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Comparison of three methods of antifungal susceptibility testing with the proposed NCCLS standard broth macrodilution assay: Lack of effect of phenol red
ELSEVIER
MYCOLOGY
Comparison of Three Methods of Antifungal Susceptibility Testing with the Proposed NCCLS Standard Broth Macrodilution Assay: Lack of Effect of Phenol Red Alan M. Sugar, M.D. and Xiuping Liu
Three microtiter plate adaptations of the NCCLS proposed standard for antifungai susceptibility testing were evaluated and compared to the NCCLS broth macrodilution method. Thirteen different fungi, including yeasts and moulds were studied. The first microtiter based method was performed exactly as described for the tube dilution assay, with the exception of performance of the assay in 100 ~1 in wells of the microtiter plate. The second assay was the same as the first, except for the deletion of phenol red from the RPM1 1640. The third microtiter assay was based on the reduction of the formazan dye, XTT, after only 24 hours of incubation. All three microtiter methods compared favorably with the macrodilution
method, when visually read after either 24 or 48 hours of incubation. Minimum inhibitory concentrations obtained by the XTT assay were usually lower than those obtained by the methods requiring a visual end point determination. Results were reproducible and comparable to those obtained with the NCCLS method. We conclude that microtiter plate adaptation of the NCCLS proposed standard is feasible and the presence of phenol red does not alter the results with the drugs tested. A 24 hour assay using XTT may provide a quicker and more quantitative method of susceptibility testing of fungi. Further investigation of this approach is warranted.
INTRODUCTION
1992; Barchiesi et al., 1994; Pfaller et al., 1994) and they appear to be reproducible and yield results similar to those obtained using the broth macrodilution assay. However, some variables still need to be evaluated and comparisons of various microplate based methods need to be correlated with the tube dilution assay. In this study, we compared three microplate based methods with the NCCLS tube dilution assay. The first microplate method was simply a miniaturization of the broth macrodilution assay using all parameters as suggested for the macro format. In the second assay, all parameters were the same, except for the elimination of phenol red from the RPM1 1640. This was done so that the third assay, one based on XTT (2,3-bis[2-methoxy-4-nitro-5sulfophenyll-2H-tetrazolium-5-carboxanilide inner salt) reduction (Stevens and Olsen 1993), could be done. Phenol red interferes with the spectrophoto-
The recent publication of a proposed standard for antifungal susceptibility testing is a big step in the history of antifungal chemotherapy (National Committee for Clinical Laboratory Standards 1992). As welcome as this standardized methodology is, however, it is labor intensive and cumbersome, since it is performed in test tubes, a format wasteful of reagents and one not amenable to automation. Several adaptations of the NCCLS methods have been proposed (Anaissie et al., 1991; Espinel-Ingroff et al., From the Evans Memorial Department of Clinical Research, Department of Medicine and the Clinical Mycology Center, Boston University Medical Center Hospital, Boston, Massachusetts. Address reprint requests to Alan M. Sugar, M.D., Boston University Medical Center Hospital, E336, 88 East Newton Street, Boston, Massachusetts 02160. DIAGN MICROBIOL INFECT DIS 1995;21:129-133 0 1995 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0732-8893/95/$9.00 SSDI 0732-8893(95)00067-Y
Sugar and Liu
130
metric reading of wells containing fungi and XTT. Thus, this study summarizes the performance of the 3 microplate formats as compared with the NCCLS proposed standard broth macrodilution assay.
MATERIALS AND METHODS Organisms Fungi were obtained from our stock collection, which are maintained on Sabouraud agar slants under sterile oil at 4°C. Subcultures of stock organisms were made prior to each experiment. The following fungi were used: a) Cundidu albicuns strain 64, an isolate used in our murine candidiasis studies (Sugar et al., 1994); b) 2 clinical strains of Cundidu ulbicuns; c) C. kefyr, obtained from the ATCC; d) a clinical isolate of C. purupsilosis, e) a clinical isolate of C. tropicalis; f) Cyptococcus neoformuns, 2 clinical strains; g) PseudulZescheriuboydii, a clinical isolate; h) Aspergillus niger, i) 2 clinical isolates of Aspergillus fumigutus, and j) Rhizopus oyzue, an isolate used in our murine mucormycosis studies (Waldorf et al., 1982, 1984; Goldani and Sugar, 1994).
Reagents RPM1 1640, with and without phenol red, and XTT were obtained from Sigma (St. Louis, MO). Fluconazole powder was obtained from Pfizer Inc. Stock solutions of 2 mg/ml were stored at -20°C and diluted to appropriate concentrations when needed. Amphotericin B was obtained as the deoxycholate formulation from Sigma. Stock suspensions were prepared in 5% dextrose and made fresh daily. Ketoconazole powder was obtained from Janssen Pharmaceutics. Stock solutions, 5 mg/ml, were prepared in 0.2 N HCI and stored at - 20°C. Appropriate dilutions were prepared from the thawed stock solutions.
Antifungal Susceptibility Testing Macrobroth dilution susceptibility testing was performed exactly by the NCCLS M27P protocol (National Committee for Clinical Laboratory Standards 1992). Microtiter plate adaptation of this method was accomplished using the same parameters, in a final volume of 100 ~1 per well. Flat bottom, 96 well microtiter plates were obtained from Costar. Two fold dilutions of each antifungal, from 256 to 0.125 pg/rnlwere used in each assay. Mould inocula were prepared by flooding slants with sterile distilled water, filtering the suspension through a sterile gauze filled syringe and recovering the conidia eluted from
the syringe. MICs were determined visually after both 24 (except for macrobroth assay) and 48 hours of incubation. After visually reading the microtiter assay performed without phenol red (after 24 hours of incubation), XTT, 0.5 mg/ml, was added to each well and the plates incubated for an additional 45 min at 37°C. Each plate was then read using a Dynatech MR5000 spectrophotometer, with the wavelength set at 450 nm. Visually determined MICs were read after agitation of the microtiter plate (Anaissie et al., 1991). The MIC as determined by microtiter assay was defined as the well containing the lowest concentration of drug without visible growth. As proposed by the NCCLS standard, azole MICs were defined in the macrobroth dilution method as the first tube showing 80% inhibition (1 + growth). In the microtiter assays, the MIC for amphotericin B and the azoles was defined as the first well showing no growth (recorded as 0). By XTT assay, a decrease in OD > 50% was used to determine the MIC.
Data Analysis All of the microtiter plate MICs were compared to those obtained by the NCCLS proposed standard broth microdilution assay. Differences among MIC endpoints of two dilutions (2 wells) or less were used to calculate the percent agreement.
RESULTS The MICs for each drug against the 13 isolates are presented in Tables l-3. Both visually read microtiter plate assays gave similar results to the NCCLS macrodilution assay. Thus after 48 hours of incubation, there was 100% agreement in MICs obtained by macrobroth dilution and both visually read microtiter plate adaptations of the standard method for each incubation time period. Results were the same in the microtiter plate assays at both 24 h and 48 h endpoints for the Cundidu strains. However, increases of 24 two-fold dilutions were seen with Aspergillus species and with both Cyptococcus neoformans isolates, with the additional 24 h of incubation, resulting in MICs equivalent to those obtained with the 48 hour incubation of the broth macrodilution method. The presence or absence of phenol red had no effect on the MICs at either time point with any of the drugs, with any of the fungi. The XTT assay was performed after a 24 hour incubation. The optical densities (ODs) of all control wells (fungi, but no drugs) were greater than 0.250, with the sole exception of C. kefyr, with ODs for all 3 assays 0.110, 0.209, and 0.210. In fact, in only 9 of
Testing with Standard Broth Microdilution Assay
TABLE 1
131
Minimum Inhibitory Concentrations” Amphotericin B MIC Range (pg/ml)
Organism (number of strains tested; number of times tested) Candida albicans (n = 3; 9)
C. kefyr (n = 1; 3) C. psrapsilosis (n = 1; 2) C. tropic&s (n = 1; 3) Cyptococcus neoformans (n = 2; 6) Pseudattescheria boydii (n = 1; 3) Aspergillus niger (n = 1; 2)
A. fumigatus (n = 2; 6) Rhizopus oyzze (n = 1; 2)
Macrobroth
Microbroth
Microbroth (no phenol red)
Xl-T
1 0.5-l
1 0.5-l
1 0.5-l
0.5-l 0.5
0.5-l 0.5
0.5-l 0.5
0.5-l 0.5
0.5-l 0.25
0.5 (16)
0.5 (16)
0.5
1
1
1
0.5
%16 8-16
1 (8-16) l-2 (8-16)
1
l-2 (8-16)
0.25-0.5
0.2SO.5
0.25-0.5
0.25-.5
0.125-0.25
16
(8-16)
0.5
“Macrobroth results were determined after 48 hours incubation. Data for other assays represent MICs at 24 hours and, if different, the 48 hour MICs are shown in parenthesis.
38 separate assays was the OD < 0.500. In several instances where the OD obtained for one isolate was >0.700 in one experiment and co.300 in another, the MICs for each of the 3 drugs was the same (within 1 two-fold dilution; data not shown). The XTT assay did not provide an MIC greater than that obtained by the visually read assays, with the sole
TABLE 2
exception of C. purupsilosis tested against fluconazole and this result was only one two-fold higher dilution. In most instances, MICs as determined by XTT assay were l->3 two-fold dilutions lower than the MICs read visually and often they were lower than the lowest concentration included on the microtiter plate, 0.125 kg/ml.
Minimum Inhibitory Concentration.9 Fluconazole MIC Range ( pg/ml)
Organism (number of strains tested; number of times tested) Candida albicans (n = 3; 9) C. kefyr (n = 1; 3) C. parapsilosis (n = 1; 3) C. tropicalis (n = 1; 3) Cryptococcus neoformans (n = 2; 6) Pseudallescheria boydii (n = 1; 3) Aspergillus niger (n = 1; 2) A. fumigatus (n = 2; 6) Rhizopus oyzae (n = 1; 3)
Macrobroth
Microbroth
Microbroth (no phenol red)
XT-l-
0.125 64
0.125 64
0.125 64
0.125 0.25
0.125 0.25
0.125 0.25
0.125 (16)
0.125 (16)
CO.125
0.25
0.25
CO.125
16 0.25 8-16 8-16 0.25-0.5
0.5-l (8-16) l-2 (4-16) 0.25-0.5
0.5-l (8-16) l-2 (4-16) 0.2545
(0.125 32 0.25 0.125-0.25
CO.125 <0.12.!%0.25 0.125-0.25
“Macrobroth results were determined after 48 hours incubation. Data for other assays represent MICs at 24 hours and, if different, the 48 hour MICs are shown in parenthesis.
Sugar and Liu
132
TABLE 3
Minimum Inhibitory Concentrations” Ketoconazole MIC Range (@ml)
Organism (number of strains tested; number of times tested) Candida albicans (n = 3; 9) C. kefyr (n = 1; 3) C. parapsilosis (n = 1; 3) C. tropicalis (n = 1; 3)
Macrobroth
Microbroth
Microbroth (no phenol red)
0.125 256
0.125 256
0.125 256
CO.125 256
0.125 0.125
0.125 0.125
0.125 0.125
co.25 CO.125
Cyptococcus neoformans (n = 2; 6)
128-256
0.5-l
(128-256)
0.5-l
(128-256)
Pseudallescheria boydii (n = 1; 3)
0.25
(n = 1; 2)
(n = 1; 3)
32-64 64-128 0.25-0.5
0.125-0.5
0.25
0.25
CO.125
2-4 (32-64) 2-4 (64.128)
2-4 (324) 2-4 (64-128)
CO.125
Aspergillus niger A. fumigatus (n = 2; 6) Rhiwpus 0yzae
X-IT
0.2ELO.5
0.2.SO.5
0.125-0.25
“Macrobroth results were determined after 48 hours incubation. Data for other assays represent MICs at 24 hours and, if different, the 48 hour MICs are shown in parenthesis.
DISCUSSION The results of this study show that microplate based methodologies can give results similar to those generated by the more cumbersome macrobroth dilution assay. Furthermore, the assay is amenable to modification by leaving out phenol red, at least with respect to consistent results being obtained with amphotericin B, fluconazole, and ketoconazole. This is an important observation, since phenol red conceivably could alter the MICs and this has been observed in several instances with other compounds (Scott Siegel, personal communication). Moreover, by eliminating phenol red from the wells and not altering the results, one can then proceed to a method amenable to spectrophotometric quantification, such as the XTT assay. It is important to note that once the MIC is read from a microtiter plate well, after a 24 hour incubation period, the same plate can then be used to generate the spectrophotometric data using XTT, after only an additional 45 minute incubation period. Generally, the XTT assay gave results l->3 two-fold dilutions lower than the visually read MIC in microplate format or the macrobroth dilution tube assay. Since appropriate breakpoints for classifying isolates as susceptible or resistant have not yet been agreed upon and correlation of specific MIC ranges with clinical outcome have not yet been established, it is not possible to come to any conclusion as to the clinical relevance of our observations from this small study. Similarly, whether the low MICs obtained with the XTT assay
can discriminate between “susceptible” and “resistant” organisms will require study of lower concentrations of drugs than were used in this study. The reasons for the low MICs, as measured by XTT reduction of the Aspergillus species and the single Rhizopus oryzae against the azoles are not clear. Sufficient inhibition of mitochondrial respiration to block XTT reduction by the azoles without significantly affecting capacity for cell growth is one obvious, but as yet unproved hypothesis. However, this may indicate some cellular damage to these fungi, effects that may be exploitable in combination therapy. Finally, it is notable that consistent results were obtained among the 4 assays when the conidia of the filamentous fungi were used as the inocula. The NCCLS proposed method is designed for testing yeasts and our observations are encouraging in that similar methods may be applicable for the testing of moulds. This would aid in the dissemination of this technology, since all fungi would then be evaluable using the same technology, one that is amenable to automation.
This study was supported by the Collaborative Medical Mycology Research Program (Pfizer Pkartnaceuticals, Janssen Pharmaceutica, Ortho Pharmaceuticals). This is publication 007 from the CMMRP. The authors acknowledge the helpful discussions with Dr. Scott Siegel, Pkytera, Inc., Worcester, Massachusetts.
Testing with Standard
Broth Microdilution
Assay
133
REFERENCES Anaissie E, Paetznick V, Bodey GP (1991) Fluconazole susceptibility testing of Cundida albicans: Microtiter method that is independent of inoculum size, temperature, and time of reading. Antimicrob Agents Ckemotker 35:1641-X46. Barchiesi F, Colombo AL, McGough DA, Rinaldi MG (1994) Comparative study of broth macrodilution and microdilution techniques for in vitro antifungal susceptibility testing of yeasts by using the National Committee for Clinical Laboratory Standards’ proposed standard. J Clin Micro 3232494-2500. Espinel-Ingroff A, Kish CW Jr, Kerkering TM, Fromtling RA, Bartizal K, Galgiani JN, Villareal K, Pfaller MA, Gerarden T, Rinaldi MG, Fothergill A (1992) Collaborative comparison of broth macrodilution and macrodilution antifungal susceptibility tests. f CZinMicro 30: 3138-3145. Goldani LZ, Sugar AM (1994) Treatment of murine pulmonary mucormycosis with SCH 42427, a broadspectrum triazole antifungal drug. J Antimicro Ckemotker 33:369-372. National Committee for Clinical Laboratory Standards (1992) Reference method for broth dilution antifungal
susceptibility testing for yeasts. Proposed standard. Document M27-P. National Committee for Clinical Laboratory Standards, Villanova, PA. Pfaller MA, Vu Q, Lancaster M, Espinel-Ingroff A, Fothergill A, Grant C, McGinnis MR, Pasarell L, Rinaldi MG, Steele-Moore L (1994) Multisite reproducibility of calorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates. J Clin Micro 32:162%1628. Stevens MG, Olsen SC (1993) Comparative analysis of using MIT and XTT in calorimetric assays for quantitating bovine neutrophil bactericidal activity. J lmmunol Methods 157:225-231. Sugar AM, Salibian M, Goldani LZ (1994) Saperconazole therapy of murine disseminated candidiasis: Efficacy and interactions with amphotericin B. Anfimicrob Agents Ckemotker 38~371-373. Waldorf AR, Halde C, Vedros NA (1982) Murine model of pulmonary mucormycosis in cortisone treated mice. Sabouraudia 20~217-224. Waldorf AR, Levitz SM, Diamond RD (1984) In vivo bronchoalveolar macrophage defense against and Aspergiilus fumigatus. 1 Infect Dis 150:752-760.
MYCOLOGY
Comparison of Three Methods of Antifungal Susceptibility Testing with the Proposed NCCLS Standard Broth Macrodilution Assay: Lack of Effect of Phenol Red Alan M. Sugar, M.D. and Xiuping Liu
Three microtiter plate adaptations of the NCCLS proposed standard for antifungai susceptibility testing were evaluated and compared to the NCCLS broth macrodilution method. Thirteen different fungi, including yeasts and moulds were studied. The first microtiter based method was performed exactly as described for the tube dilution assay, with the exception of performance of the assay in 100 ~1 in wells of the microtiter plate. The second assay was the same as the first, except for the deletion of phenol red from the RPM1 1640. The third microtiter assay was based on the reduction of the formazan dye, XTT, after only 24 hours of incubation. All three microtiter methods compared favorably with the macrodilution
method, when visually read after either 24 or 48 hours of incubation. Minimum inhibitory concentrations obtained by the XTT assay were usually lower than those obtained by the methods requiring a visual end point determination. Results were reproducible and comparable to those obtained with the NCCLS method. We conclude that microtiter plate adaptation of the NCCLS proposed standard is feasible and the presence of phenol red does not alter the results with the drugs tested. A 24 hour assay using XTT may provide a quicker and more quantitative method of susceptibility testing of fungi. Further investigation of this approach is warranted.
INTRODUCTION
1992; Barchiesi et al., 1994; Pfaller et al., 1994) and they appear to be reproducible and yield results similar to those obtained using the broth macrodilution assay. However, some variables still need to be evaluated and comparisons of various microplate based methods need to be correlated with the tube dilution assay. In this study, we compared three microplate based methods with the NCCLS tube dilution assay. The first microplate method was simply a miniaturization of the broth macrodilution assay using all parameters as suggested for the macro format. In the second assay, all parameters were the same, except for the elimination of phenol red from the RPM1 1640. This was done so that the third assay, one based on XTT (2,3-bis[2-methoxy-4-nitro-5sulfophenyll-2H-tetrazolium-5-carboxanilide inner salt) reduction (Stevens and Olsen 1993), could be done. Phenol red interferes with the spectrophoto-
The recent publication of a proposed standard for antifungal susceptibility testing is a big step in the history of antifungal chemotherapy (National Committee for Clinical Laboratory Standards 1992). As welcome as this standardized methodology is, however, it is labor intensive and cumbersome, since it is performed in test tubes, a format wasteful of reagents and one not amenable to automation. Several adaptations of the NCCLS methods have been proposed (Anaissie et al., 1991; Espinel-Ingroff et al., From the Evans Memorial Department of Clinical Research, Department of Medicine and the Clinical Mycology Center, Boston University Medical Center Hospital, Boston, Massachusetts. Address reprint requests to Alan M. Sugar, M.D., Boston University Medical Center Hospital, E336, 88 East Newton Street, Boston, Massachusetts 02160. DIAGN MICROBIOL INFECT DIS 1995;21:129-133 0 1995 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0732-8893/95/$9.00 SSDI 0732-8893(95)00067-Y
Sugar and Liu
130
metric reading of wells containing fungi and XTT. Thus, this study summarizes the performance of the 3 microplate formats as compared with the NCCLS proposed standard broth macrodilution assay.
MATERIALS AND METHODS Organisms Fungi were obtained from our stock collection, which are maintained on Sabouraud agar slants under sterile oil at 4°C. Subcultures of stock organisms were made prior to each experiment. The following fungi were used: a) Cundidu albicuns strain 64, an isolate used in our murine candidiasis studies (Sugar et al., 1994); b) 2 clinical strains of Cundidu ulbicuns; c) C. kefyr, obtained from the ATCC; d) a clinical isolate of C. purupsilosis, e) a clinical isolate of C. tropicalis; f) Cyptococcus neoformuns, 2 clinical strains; g) PseudulZescheriuboydii, a clinical isolate; h) Aspergillus niger, i) 2 clinical isolates of Aspergillus fumigutus, and j) Rhizopus oyzue, an isolate used in our murine mucormycosis studies (Waldorf et al., 1982, 1984; Goldani and Sugar, 1994).
Reagents RPM1 1640, with and without phenol red, and XTT were obtained from Sigma (St. Louis, MO). Fluconazole powder was obtained from Pfizer Inc. Stock solutions of 2 mg/ml were stored at -20°C and diluted to appropriate concentrations when needed. Amphotericin B was obtained as the deoxycholate formulation from Sigma. Stock suspensions were prepared in 5% dextrose and made fresh daily. Ketoconazole powder was obtained from Janssen Pharmaceutics. Stock solutions, 5 mg/ml, were prepared in 0.2 N HCI and stored at - 20°C. Appropriate dilutions were prepared from the thawed stock solutions.
Antifungal Susceptibility Testing Macrobroth dilution susceptibility testing was performed exactly by the NCCLS M27P protocol (National Committee for Clinical Laboratory Standards 1992). Microtiter plate adaptation of this method was accomplished using the same parameters, in a final volume of 100 ~1 per well. Flat bottom, 96 well microtiter plates were obtained from Costar. Two fold dilutions of each antifungal, from 256 to 0.125 pg/rnlwere used in each assay. Mould inocula were prepared by flooding slants with sterile distilled water, filtering the suspension through a sterile gauze filled syringe and recovering the conidia eluted from
the syringe. MICs were determined visually after both 24 (except for macrobroth assay) and 48 hours of incubation. After visually reading the microtiter assay performed without phenol red (after 24 hours of incubation), XTT, 0.5 mg/ml, was added to each well and the plates incubated for an additional 45 min at 37°C. Each plate was then read using a Dynatech MR5000 spectrophotometer, with the wavelength set at 450 nm. Visually determined MICs were read after agitation of the microtiter plate (Anaissie et al., 1991). The MIC as determined by microtiter assay was defined as the well containing the lowest concentration of drug without visible growth. As proposed by the NCCLS standard, azole MICs were defined in the macrobroth dilution method as the first tube showing 80% inhibition (1 + growth). In the microtiter assays, the MIC for amphotericin B and the azoles was defined as the first well showing no growth (recorded as 0). By XTT assay, a decrease in OD > 50% was used to determine the MIC.
Data Analysis All of the microtiter plate MICs were compared to those obtained by the NCCLS proposed standard broth microdilution assay. Differences among MIC endpoints of two dilutions (2 wells) or less were used to calculate the percent agreement.
RESULTS The MICs for each drug against the 13 isolates are presented in Tables l-3. Both visually read microtiter plate assays gave similar results to the NCCLS macrodilution assay. Thus after 48 hours of incubation, there was 100% agreement in MICs obtained by macrobroth dilution and both visually read microtiter plate adaptations of the standard method for each incubation time period. Results were the same in the microtiter plate assays at both 24 h and 48 h endpoints for the Cundidu strains. However, increases of 24 two-fold dilutions were seen with Aspergillus species and with both Cyptococcus neoformans isolates, with the additional 24 h of incubation, resulting in MICs equivalent to those obtained with the 48 hour incubation of the broth macrodilution method. The presence or absence of phenol red had no effect on the MICs at either time point with any of the drugs, with any of the fungi. The XTT assay was performed after a 24 hour incubation. The optical densities (ODs) of all control wells (fungi, but no drugs) were greater than 0.250, with the sole exception of C. kefyr, with ODs for all 3 assays 0.110, 0.209, and 0.210. In fact, in only 9 of
Testing with Standard Broth Microdilution Assay
TABLE 1
131
Minimum Inhibitory Concentrations” Amphotericin B MIC Range (pg/ml)
Organism (number of strains tested; number of times tested) Candida albicans (n = 3; 9)
C. kefyr (n = 1; 3) C. psrapsilosis (n = 1; 2) C. tropic&s (n = 1; 3) Cyptococcus neoformans (n = 2; 6) Pseudattescheria boydii (n = 1; 3) Aspergillus niger (n = 1; 2)
A. fumigatus (n = 2; 6) Rhizopus oyzze (n = 1; 2)
Macrobroth
Microbroth
Microbroth (no phenol red)
Xl-T
1 0.5-l
1 0.5-l
1 0.5-l
0.5-l 0.5
0.5-l 0.5
0.5-l 0.5
0.5-l 0.5
0.5-l 0.25
0.5 (16)
0.5 (16)
0.5
1
1
1
0.5
%16 8-16
1 (8-16) l-2 (8-16)
1
l-2 (8-16)
0.25-0.5
0.2SO.5
0.25-0.5
0.25-.5
0.125-0.25
16
(8-16)
0.5
“Macrobroth results were determined after 48 hours incubation. Data for other assays represent MICs at 24 hours and, if different, the 48 hour MICs are shown in parenthesis.
38 separate assays was the OD < 0.500. In several instances where the OD obtained for one isolate was >0.700 in one experiment and co.300 in another, the MICs for each of the 3 drugs was the same (within 1 two-fold dilution; data not shown). The XTT assay did not provide an MIC greater than that obtained by the visually read assays, with the sole
TABLE 2
exception of C. purupsilosis tested against fluconazole and this result was only one two-fold higher dilution. In most instances, MICs as determined by XTT assay were l->3 two-fold dilutions lower than the MICs read visually and often they were lower than the lowest concentration included on the microtiter plate, 0.125 kg/ml.
Minimum Inhibitory Concentration.9 Fluconazole MIC Range ( pg/ml)
Organism (number of strains tested; number of times tested) Candida albicans (n = 3; 9) C. kefyr (n = 1; 3) C. parapsilosis (n = 1; 3) C. tropicalis (n = 1; 3) Cryptococcus neoformans (n = 2; 6) Pseudallescheria boydii (n = 1; 3) Aspergillus niger (n = 1; 2) A. fumigatus (n = 2; 6) Rhizopus oyzae (n = 1; 3)
Macrobroth
Microbroth
Microbroth (no phenol red)
XT-l-
0.125 64
0.125 64
0.125 64
0.125 0.25
0.125 0.25
0.125 0.25
0.125 (16)
0.125 (16)
CO.125
0.25
0.25
CO.125
16 0.25 8-16 8-16 0.25-0.5
0.5-l (8-16) l-2 (4-16) 0.25-0.5
0.5-l (8-16) l-2 (4-16) 0.2545
(0.125 32 0.25 0.125-0.25
CO.125 <0.12.!%0.25 0.125-0.25
“Macrobroth results were determined after 48 hours incubation. Data for other assays represent MICs at 24 hours and, if different, the 48 hour MICs are shown in parenthesis.
Sugar and Liu
132
TABLE 3
Minimum Inhibitory Concentrations” Ketoconazole MIC Range (@ml)
Organism (number of strains tested; number of times tested) Candida albicans (n = 3; 9) C. kefyr (n = 1; 3) C. parapsilosis (n = 1; 3) C. tropicalis (n = 1; 3)
Macrobroth
Microbroth
Microbroth (no phenol red)
0.125 256
0.125 256
0.125 256
CO.125 256
0.125 0.125
0.125 0.125
0.125 0.125
co.25 CO.125
Cyptococcus neoformans (n = 2; 6)
128-256
0.5-l
(128-256)
0.5-l
(128-256)
Pseudallescheria boydii (n = 1; 3)
0.25
(n = 1; 2)
(n = 1; 3)
32-64 64-128 0.25-0.5
0.125-0.5
0.25
0.25
CO.125
2-4 (32-64) 2-4 (64.128)
2-4 (324) 2-4 (64-128)
CO.125
Aspergillus niger A. fumigatus (n = 2; 6) Rhiwpus 0yzae
X-IT
0.2ELO.5
0.2.SO.5
0.125-0.25
“Macrobroth results were determined after 48 hours incubation. Data for other assays represent MICs at 24 hours and, if different, the 48 hour MICs are shown in parenthesis.
DISCUSSION The results of this study show that microplate based methodologies can give results similar to those generated by the more cumbersome macrobroth dilution assay. Furthermore, the assay is amenable to modification by leaving out phenol red, at least with respect to consistent results being obtained with amphotericin B, fluconazole, and ketoconazole. This is an important observation, since phenol red conceivably could alter the MICs and this has been observed in several instances with other compounds (Scott Siegel, personal communication). Moreover, by eliminating phenol red from the wells and not altering the results, one can then proceed to a method amenable to spectrophotometric quantification, such as the XTT assay. It is important to note that once the MIC is read from a microtiter plate well, after a 24 hour incubation period, the same plate can then be used to generate the spectrophotometric data using XTT, after only an additional 45 minute incubation period. Generally, the XTT assay gave results l->3 two-fold dilutions lower than the visually read MIC in microplate format or the macrobroth dilution tube assay. Since appropriate breakpoints for classifying isolates as susceptible or resistant have not yet been agreed upon and correlation of specific MIC ranges with clinical outcome have not yet been established, it is not possible to come to any conclusion as to the clinical relevance of our observations from this small study. Similarly, whether the low MICs obtained with the XTT assay
can discriminate between “susceptible” and “resistant” organisms will require study of lower concentrations of drugs than were used in this study. The reasons for the low MICs, as measured by XTT reduction of the Aspergillus species and the single Rhizopus oryzae against the azoles are not clear. Sufficient inhibition of mitochondrial respiration to block XTT reduction by the azoles without significantly affecting capacity for cell growth is one obvious, but as yet unproved hypothesis. However, this may indicate some cellular damage to these fungi, effects that may be exploitable in combination therapy. Finally, it is notable that consistent results were obtained among the 4 assays when the conidia of the filamentous fungi were used as the inocula. The NCCLS proposed method is designed for testing yeasts and our observations are encouraging in that similar methods may be applicable for the testing of moulds. This would aid in the dissemination of this technology, since all fungi would then be evaluable using the same technology, one that is amenable to automation.
This study was supported by the Collaborative Medical Mycology Research Program (Pfizer Pkartnaceuticals, Janssen Pharmaceutica, Ortho Pharmaceuticals). This is publication 007 from the CMMRP. The authors acknowledge the helpful discussions with Dr. Scott Siegel, Pkytera, Inc., Worcester, Massachusetts.
Testing with Standard
Broth Microdilution
Assay
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