Antifungal Susceptibility Testing Of Yeasts Research Articles

Reliability of the WIDERYST Susceptibility Testing System for Detection of In Vitro Antifungal Resistance in Yeasts

This study evaluated the WIDERYST system, a commercially available computer-assisted image-processing device for the antifungal susceptibility testing of yeasts. A collection of 90 clinical isolates selected to represent ranges of susceptibilities in vitro as broad as possible was tested. An evaluation compared the results obtained by the new system with those achieved by both the Clinical and Laboratory Standards Institute (CLSI) microdilution reference procedure and the antifungal susceptibility standard of the European Committee for Antimicrobial Susceptibility Testing (EUCAST). Overall, the agreement and the correlation index between results obtained by the EUCAST method and the WIDERYST system were 89% and 0.84 (P < 0.01), respectively, and agreement and correlation index between data obtained by the CLSI procedure and the WIDERYST system were 90% and 0.86 (P < 0.01), respectively. The system was able to detect amphotericin B-resistant isolates. All Candida sp. isolates with resistance in vitro to azole agents were detected as well. The system misclassified some isolates belonging to the slowly growing genera Dipodascus and Pichia. A total of 2.7% very major errors were detected for fluconazole. The WIDERYST system is an alternative to reference procedures for antifungal susceptibility testing of clinical isolates of yeasts, particularly for Candida and Cryptococcus species.

Multicentre determination of quality control strains and quality control ranges for antifungal susceptibility testing of yeasts and filamentous fungi using the methods of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST)

A multicentre study involving seven laboratories was performed using techniques recommended by the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST) to evaluate and propose quality control ranges and strains for susceptibility testing of fermentative yeasts and filamentous fungi. Participating laboratories tested the susceptibilities of a panel of 12 encoded isolates to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole. In total, 15 lots of assay medium were tested, with one lot being common to all laboratories, and 18 144 MIC values were determined. Intra- and inter-laboratory agreements and intra-class correlation coefficients (ICCs) of the results for each drug/strain/lot combination were calculated. An average value of 85% agreement was selected for validation purposes. The average percentage of intra-laboratory agreement was 90-95%, with ICC values of 0.90-0.95 (p <0.01). Inter-laboratory reproducibility was also high, with 92% agreement and an ICC of 0.97 (p <0.01). The reproducibility was somewhat better with the common lot of assay medium (96% agreement) than with the different lots (91% agreement), but this difference was not significant. Two isolates that showed trailing growth had agreement percentages below the 85% limit selected for validation purposes and were therefore excluded from the panel of quality control strains. The recommended EUCAST methodologies were found to be highly reproducible and reliable for susceptibility testing of yeasts and filamentous fungi. Ten isolates are proposed for use as quality control strains with these EUCAST procedures.

Statistical analyses of correlation between fluconazole MICs for Candida spp. assessed by standard methods set forth by the European Committee on Antimicrobial Susceptibility Testing (E.Dis. 7.1) and CLSI (M27-A2).

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) Subcommittee on Antifungal Susceptibility Testing recently published a standard for determining the susceptibility of fermentative yeasts to antifungals. From the beginning, the EUCAST and its North American counterpart, the CLSI, decided to work together in order to establish common standards. As part of this exercise, the susceptibility of a set of 475 yeast isolates was tested by both standards. The intraclass correlation coefficient and the equations defining the linear regression between both methods were estimated. Both methods produced very similar results, with an intraclass correlation coefficient of 0.954 (0.945 to 0.962), although linear regression analysis shows that the EUCAST standard resulted in slightly lower MICs. There were only eight isolates showing at least four twofold dilution MIC differences between both standards. After 24 h of incubation, the MICs obtained by the CLSI method were equivalent to those obtained by the EUCAST standard. In summary, both methods produce very similar MICs, indicating that methodology does not pose any obstacle to obtaining uniform standards for antifungal susceptibility testing of yeasts.

Antifungal activity of some Tanzanian plants used traditionally for the treatment of fungal infections

Using the ethnobotanical approach, some Tanzanian plants reported to be used by traditional healers for the treatment of oral candidiasis and fungal infections of the skin were collected and screened for their antifungal activity against Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei and Cryptococcus neoformans. A total of 65 crude methanol extracts belonging to 56 plant species and 38 families were screened using the broth microdilution method, according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI) (formerly, National Committee for Clinical and Laboratory Standards) [National Committee for Clinical Laboratory Standards, 2002. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard-2nd Edition M27-A2, National Committee for Clinical Laboratory Standards, Wayne, PA, USA]. Among the tested plant species, 45% (25 species) showed antifungal activity against one or more of the test fungi. The most susceptible yeasts were Cryptococcus neoformans, followed by Candida krusei, Candida tropicalis, and Candida parapsilosis. The least susceptible were Candida albicans and Candida glabrata. Strong antifungal activity was exhibited by extracts of Clausena anisata Oliv., Sclerocariya birrea Sond, Turraea holstii Gurk, Sterculia africana (Lour) Fiori, Acacia robusta subsp. Usambarensis (Taub) Brenan, Cyphosterma hildebrandti (Gilg), Desc, Elaeodendron buchannanii (Lows), Acacia nilotica (L.) Wild ex Del, Jatropha multifida L., and Pteridium aquilinum (L.) Kuhn .

Multicenter Evaluation of Commercial Frozen Plates for Microdilution Broth Antifungal Susceptibility Testing of Yeasts and Comparison of MIC Limits Recommended in NCCLS M27–A2

A commercial kit, Frozen Plate for Antifungal Susceptibility Testing of Yeasts, Eiken (Eiken Chemical Co., Ltd., Tokyo), was tested in a multi-institute study to evaluate the agreement between interinstitute MICs and National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 recommendation limits of MIC value. The kit was reported as a method equivalent to the standardized guidelines for antifungal susceptibility testing by the Committee for Clinical Laboratory Standards-1994, the Japanese Society for Medical Mycology, and which is widely used in Japan for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole. The degrees of inter-institute and NCCLS agreements were good to excellent especially with 48-hr incubation for all antifungal agents. However, the percent agreements to NCCLS recommendations against itraconazole were poor. Overall, MIC values obtained using the frozen plate antifungal susceptibility testing kit, with 48-hr incubation, were thought to be reliable and convenient alternatives to the data obtained by the NCCLS M27-A2 reference macrodilution and microdilution method. This kit will allow matching of results between international laboratories. However, the MIC value for itraconazole requires careful interpretation.

Comparative Evaluation of Standard Dilution Method and Commercial Kit for Frozen Plate Antifungal Susceptibility Testing of Yeasts Using 200 Clinical Isolates

A comparative evaluation of standard microdilution methods and a commercial kit for frozen plate antifungal susceptibility testing of yeasts was performed using amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole on 200 yeast isolates. The isolates included 100 strains of Candida albicans, eight of C. tropicalis, twelve of C. parapsilosis, eight of C. glabrata, five of Cryptococcus neoformans, thirteen of Trichosporon asahii, and 54 other strains of seven other species of ascomycotic yeasts. Microdilution testing was performed according to the standard method for antifungal susceptibility testing published by the Japanese Society for Medical Mycology (JSMM), which are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) M27-P. The commercial kit was prepared according to the manufacturer's instructions. The degree of agreement within +/-1 dilution for 200 clinical isolates against five antifungal agents was excellent with values for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole of 100%, 99.0%, 97.5%, 97.0%, and 97.0%, respectively. Overall, the frozen plate antifungal susceptibility testing kit provided convenient and reproducible results comparable to those obtained with the JSMM standard method.

Commercial systems for fluconazole susceptibility testing of yeasts: comparison with the broth microdilution method

Fluconazole susceptibility was tested in 100 clinical yeast isolates (65 Candida albicans, 13 C. glabrata, 8 C. tropicalis, 7 C. parapsilosis, 3 Saccharomyces cerevisiae, 1 each of C. krusei, C. lusitaniae, Cryptococcus neoformans, Rhodotorula glutinis) and two control strains ( Candida krusei ATCC® 6258, C. parapsilosis ATCC® 22019) using broth microdilution (reference method), disk diffusion, Etest strips, Sensititre YeastOne, Candifast, Integral System Yeasts. Using M27-A breakpoints, isolates were classified as susceptible (81%), susceptible-dose dependent or Resistant with broth dilution. Rates of concordance with the reference method were good for Sensititre YeastOne, Etest and disc-diffusion (81.2%–94.7%) but very low for the Candifast® (3.1%) and Integral System (16.6%), which classified most susceptible isolates as resistant. Lack of standardisation (inoculum, medium composition) and non-objective interpretation schemes may be the cause of their poor performance. Sensititre YeastOne, Etest and disc-diffusion are potentially useful for fluconazole antifungal susceptibility testing of yeasts in clinical laboratories.

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.

A global evaluation of the susceptibility of Candida species to fluconazole by disk diffusion

An improved fluconazole 25-mg disk diffusion method was used to test the susceptibility of 20,900 consecutively isolated clinical strains of Candida species from 40 hospital laboratories in 26 countries. The procedure is similar to the National Committee for Clinical Laboratory Standards (NCCLS) M2-A6 method for testing bacteria, except Mueller-Hinton agar is supplemented with 2% glucose and 0.5 mcg/mL methylene blue. Plates were incubated at 35°C and read after 18 to 24 h. Tentative zone interpretive criteria were based on the correlation by regression analysis with the NCCLS M27-A Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: >=19 mm Susceptible, <=12 mm Resistant, and 13–18 mm Susceptible-Dose Dependent. Of 14,368 isolates of Candida albicans, 2,073 C. glabrata, 869 C. tropicalis, 752 C. parapsilosis, and 351 C. krusei, 99%, 67%, 90%, 94%, and 26%, respectively, were susceptible in vitro to fluconazole. All study sites used the BIOMIC System to electronically read zones on plates, interpret, record test results and verify quality control data. This is the largest study to date that evaluated a broad range of sequentially collected yeasts from various infections and different types of hospitals. The modified disk testing procedure is facile and economical to perform and offers a reproducible and accurate means to assess the in vitro susceptibility of Candida species to fluconazole.

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.

Antifungal susceptibility testing of yeasts: uses and limitations.

With recent developments in the field of mycology, such as increased incidence of fungal infections, the introduction of newer, safer antifungals, and the emergence of resistance, the need for clinically relevant antifungal susceptibility testing methods is obvious. Studies performed over the past decade have allowed the National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Testing to achieve consensus on a new standardized broth dilution method for in vitro susceptibility testing of yeasts (NCCLS M27-A). Once the reproducibility of the M27-A document was established, tentative breakpoints for fluconazole and itraconazole were derived. The availability of a standardized procedure for determining the minimum inhibitory concentrations (MICs) of antifungal agents is an important tool in drug discovery and development. In addition, it provides means for detection of resistant strains and, in the case of oropharyngeal candidiasis, means for patient management. Copyright 2000 Harcourt Publishers Ltd.

In vitro antifungal susceptibility methods and clinical implications of antifungal resistance

As new antifungal agents are introduced for the treatment of infections caused by yeasts and filamentous fungi (moulds), it is important that reliable methods are available for the in vitro testing of both new and established agents. The ultimate goal of in vitro testing is the prediction of the clinical outcome of therapy. The use of the M27-A procedures that were developed by the US National Committee for Clinical Laboratory Standards (NCCLS) has led to increased interlaboratory agreement of minimum inhibitory concentrations (MICs) for yeasts and has facilitated the establishment of interpretive breakpoints for fluconazole and itraconazole. The clinical relevance and limitations of these breakpoints are discussed elsewhere. The focus of this paper is to review the advantages and disadvantages of the available methods for antifungal susceptibility testing of yeasts and moulds as well as the clinical implications of in vitro antifungal resistance.

Amphotericin B susceptibility testing of Candida lusitaniae isolates by flow cytofluorometry: comparison with the Etest and the NCCLS broth macrodilution method

A flow cytofluorometric susceptibility test (FCST) was used for rapid determination of the susceptibility of Candida lusitaniae isolates to amphotericin B. The test is based on the decrease in fluorescence intensity of cells stained with 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)), a membrane potential-sensitive cationic dye, after drug treatment. A total of 58 C. lusitaniae clinical isolates including strains known to be amphotericin B-resistant on the basis of in-vivo and/or in-vitro data were tested. MICs were determined concurrently by the NCCLS broth macrodilution method and the Etest, both with antibiotic medium 3. Regression analysis demonstrated that the data from the FCST and the Etest were better correlated (r = 0.93, n = 59, P < 0.001) than those from the FCST and the NCCLS method (r = 0.63, n = 59, P < 0.001). The FCST readily identified a series of putatively susceptible and resistant isolates. Our study points out the advantages of the flow cytometry approach in antifungal susceptibility testing of yeasts, since speed remains a major problem in conventional tests.

NCCLS announces new standard on broth dilution antifungal susceptibility testing of yeasts

NCCLS announces new standard on broth dilution antifungal susceptibility testing of yeasts

Development of reference procedures for broth microdilution antifungal susceptibility testing of yeasts with standardized endpoint determination.

Standard guidelines for the broth microdilution antifungal susceptibility testing of amphotericin B, flucytosine, fluconazole, miconazole and itraconazole are reported. These are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) on the following two points: standardization of the means of endpoint determination and the inclusion of miconazole and itraconazole in the testing. MIC was determined to be when the positive control had a turbidity of 0.2 at the 630 nm wavelength. The endpoint was 80% inhibition for azoles and 100% inhibition for other drugs. The method provided good reproducibility, and a wide range of MIC distribution was observed in all antifungal agents except amphotericin B.

Comparison of the E Test with Broth Microdilution for Antifungal Susceptibility Testing of Yeasts

The susceptibilities of 26 recent invasive clinical isolates to amphotericin B (AMP), 5-flucytosine (5FC), fluconazole (FLU) and itraconazole (ITR) were determined by a broth microdilution modification of the NCCLS M27P method and also by E test. Using breakpoint criteria each result was classified as either sensitive (S), intermediate (I) or resistant (R). Taking the optical density (OD)80 as the standard, the results were further classified into major (M) or minor (m) errors. E test: AMP = 0M 0m, 5FC = 0M 5m, FLU = 1M 12m, ITR = 1M 5m errors. Minimal inhibitory concentrations (MIC): AMP = 0M 2m, 5FC = 0M 0m, FLU = 3M 4m, ITR = 1M 7m errors. The E test was quick and relatively simple to perform. Results using the E test compared favourably with those of the OD80 and it should be suitable for the routine susceptibility testing of yeasts to antifungal agents.

Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans

The National Committee for Clinical Laboratory Standards has proposed a reference broth macrodilution method for in vitro antifungal susceptibility testing of yeasts (the M27-P method). This method is cumbersome and time-consuming and includes MIC endpoint determination by the visual and subjective inspection of growth inhibition after 48 h of incubation. Two alternative microdilution procedures for MIC endpoint determination, a spectrophotometric MIC endpoint test that evaluates 80% growth inhibition by the drug and a colorimetric method with an oxidation-reduction indicator (Alamar Blue), were compared with the M27-P method for fluconazole susceptibility testing of 45 susceptible and resistant isolates of Candida albicans. The spectrophotometric method was performed with RPMI 1640 medium with 2% glucose, and the other two tests were performed with plain RPMI 1640 medium. All tests were incubated at 35 degrees C. Excellent agreement was demonstrated between the M27-P method and both 24-h microdilution tests (97.7%) as well as between the two microdilution tests (95.5%). Also, there was agreement in the detection in vivo of fluconazole resistance by the three methods. These preliminary data indicate that both microdilution methods may serve as less subjective alternatives to the M27-P method for the determination of fluconazole MIC endpoints.

Antifungal susceptibility testing of yeasts: evaluation of technical variables for test automation

The technical parameters for antifungal susceptibility testing with Candida species were reexamined to determine the optimal conditions for testing with semiautomated preparations of broth microdilution cultures, automated spectrophotometric readings of the cultures, and dose-response and endpoint determinations by means of a computer spreadsheet. Tests were based on proposed standard method M27P of the National Committee for Clinical Laboratory Standards for antifungal agents. RPMI 1640 broth with extra glucose to a final concentration of 2% gave higher and more reproducible drug-free control readings without affecting susceptibility endpoint readings. An inoculum of 8 x 10(4) yeasts per ml prepared from a carbon-limiting broth culture without further standardization was found to give optimal control readings after 48 h of incubation at 37 degrees C. For flucytosine, fluconazole, itraconazole, and ketoconazole, endpoints based on 50% growth inhibition (50% inhibitory concentration) gave the minimum variation with inoculum size and the fewest endpoint differences with RPMI 1640 medium obtained from two different suppliers. The 50% inhibitory concentration was also the optimal endpoint for fluconazole and ketoconazole susceptibilities in comparison with broth macrodilution MICs determined by the method of the National Committee for Clinical Laboratory Standards. Intralaboratory reproducibility was determined by retrospective analysis of replicate results for isolates retested at random over a 2-year period. This approach showed less favorable reproducibility than has been reported from purpose-designed, prospective antifungal susceptibility studies, but it may better reflect real-life test reproducibility. Susceptibility data for 616 clinical isolates of yeasts, representing 16 Candida and Saccharomyces spp., confirmed the tendency of Candida lusitaniae isolates to show relatively low susceptibilities to amphotericin B, the tendency of Candida krusei isolates to show low flucytosine and fluconazole susceptibilities, and the presence of some isolates in the species Candida albicans, Candida glabrata, and Candida tropicalis with low susceptibilities to azole derivative antifungal agents. The study demonstrates the value of automation and standardization in all stages of yeast susceptibility testing, from plate preparation to data analysis.

Etest for antifungal susceptibility testing of yeasts

Comparison [+/- 1 dilution between minimum inhibitory concentrations (MICs)] of fluconazole and flucytosine Etest MICs of 10 candidate quality control yeast isolates and 78 clinical isolates demonstrated good agreement (> or = 90%) with the reference method (National Committee for Clinical Laboratory Standards document M27-P). This study suggests that the Etest is a promising alternative method for antifungal susceptibility testing in the clinical laboratory, but that further evaluations are needed.

Antifungal Susceptibility Testing of Yeasts: A Brief Overview

Antifungal susceptibility testing of yeasts has lacked reproducibility because of the absence of universally accepted guidelines. The major sources of variation in susceptibility testing have been attributed to the choice of medium and pH, inoculum preparation and size, incubation temperature and time, and end-point criteria. These parameters have been addressed by the National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Testing, and proposed guidelines have recently been published. Although major advances in antifungal susceptibility testing have been achieved, particularly through the efforts of the NCCLS subcommittee, the results of studies to date do not support a correlation between the results of in vitro susceptibility testing and in vivo response. Routine antifungal susceptibility testing of yeasts should therefore be discouraged until the methodology is standardized such that it predicts clinical outcome.