biomass for A. fumigatus (both drugs) and A. terreus (amphotericin B), suggests that concentrations lower than CLSI MIC by 1–2 dilutions still inhibit metabolic activity at early time points for these species and agents. The higher bottom values obtained for voriconazole as compared with amphotericin B for all species probably signifies the inability of inhibitory concentrations of this azole to fully suppress the metabolic activity of Aspergillus spp. at early time points after inoculation, as has been reported both for voriconazole and posaconazole in similar studies with Zygomycetes.21 This ‘lag’ in complete metabolic inhibition could be related to the mechanism of action of azoles, which involves inhibition of ergosterol biosynthesis29 and probably not an immediate cessation of metabolic activity in the fungal cell; in support of this notion are previous reports that azole-induced complete inhibition of ergosterol synthesis requires at least 1 h and complete exchange of ergosterol by its methylated precursors under the influence of azoles occurs after about 6 h of exposure.30,31
The high percentages of relative agreement with the CLSI method allow the use of the XTT method, performed as early as 6 h after inoculation, for rapid susceptibility testing of A. fumigatus and A. flavus. The somewhat higher agreement provided with the 8 h assay should of course be balanced with the longer time to MIC determination. For A. terreus it should be noted that the 8 h MIC determination was not feasible for 4 of 12 isolates, due to insufficient XTT conversion, and also demonstrated lower reproducibility and agreement with the CLSI method as opposed to the 12 h XTT assay.
The high reproducibility of the rapid XTT method in this study could be due, at least in part, to the use of a haematocytometer for inoculum preparation. Notably, the use of higher inocula and the haematocytometer employed in this XTT assay are also proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method for susceptibility testing against Aspergillus that is currently in development.32
It should be emphasized that the various cutoff levels for MIC determination, for different species, drugs or time points, do not really signify that the XTT method is complex, as they only refer to interpretation of the results (i.e. what percentage of metabolic activity compared with drug-free control corresponds to the MIC), and could be easily summarized and reviewed in the clinical laboratory in a table similar to Table 2. The XTT assay itself remains simple and easy to perform in most clinical laboratories and most emphasis, in the case of Aspergillus, should in fact be given to the use of appropriate inoculum size.
In summary, the use of higher inocula allows rapid susceptibility testing of A. fumigatus, A. flavus and A. terreus with the XTT methodology. Understanding and quantifying inter-species differences in drug-induced changes of early metabolic activity provides appropriate cutoff levels for MIC determination with the XTT assay, yielding reproducible results that are in agreement with the CLSI method. A comparison of this rapid susceptibility assay with the EUCAST method should also be considered. Further validation of the suggested cutoff values, the sustainability of high reproducibility, and agreement levels is warranted through a multicentre study.
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None to declare.
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Acknowledgements |
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This work was supported by the Intramural Research Program of the National Cancer Institute.
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References |
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1 Hope WW and Denning DW. (2004) Invasive aspergillosis: current and future challenges in diagnosis and therapy. Clin Microbiol Infect 10:2–4.[CrossRef][ISI][Medline]
2 Marr KA, Carter RA, Crippa F, et al. (2002) Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin Infect Dis 34:909–17.[CrossRef][ISI][Medline]
3
Lewis RE, Wiederhold NP, Lionakis MS, et al. (2005) Frequency and species distribution of gliotoxin-producing Aspergillus isolates recovered from patients at a tertiary-care cancer center. J Clin Microbiol 43:6120–2.
4 Trullas JC, Cervera C, Benito N, et al. (2005) Invasive pulmonary aspergillosis in solid organ and bone marrow transplant recipients. Transplant Proc 37:4091–3.[CrossRef][ISI][Medline]
5
Pfaller MA and Diekema DJ. (2004) Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus.. J Clin Microbiol 42:4419–31.
6
Warris A, Weemaes CM, Verweij PE. (2002) Multidrug resistance in Aspergillus fumigatus. N Engl J Med 347:2173–4.
7
Mellado E, Garcia-Effron G, Alcazar-Fuoli L, et al. (2004) Substitutions at methionine 220 in the 14
-sterol demethylase (Cyp51A) of Aspergillus fumigatus are responsible for resistance in vitro to azole antifungal drugs. Antimicrob Agents Chemother 48:2747–50.
8
Balajee SA, Weaver M, Imhof A, et al. (2004) Aspergillus fumigatus variant with decreased susceptibility to multiple antifungals. Antimicrob Agents Chemother 48:1197–203.
9
Balajee SA, Gribskov JL, Hanley E, et al. (2005) Aspergillus lentulus sp. nov, a new sibling species of A. fumigatus. Eukaryot Cell 4:625–32.
10 Howard SJ, Webster I, Moore CB, et al. (2006) Multi-azole resistance in Aspergillus fumigatus. Int J Antimicrob Agents 28:450–3.[CrossRef][Medline]
11 Mellado E, Alcazar-Fuoli L, Garcia-Effron G, et al. (2006) New resistance mechanisms to azole drugs in Aspergillus fumigatus and emergence of antifungal drugs-resistant A. fumigatus atypical strains. Med Mycol 44:367–71.
12 Chamilos G and Kontoyiannis DP. (2005) Update on antifungal drug resistance mechanisms of Aspergillus fumigatus. Drug Resist Updat 8:344–58.[CrossRef][ISI][Medline]
13 National Committee for Clinical Laboratory Standards. (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi: Approved Standard (NCCLS, Pennsylvania, PA, USA).
14 Espinel-Ingroff A, Bartlett M, Bowden R, et al. (1997) Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi. J Clin Microbiol 35:139–43.[Abstract]
15
Merz WG, Fay D, Thumar B, et al. (1984) Susceptibility testing of filamentous fungi to amphotericin B by a rapid radiometric method. J Clin Microbiol 19:54–6.
16
Balajee SA and Marr KA. (2002) Conidial viability assay for rapid susceptibility testing of Aspergillus species. J Clin Microbiol 40:2741–5.
17
Balajee SA, Imhof A, Gribskov JL, et al. (2005) Determination of antifungal drug susceptibilities of Aspergillus species by a fluorescence-based microplate assay. J Antimicrob Chemother 55:102–5.
18
Wetter TJ, Hazen KC, Cutler JE. (2003) Modification of rapid susceptibility assay for antifungal susceptibility testing of Aspergillus fumigatus. J Clin Microbiol 41:4252–8.
19
Meletiadis J, te Dorsthorst DT, Verweij PE. (2003) Use of turbidimetric growth curves for early determination of antifungal drug resistance of filamentous fungi. J Clin Microbiol 41:4718–25.
20 Antachopoulos C, Meletiadis J, Roilides E, et al. (2006) Relationship between metabolism and biomass of medically important zygomycetes. Med Mycol 44:429–38.[CrossRef][ISI][Medline]
21
Antachopoulos C, Meletiadis J, Roilides E, et al. (2006) Rapid susceptibility testing of medically important zygomycetes by XTT assay. J Clin Microbiol 44:553–60.
22
Meletiadis J, Mouton JW, Meis JF, et al. (2001) Comparison of spectrophotometric and visual readings of NCCLS method and evaluation of a colorimetric method based on reduction of a soluble tetrazolium salt, 2,3-bis [2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide], for antifungal susceptibility testing of Aspergillus species. J Clin Microbiol 39:4256–63.
23
Meletiadis J, Mouton JW, Meis JF, et al. (2001) Colorimetric assay for antifungal susceptibility testing of Aspergillus species. J Clin Microbiol 39:3402–8.
24 Klohn PC and Neumann HG. (1997) Impairment of respiration and oxidative phosphorylation by redox cyclers 2-nitrosofluorene and menadione. Chem Biol Interact 106:15–28.[CrossRef][ISI][Medline]
25
Chen Q and Cederbaum AI. (1997) Menadione cytotoxicity to Hep G2 cells and protection by activation of nuclear factor-
B. Mol Pharmacol 52:648–57.
26 Niemczyk E, Majczak A, Hallmann A, et al. (2004) A possible involvement of plasma membrane NAD(P)H oxidase in the switch mechanism of the cell death mode from apoptosis to necrosis in menadione- induced cell injury. Acta Biochim Pol 51:1015–22.[ISI][Medline]
27 Zadzinski R, Fortuniak A, Bilinski T, et al. (1998) Menadione toxicity in Saccharomyces cerevisiae cells: activation by conjugation with glutathione. Biochem Mol Biol Int 44:747–59.[ISI][Medline]
28
Tunney MM, Ramage G, Field TR, et al. (2004) Rapid colorimetric assay for antimicrobial susceptibility testing of Pseudomonas aeruginosa. Antimicrob Agents Chemother 48:1879–81.
29 Groll AH, Gea-Banacloche JC, Glasmacher A, et al. (2003) Clinical pharmacology of antifungal compounds. Infect Dis Clin North Am 17:ix–91.
30 Scheven M and Schwegler F. (1995) Antagonistic interactions between azoles and amphotericin B with yeasts depend on azole lipophilia for special test conditions in vitro. Antimicrob Agents Chemother 39:1779–83.[Abstract]
31 Vanden Bossche H, Marichal P, Gorrens J, et al. (1989) Biochemical approaches to selective antifungal activity. Focus on azole antifungals. Mycoses 32:Suppl 1, 35–52.
32 Lass-Florl C, Denning D, Rodriguez-Tudela JL. (2006) Status of susceptibility testing in Aspergillus. Abstracts of the Second Advances Against Aspergillosis, Athens, Greece pp. 51–2.
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