Antifungal susceptibility testing of Candida species by flow cytometry

The feasibility of flow cytometric antifungal susceptibility testing has been studied using the fluorescent anionic membrane potential probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. The in vitro antifungal susceptibility testing of amphotericin B was performed on 8 Candida isolates from clinical specimens and 2 ATCC strains by flow cytometry with the results compared to those of the National Committee of Clinical Laboratory Standards (NCCLS) M27-T, broth macrodilution method. The flow cytometric method is based on an increase of fluorescence given out by DiBAC4(3) in fungi when they are killed by antifungal agents. Minimum inhibitory concentration (MIC) of amphotericin B ranged from 0.25 to 1 microg/mL. All results agreed within +/-2 dilution between the flow cytometric method and the M27-T method. MIC with ATCC strains were within recommended ranges of M27-T. The new flow cytometric method revealed a clear and distinct reproducible test end point. A four hr of incubation was sufficient for the test. In conclusion, flow cytometry using DiBAC4(3) is a rapid and accurate in vitro antifungal susceptibility testing method.     

Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates.

A multicenter study was performed to establish the interlaboratory reproducibility of Etest, to provide an additional comparison of Etest MICs with reference broth macrodilution MICs, and to develop some tentative quality control (QC) guidelines for using Etest for antifungal susceptibility testing of Candida spp. Two QC strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were tested by Etest against amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole in each of four laboratories. The QC strains were tested 20 times each against the five antifungal agents by using a common lot of RPMI agar. A total of 80 MICs per drug per strain were generated during the study. Overall, 98 to 100% of the MICs fell within a 3 log2 dilution range for the respective yeast-antifungal agent combinations. The level of agreement of Etest MICs with broth macrodilution MICs was 86 to 100% with amphotericin B (C. krusei and C. parapsilosis), itraconazole (C. krusei and C. parapsilosis), flucytosine (C. parapsilosis), and fluconazole (C. parapsilosis). A lower level of agreement was observed with ketoconazole (C. krusei and C. parapsilosis). Although all participants reported identical Etest MICs, the MICs of flucytosine and fluconazole when tested against C. krusei fell well above the upper limits of the reference range for this strain. The tentative QC limits for the two QC strains and five antifungal agents when tested by the Etest methodology are the same as the QC limits when tested by the reference broth macrodilution method for amphotericin B and C. krusei, itraconazole and C. krusei, flucytosine and C. parapsilosis, fluconazole and C. parapsilosis, and itraconazole and C. parapsilosis. The Etest QC ranges are 1 dilution broader (4-dilution range) than the reference macrodilution method QC ranges for ketoconazole and C. krusei, amphotericin B and C. parapsilosis, and ketoconazole and C. parapsilosis.     

Preliminary evaluation of a semisolid agar antifungal susceptibility test for yeasts and molds

This report presents a semisolid agar antifungal susceptibility (SAAS) method for the rapid susceptibility screening of yeasts and molds. The reproducibility and accuracy of the SAAS method were assessed by comparing the MICs of amphotericin B and fluconazole obtained for 10 candidate quality control (QC) American Type Culture Collection yeast strains in >/=15 replicates with those found by six independent laboratories using the National Committee for Clinical Laboratory Standards (NCCLS) M27-P broth macrodilution method (M. A. Pfaller et al., J. Clin. Microbiol. 33:1104-1107, 1995). Overall, 96% of MICs for both drugs fell within 1 log(2) dilution of the modal MIC for each strain. The MICs for amphotericin B showed 99% agreement with the NCCLS proposed QC ranges within 1 log(2) dilution. Likewise, the MICs for fluconazole at >/=75% growth reduction showed 99% agreement for seven strains. Three strains, Candida albicans ATCC 24333 and ATCC 76615 and Candida tropicalis ATCC 750, showed a less sharp fluconazole endpoint at >/=75% growth reduction, but at >50% growth reduction, the agreement was 98% within 1 log(2) dilution of the proposed range. The MIC agreement within the proposed range for the suggested QC strains Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 was 100% for fluconazole and 100% within 1 log(2) dilution of the proposed range for amphotericin B. The SAAS method demonstrated the susceptibility or resistance of 25 clinical isolates of filamentous fungi such as Aspergillus fumigatus to amphotericin B, itraconazole, and fluconazole, usually within 48 h. Although the results are preliminary, this SAAS method is promising as a rapid and cost-effective screen and is worthy of concerted investigation.     

Effects of incubation time and buffer concentration on in vitro activities of antifungal agents against Candida albicans.

Nine selected isolates of Candida albicans were tested for their susceptibilities to amphotericin B and fluconazole by using three methods to assess the effect of incubation time and buffer concentration. By using a microdilution method with 0.0165 M 3-(N-morpholino)propanesulfonic acid (MOPS) and a 24-h incubation time, all of the isolates were found to be susceptible to amphotericin B and fluconazole. After 48 h of incubation, all isolates were still susceptible to amphotericin B. Seven of the nine isolates were resistant to fluconazole, and for the remaining two isolates, MICs increased by fourfold or more but the isolates remained susceptible (MIC, < or = 10 microg/ml). The nine isolates, along with three control strains, were further tested against amphotericin B and fluconazole by a standard broth macrodilution method with both 0.165 and 0.0165 M MOPS. The susceptibility results for fluconazole by the broth macrodilution method with the lower MOPS concentration correlated with the results of the 24-h broth microdilution method for determination of susceptibility or resistance in eight of nine tests and with the results of the 48 h broth microdilution method in three of nine tests. The results of the broth macrodilution method with the standard MOPS concentration did not correlate with any of the results obtained by the 24-h broth microdilution but correlated with results of seven of nine tests by the 48-h broth microdilution method. All nine test strains appeared to be susceptible when they were examined by a flow cytometric method. For clinical yeast susceptibility testing in microdilution panels, the 0.0165 M MOPS concentration combined with 24 h of incubation appeared to be the method of choice. The lower MOPS concentration may also be a useful modification to the tentative broth macrodilution method of the National Committee for Clinical Laboratory Standards. Use of the higher buffer concentration or longer incubation time may lead to false in vitro resistance for agents like fluconazole.     

Comparative evaluation of macrodilution and alamar colorimetric microdilution broth methods for antifungal susceptibility testing of yeast isolates.

A comparative evaluation of the macrodilution method and the Alamar colorimetric method for the susceptibility testing of amphotericin B, fluconazole, and flucytosine was conducted with 134 pathogenic yeasts. The clinical isolates included 28 Candida albicans, 17 Candida tropicalis, 15 Candida parapsilosis, 12 Candida krusei, 10 Candida lusitaniae, 9 Candida guilliermondii, 18 Torulopsis glabrata, and 25 Cryptococcus neoformans isolates. The macrodilution method was performed and interpreted according to the recommendations of the National Committee for Clinical Laboratory Standards (document M27-P), and the Alamar colorimetric method was performed according to the manufacturer's instructions. For the Alamar colorimetric method, MICs were determined at 24 and 48 h of incubation for Candida species and T. glabrata and at 48 and 72 h of incubation for C. neoformans. The overall agreement within +/- 1 dilution for Candida species and T. glabrata against the three antifungal agents was generally good, with the values for amphotericin B, fluconazole, and flucytosine being 85.3, 77.9, and 86.2%, respectively, at the 24-h readings and 69.3, 65.2, and 97.2%, respectively, at the 48-h readings. Most disagreement was noted with fluconazole against C. tropicalis and T. glabrata. Our studies indicate that determination of MICs at 24 h by the Alamar colorimetric method is a valid alternate method for testing amphotericin B, fluconazole, and flucytosine against Candida species but not for testing fluconazole against C. tropicalis and T. glabrata. For flucytosine, much better agreement can be demonstrated against Candida species and T. glabrata at the 48-h readings by the Alamar method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison study of broth macrodilution and microdilution antifungal susceptibility tests.

An evaluation of broth dilution antifungal susceptibility tests was performed by determining both the micro- and macrodilution MICs of amphotericin B, flucytosine, fluconazole, ketoconazole, and cilofungin against 38 isolates of Candida albicans, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and Torulopsis glabrata. The following preliminary antifungal working group recommendations of the National Committee for Clinical Laboratory Standards for broth macrodilution tests with antifungal agents were used: inocula standardized to 1 x 10(4) to 5 x 10(4) CFU/ml with a spectrophotometer, RPMI 1640 medium buffered with morpholinopropanesulfonic acid (pH 7.0), incubation at 35 degrees C for 24 to 48 h, and an additive drug dilution procedure. Broth microdilution MICs were higher (two or more dilutions) than broth macrodilution MICs for all isolates tested with amphotericin B and for most isolates tested with ketoconazole, fluconazole, and cilofungin. MICs of flucytosine were the same by both techniques or lower by the broth microdilution test except in tests with C. neoformans. However, the only statistically significant differences between the two tests were observed with amphotericin B against all isolates (P = 0.01 to 0.07), ketoconazole against C. neoformans (P = 0.01 to 0.02), and cilofungin against C. albicans (P = 0.05 to 0.14). Tests performed with less dense inocula (1 x 10(3) to 5 x 10(3] produced similar results.     

In vitro antifungal susceptibility testing of Candida blood isolates and evaluation of the E-test method.

Fungal infections have dramatically increased in recent years, along with the increase of drug-resistant isolates in immunocompromised patients. Ninety eight Candida species obtained from blood cultures at the Tri-Service General Hospital, Taiwan, from 1998 to 2000 were studied. These included 50 Candida albicans, 13 Candida glabrata, 24 Candida tropicalis and 11 Candida parapsilosis isolates. To investigate their susceptibility to commonly used antifungal drugs, minimum inhibitory concentrations (MIC) of amphotericin B, fluconazole, flucytosine, and ketoconazole were determined. Both the National Committee for Clinical Laboratory Standards reference broth macrodilution method and E-test were used in parallel. Ninety five isolates (95/98, 96.94%) were susceptible to amphotericin B at a concentration < or = 1 microg/mL. All isolates (100%, 98/98) were susceptible to flucytosine. Approximately 30% of these Candida isolates were resistant to fluconazole. The MIC for 90% of isolates (MIC90) values for both methods for these isolates were 0.5 microg/mL for amphotericin B, 32 microg/mL for fluconazole, 0.25 microg/mL for flucytosine (0.125 microg/mL by E-test method), and 4 microg/mL for ketoconazole. MIC for 50% of isolates (MIC50) values for these agents were 0.25, 2, 0.06, and 0.06 microg/mL, respectively. The essential agreement of MIC values within 2 dilutions for the 2 methods was 99.0% for amphotericin B, 90.8% for ketoconazole, 92.9% for fluconazole, and 91.8% for flucytosine. This study showed that E-test has equivalent performance to the broth macrodilution method and can be used as an alternative MIC technique for antifungal susceptibility testing.     

Comparison of broth macrodilution, broth microdilution, and E test antifungal susceptibility tests for fluconazole.

A comparison of the E test, the broth microdilution test, and the reference broth macrodilution susceptibility test of the National Committee for Clinical Laboratory Standards for fluconazole susceptibility testing was performed with 238 clinical isolates of Candida species and Torulopsis (Candida) glabrata. An 80% inhibition endpoint MIC was determined by the reference broth macrodilution method after 48 h of incubation. The MICs obtained by the two study methods were read after 24 and 48 h of incubation. Overall, excellent agreement within 2 doubling dilutions was obtained between the broth microdilution and the broth macrodilution methods for the combined results for all species at both 24 h (93%) and 48 h (94%). The correlation of 24-h MIC endpoints between the E test and the broth macrodilution methods was 37% for T. glabrata, 56% for Candida tropicalis, 93% for Candida albicans, and 90% for other Candida species. The percent agreement at 48 h ranged from 34% for T. glabrata to 97% for Candida species other than C. albicans and C. tropicalis. These initial results support the further evaluation of the E test as an alternative method for fluconazole susceptibility testing of Candida species.     

Selection of candidate quality control isolates and tentative quality control ranges for in vitro susceptibility testing of yeast isolates by National Committee for Clinical Laboratory Standards proposed standard methods.

The National Committee for Clinical Laboratory Standards has developed a proposed standard method for in vitro antifungal susceptibility testing of yeast isolates (National Committee for Clinical Laboratory Standards, document M27-P, 1992). In order for antifungal testing by the M27-P method to be accepted, reliable quality control (QC) performance criteria must be developed. In the present study, five laboratories tested 10 candidate QC strains 20 times each against three antifungal agents: amphotericin B, fluconazole, and 5-fluorocytosine. All sites conformed to the M27-P standards and used a common lot of tube dilution reagents and RPMI 1640 broth medium. Overall, 98% of MIC results with amphotericin B, 95% with fluconazole, and 92% with 5-fluorocytosine fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). Excellent performance with all three antifungal agents was observed for six strains: Candida albicans ATCC 90028, Candida parapsilosis ATCC 90018, C. parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida tropicalis ATCC 750, and Saccharomyces cerevisiae ATCC 9763. With these strains, 3-log2 dilution ranges encompassing 94 to 100% of MICs for all three drugs were established. Additional studies with multiple lots of RPMI 1640 test medium will be required to establish definitive QC ranges.     

Emergence of resistance of Candida albicans to clotrimazole in human immunodeficiency virus-infected children: in vitro and clinical correlations

Oropharyngeal candidiasis (OPC) is a common opportunistic infection in human immunodeficiency virus (HIV)-infected patients and other immunocompromised hosts. Clotrimazole troches are widely used in the treatment of mucosal candidiasis. However, little is known about the potential contribution of clotrimazole resistance to the development of refractory mucosal candidiasis. We therefore investigated the potential emergence of resistance to clotrimazole in a prospectively monitored HIV-infected pediatric population receiving this azole. Adapting the National Committee for Clinical Laboratory Standards M27-A reference method for broth antifungal susceptibility testing of yeasts to clotrimazole, we compared MICs in macrodilution and microdilution assays. We further analyzed the correlation between these in vitro findings and the clinical response to antifungal therapy. One isolate from each of 87 HIV-infected children was studied by the macrodilution and microdilution methods. Two inoculum sizes were tested by the macrodilution method (10(3) and 10(4) CFU/ml) in order to assess the effect of inoculum size on clotrimazole MICs. The same isolates also were tested using a noncolorimetric microdilution method. Clotrimazole concentrations ranged from 0.03 to 16 microg/ml. Readings were performed after incubation for 24 and 48 h at 35 degrees C. For 62 (71.2%) of 87 clinical isolates, the MICs were low (< or =0.06 microg/ml). The MIC for 90% of the strains tested was 0.5 microg/ml, and the highest MIC was 8 microg/ml. There was no significant difference between MICs at the two inoculum sizes. There was 89% agreement (+/-1 tube) between the microdilution method at 24 h and the macrodilution method at 48 h. If the MIC of clotrimazole for an isolate of C. albicans was > or =0.5 microg/ml, there was a significant risk (P < 0.001) of cross-resistance to other azoles: fluconazole, > or = 8 microg/ml (relative risk [RR] = 8.9); itraconazole, > or =1 microg/ml (RR = 10). Resistance to clotrimazole was highly associated with clinically overt failure of antifungal azole therapy. Six (40%) of 15 patients for whom the clotrimazole MIC was > or =0.5 microg/ml required amphotericin B for refractory mucosal candidiasis versus 4 (5.5%) of 72 for whom the MIC was <0.5 microg/ml (P = 0.001; 95% confidence interval = 2.3 to 22; RR = 7.2). These findings suggest that an interpretive breakpoint of 0.5 microg/ml may be useful in defining clotrimazole resistance in C. albicans. The clinical laboratory's ability to determine MICs of clotrimazole may help to distinguish microbiologic resistance from the other causes of refractory OPC, possibly reducing the usage of systemic antifungal agents. We conclude that resistance to clotrimazole develops in isolates of C. albicans from HIV-infected children, that cross-resistance to other azoles may develop concomitantly, and that this resistance correlates with refractory mucosal candidiasis.     

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.

A comparative study of broth macro- and microdilution methods for susceptibility testing of fluconazole, itraconazole, flucytosine, and amphotericin B was conducted with 273 yeasts. The clinical isolates included 100 Candida albicans, 28 Candida tropicalis, 25 Candida parapsilosis, 15 Candida lusitaniae, 15 Candida krusei, 50 Cryptococcus neoformans var. neoformans, 25 Torulopsis (Candida) glabrata, and 15 Trichosporon beigelii strains. Both methods were performed according to the National Committee for Clinical Laboratory Standards' (NCCLS) recommendations (document M27-P). For fluconazole, itraconazole, and flucytosine, the endpoint was the tube that showed 80% growth inhibition compared with the growth control for the macrodilution method and the well with slightly hazy turbidity (score 1) compared with the growth control for the microdilution method. For amphotericin B, the endpoint was the tube and/or well in which there was absence of growth. For the reference macrodilution method, the MICs were determined after 48 h of incubation for Candida spp., T. glabrata, and T. beigelii and after 72 h for C. neoformans var. neoformans. For the microdilution method, either the first-day MICs (24 h for all isolates other than C. neoformans and 48 h for C. neoformans var. neoformans) or the second-day MICs (48 and 72 h, respectively) were evaluated. The agreement within one doubling dilution of the macrodilution reference for all drugs was higher with the second-day MICs than with the first-day MICs for the microdilution test for most of the tested strains. General agreement was 92% for fluconazole, 85.7% for itraconazole, 98.3% for flucytosine, and 96.4% for amphotericin B. For C. neoformans var. neoformans and T. beigelii, the agreement of the first-day reading was higher than that of the second-day reading for fluconazole (94 versus 92%, respectively, for C. neoformans var. neoformans, and 86.7 versus 80%, respectively, for T. beigelii). Our studies indicate that the microdilution technique performed following the NCCLS guidelines with a second-day reading is a valid alternative method for testing fluconazole, itraconazole, flucytosine, and amphotericin B against these eight species of yeasts.     

Evaluation of Etest for direct antifungal susceptibility testing of yeasts in positive blood cultures

The performance of the Etest (AB BIODISK, Solna, Sweden) for direct antifungal susceptibility testing of yeasts in positive blood cultures was compared with that of the macrodilution method for determining the MICs of five antifungal agents. Culture broths with blood from bottles positive for yeasts were inoculated directly onto plates for susceptibility testing with the Etest, and the MICs were read after 24 and 48 h of incubation. A total of 141 positive blood cultures (72 cultures of Candida albicans, 31 of Candida tropicalis, 14 of Candida glabrata, 11 of Candida parapsilosis, 3 of Candida krusei, and 3 of Cryptococcus neoformans, 4 miscellaneous yeast species, and 3 mixed cultures) were tested, and the rates of MIC agreement (+/-1 log(2) dilution) between the direct Etest (at 24 and 48 h, respectively) and macrodilution methods were as follows: amphotericin B, 81.8 and 93.5%; flucytosine, 84.8 and 87.7%; fluconazole, 89.4 and 85.5%; itraconazole, 69.7 and 63.8%; ketoconazole, 87.9 and 79.0%. By a large-sample t test, the difference in log(2) dilution between the direct Etest and the macrodilution method was found to be small (P < 0.05). The lone exceptions were ketoconazole at 48 h of incubation and itraconazole at both 24 and 48 h of incubation (P > 0.05). By Tukey's multiple comparisons, the difference between the direct Etest (48 h) and reference methods among different species was found to be less than 1 log(2) dilution. When the MICs were translated into interpretive susceptibility, the minor errors caused by the direct Etest (at 24 and 48 h, respectively) were as follows: flucytosine, 2.3 and 1.4%; fluconazole, 3.0 and 3.6%; itraconazole, 21.2 and 21.3%. Itraconazole also produced an additional 3.0 and 3.6% major errors as determined by the direct Etest at 24 and 48 h, respectively. It was concluded that, except for itraconazole, the Etest method was feasible for direct susceptibility testing of blood cultures positive for yeasts. The method is simple, and the results could be read between 24 and 48 h after direct inoculation, whenever the inhibition zones were discernible.     

Trends in Antifungal Susceptibility among Swedish Candida Species Bloodstream Isolates from 1994 to 1998: Comparison of the E-test and the Sensititre YeastOne Colorimetric Antifungal Panel with the NCCLS M27-A Reference Method

A comparative evaluation of the NCCLS macrodilution method, the E-test, and the Sensititre YeastOne Colorimetric Antifungal Panel for the susceptibility testing of fluconazole, itraconazole, amphotericin B, and flucytosine was conducted with 233 blood isolates of Candida species collected between 1994 and 1998 in Sweden. Antifungal susceptibility profiles of Candida albicans and non-C. albicans Candida species remained essentially unchanged within the 5-year study period. The overall agreement rates for the E-test and the NCCLS MICs and for the YeastOne and the NCCLS MICs were > or =86 and > or =87%, respectively, within +/-1 dilution for fluconazole, amphotericin B, and flucytosine, and > or =66 and > or =57%, respectively, for itraconazole. The E-test and the YeastOne panels are equivalent, and both are convenient methods for routine use.     

Evaluation of a capacitance method for direct antifungal susceptibility testing of yeasts in positive blood cultures

The feasibility of using a capacitance method (CM) for direct antifungal susceptibility testing of yeasts in positive blood cultures was evaluated. The CM used the same test conditions as those recommended by the National Committee for Clinical Laboratory Standards. After direct inoculation of positive culture broths into module wells (Bactometer; bioMérieux, Inc., Hazelwood, Mo.), the end-point determination was made by monitoring the capacitance change in the culture broths with Bactometer. The MIC of amphotericin B was the lowest concentration at which yeast growth was completely inhibited, while the MICs of ketoconazole, flucytosine, and fluconazole were the concentrations at which a >/=80% reduction in capacitance change was observed. The MICs of the four drugs against each blood isolate obtained on subculture plates were also determined by the macrodilution method. For 51 positive blood cultures tested, the percent agreement (+/-2 log(2) dilutions) between the CM and the macrodilution method were as follows: amphotericin B (98%), ketoconazole (92%), flucytosine (84%), and fluconazole (96%). The CM was further used for breakpoint susceptibility testing of fluconazole (8 and 64 microg/ml) and flucytosine (4 and 32 microg/ml) against yeasts in positive blood cultures. After testing of 74 specimens by the CM, flucytosine and fluconazole produced one (1.4%) major error and two (2.8%) minor errors, respectively. All yeasts that displayed resistance to flucytosine or fluconazole were detected within 24 h after direct inoculation of the positive broths into Bactometer. The CM may be useful for the rapid detection of antifungal resistance in positive blood cultures containing yeasts.     

Rapid susceptibility testing of Candida albicans by flow cytometry.

The emerging magnitude of human fungal infections has renewed interest in developing rapid and standardized methods for susceptibility testing. We demonstrated that susceptibility testing of Candida albicans can be accomplished rapidly by using flow cytometry. Test results were available within 8 to 24 h after C. albicans isolates were incubated with amphotericin B, itraconazole, and flucytosine. This is an improvement of 24 to 60 h in the time to availability of susceptibility test results compared to the time to availability of National Committee for Clinical Laboratory Standards-recommended broth macrodilution test results. In addition, the flow cytometric endpoints, mean channel fluorescence, and number of fluorescence-labeled C. albicans cells were easy to interpret for greater sensitivity and reliability. Flow cytometry provides a more accurate means of obtaining antifungal susceptibility test results.     

Transient fungemia caused by an amphotericin B-resistant isolate of Candida haemulonii

A bloodstream infection due to Candida haemulonii afflicting a patient with fever and a medical history of megaloblastic anemia is reported. The clinical isolate was misidentified by the API 20C and VITEK identification systems. The results of susceptibility tests showed that the MIC of amphotericin B for C. haemulonii was 4 microg/ml. Additional susceptibility testing procedures based on the use of antibiotic medium 3 and Iso-Sensitest broth were performed, and killing curves were determined. Two collection strains of C. haemulonii were employed as controls. The three isolates exhibited resistance to amphotericin B in vitro regardless of the antifungal susceptibility testing method employed. In addition, the MICs of fluconazole for the three isolates were high. Further studies are needed in order to ascertain whether this species exhibits innate or acquired resistance to amphotericin B and other antifungal agents.     

Improved detection of amphotericin B-resistant isolates of Candida lusitaniae by Etest

Both intrinsic and acquired resistance to amphotericin B have been documented for Candida lusitaniae. Amphotericin B remains the drug of choice for many critical fungal infections, and the detection of resistance is essential to monitor treatment effectively. The limitations of the National Committee for Clinical Laboratory Standards (NCCLS) reference methodology for detection of amphotericin B resistance are well documented, and several alternative methods have been proposed. Etest assays with RPMI and antibiotic medium 3 (AM3) agar were compared to the NCCLS M27-A broth macrodilution method using AM3 for amphotericin B resistance testing with 49 clinical isolates of C. lusitaniae. The panel included nine isolates with known or presumed resistance to amphotericin B on the basis of in vivo and/or in vitro data. The distribution of amphotericin B MICs by Etest with RPMI ranged from 0. 032 to 16 microg/ml and was bimodal. All of the putatively resistant isolates were inhibited by amphotericin B at >/=0.38 microg/ml and could be categorized as resistant using this breakpoint. Etest with AM3 yielded a broader amphotericin B MIC range (0.047 to 32 microg/ml), and there were six putatively resistant isolates for which MICs were >1 microg/ml. The separation of putatively susceptible and resistant isolates was less obvious. Broth macrodilution with AM3 generated a unimodal distribution of MICs (ranging from 0.032 to 2 microg/ml) and failed to discriminate most of the putatively resistant isolates at both 24 and 48 h. Etest using RPMI and, to a lesser extent, using AM3 provided better discrimination between amphotericin B-resistant and -susceptible isolates of C. lusitaniae.     

Comparison of a new colorimetric assay with the NCCLS broth microdilution method (M-27A) for antifungal drug MIC determination

We evaluated a new microtiter assay for antifungal susceptibility testing based on a colorimetric reaction to monitor fungal substrate utilization. This new method (rapid susceptibility assay [RSA]) provides quantitative endpoint readings in less than 8 h compared with visual determination of MIC by the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method, which requires a minimum of 48 h of incubation. In this study, we tested clinical isolates from each of the following species: Candida albicans (20 isolates), C. glabrata (20 isolates), C. krusei (19 isolates), C. tropicalis (19 isolates), and C. parapsilosis (28 isolates). RSA and NCCLS broth dilution methods were used to determine the MICs of amphotericin B, fluconazole, itraconazole, and 5-flucytosine for all 106 isolates. RPMI 1640 medium buffered with morpholinopropanesulfonic acid was used for both methods; however, glucose and inoculum concentrations in the RSA were modified. RSA MICs were determined as the lowest drug concentration that prevented glucose consumption by the organism after 6 h of incubation. MICs obtained from the RSA were compared with those obtained from the NCCLS M-27A method read at 24 and 48 h. MIC pairs were considered in agreement when the difference between the pairs was within 2 twofold dilutions. For the 106 isolates tested, amphotericin B and 5-flucytosine demonstrated the highest agreement in MICs between the two methods (100 and 98%, respectively), whereas fluconazole and itraconazole produced less favorable MIC agreement (63.2 and 61.3%, respectively). The azole MIC differences between the two methods were significantly reduced when lower inocula were used with a prolonged incubation time. This preliminary comparison suggests that this rapid procedure may be a reliable tool for the in vitro determination of MICs of amphotericin B and 5-flucytosine and warrants further evaluation.     

Rapid susceptibility testing of fungi by flow cytometry using vital staining.

A 1-h assay for antifungal susceptibility testing measuring the impairment of fungal metabolic activity was developed. Yeast viability was analyzed by flow cytometry with a novel fluorescent probe, FUN-1, which emits a red fluorescence when the yeast is metabolically active. For nine Candida albicans strains tested, this method yielded results comparable to those obtained by the standard M27 procedure for amphotericin B, flucytosine, fluconazole, and ketoconazole. Whether the flow cytometry antifungal susceptibility test results correlate with the in vivo activities of the drugs remains to determined.     

Comparative evaluation of alternative methods for broth dilution susceptibility testing of fluconazole against Candida albicans.

A comparative evaluation of methods for broth macro- and microdilution susceptibility testing of fluconazole was conducted with 119 clinical isolates of Candida albicans. Macro- and microdilution testing were performed according to National Committee for Clinical Laboratory Standards recommendations. For reference macrodilution testing, an 80% inhibition endpoint (MIC 80%) was determined after 48 h of incubation in accordance with National Committee for Clinical Laboratory Standards proposed standard M27-P. Microdilution endpoints were scored as the first tube or well in which a prominent reduction in turbidity (score 2 out of a possible 4) was observed compared with the growth control (Micro MIC-2). Alternative endpoint criteria were assessed independently of the reference MIC 80% and Micro MIC-2 values and included a colorimetric microdilution endpoint determined by using an oxidation-reduction indicator (Alamar Blue; Alamar Bio-sciences Inc., Sacramento, Calif.). The MICs for the two microdilution test systems were read after 24 and 48 h of incubation. The percentage of fluconazole MICs within 2 doubling dilutions of the macrodilution reference values was 94% for both microdilution tests read at 24 h. Agreement was slightly lower at 48 h and ranged from 91 to 93%. Comparison of Micro MIC-2 and colorimetric microdilution MICs resulted in agreements of 97 and 93% at 24 and 48 h, respectively. These results show excellent agreement among alternative methods for fluconazole susceptibility testing.