Process Analysis of Variables for Standardization of Antifungal Susceptibility Testing of Nonfermentative Yeasts

ABSTRACTNonfermentative yeasts, such asCryptococcusspp., have emerged as fungal pathogens during the last few years. However, standard methods to measure their antifungal susceptibility (antifungal susceptibility testing [AST]) are not completely reliable due to the impaired growth of these yeasts in standard media. In this work, we have compared the growth kinetics and the antifungal susceptibilities of representative species of nonfermentative yeasts such asCryptococcus neoformans,Cryptococcus gattii,Cryptococcus albidus,Rhodotorulaspp.,Yarrowia lipolytica,Geotrichumspp., andTrichosporonspp. The effect of the growth medium (RPMI medium versus yeast nitrogen base [YNB]), glucose concentration (0.2% versus 2%), nitrogen source (ammonium sulfate), temperature (30°C versus 35°C), shaking, and inoculum size (103, 104, and 105cells) were analyzed. The growth rate, lag phase, and maximum optical density were obtained from each growth experiment, and after multivariate analysis, YNB-based media demonstrated a significant improvement in the growth of yeasts. Shaking, an inoculum size of 105CFU/ml, and incubation at 30°C also improved the growth kinetics of organisms. Supplementation with ammonium sulfate and with 2% glucose did not have any effect on growth. We also tested the antifungal susceptibilities of all the isolates by the reference methods of the CLSI and EUCAST, the EUCAST method with shaking, YNB under static conditions, and YNB with shaking. MIC values obtained under different conditions showed high percentages of agreement and significant correlation coefficient values between them. MIC value determinations according to CLSI and EUCAST standards were rather complicated, since more than half of isolates tested showed a limited growth index, hampering endpoint determinations. We conclude that AST conditions including YNB as an assay medium, agitation of the plates, reading after 48 h of incubation, an inoculum size of 105CFU/ml, and incubation at 30°C made MIC determinations easier without an overestimation of MIC values.

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Influence of Glucose Supplementation and Inoculum Size on Growth Kinetics and Antifungal Susceptibility Testing of Candida spp.

Journal of Clinical Microbiology ◽

10.1128/jcm.39.2.525-532.2001 ◽

2001 ◽

Vol 39 (2) ◽

pp. 525-532 ◽

Cited By ~ 40

Author(s):

M. Cuenca-Estrella ◽

T. M. Diaz-Guerra ◽

E. Mellado ◽

J. L. Rodriguez-Tudela

Keyword(s):

Growth Kinetics ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Inoculum Size ◽

Antifungal Susceptibility Testing ◽

Candida Spp ◽

Glucose Supplementation

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Flow Cytometry Antifungal Susceptibility Testing of Pathogenic Yeasts other than Candida albicans and Comparison with the NCCLS Broth Microdilution Test

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.10.2752-2758.2000 ◽

2000 ◽

Vol 44 (10) ◽

pp. 2752-2758 ◽

Cited By ~ 44

Author(s):

Rama Ramani ◽

Vishnu Chaturvedi

Keyword(s):

Flow Cytometry ◽

Candida Albicans ◽

Amphotericin B ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Antifungal Susceptibility Testing ◽

Broth Microdilution ◽

Nitrogen Base ◽

Microdilution Method ◽

Broth Microdilution Method

ABSTRACT Candida species other than Candida albicansfrequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing ofCandida glabrata, Candida guilliermondii,Candida krusei, Candida lusitaniae,Candida parapsilosis, Candida tropicalis, andCryptococcus neoformans based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except C. lusitaniae were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired ttest values were not statistically significant (P = 0.377 for amphotericin B; P = 0.383 for fluconazole). Exceptionally, C. lusitaniae isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.

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Antifungal susceptibility testing.

Clinical Microbiology Reviews ◽

10.1128/cmr.6.4.367 ◽

1993 ◽

Vol 6 (4) ◽

pp. 367-381 ◽

Cited By ~ 139

Author(s):

J H Rex ◽

M A Pfaller ◽

M G Rinaldi ◽

A Polak ◽

J N Galgiani

Keyword(s):

Susceptibility Testing ◽

Incubation Temperature ◽

Clinical Laboratory ◽

Antifungal Susceptibility ◽

Inoculum Size ◽

Antifungal Susceptibility Testing ◽

National Committee ◽

Candida Spp ◽

Interlaboratory Variability ◽

Future Work

Unlike antibacterial susceptibility testing, reliable antifungal susceptibility testing is still largely in its infancy. Many methods have been described, but they produce widely discrepant results unless such factors as pH, inoculum size, medium formulation, incubation time, and incubation temperature are carefully controlled. Even when laboratories agree upon a common method, interlaboratory agreement may be poor. As a result of numerous collaborative projects carried out both independently and under the aegis of the Subcommittee on Antifungal Susceptibility Testing of the National Committee for Clinical Laboratory Standards, the effects of varying these factors have been extensively studied and a standard method which minimizes interlaboratory variability during the testing of Candida spp. and Cryptococcus neoformans has been proposed. This review summarizes this work, reviews the strengths and weaknesses of the proposed susceptibility testing standard, and identifies directions for future work.

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Corrigendum: Growth kinetics in Candida spp.: Differences between species and potential impact on antifungal susceptibility testing as described by the EUCAST.

Medical Mycology ◽

10.1093/mmy/myy155 ◽

2019 ◽

Vol 57 (5) ◽

pp. e8-e10 ◽

Cited By ~ 1

Author(s):

María Ángeles Bordallo-Cardona ◽

Carlos Sánchez-Carrillo ◽

Patricia Muñoz ◽

Emilio Bouza ◽

Pilar Escribano ◽

...

Keyword(s):

Growth Kinetics ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Antifungal Susceptibility Testing ◽

Candida Spp ◽

Potential Impact

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Influence of Shaking on Antifungal Susceptibility Testing of Cryptococcus neoformans: a Comparison of the NCCLS Standard M27A Medium, Buffered Yeast Nitrogen Base, and RPMI–2% Glucose

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.2.400-404.2000 ◽

2000 ◽

Vol 44 (2) ◽

pp. 400-404 ◽

Cited By ~ 25

Author(s):

Juan L. Rodríguez-Tudela ◽

Francisco Martín-Díez ◽

Manuel Cuenca-Estrella ◽

Laura Rodero ◽

Yolanda Carpintero ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Clinical Laboratory ◽

Culture Media ◽

Antifungal Susceptibility ◽

Optimal Growth ◽

Inoculum Size ◽

National Committee ◽

Yeast Nitrogen Base ◽

Nitrogen Base ◽

Antifungal Susceptibility Test

ABSTRACT Cryptococcus neoformans is a nonfermentative yeast that requires oxygen for growth. The shaking of culture media achieves good oxygenation, promoting the growth of cryptococci. In this study, three test media (RPMI 1640, RPMI 1640–2% glucose, and buffered yeast nitrogen base [BYNB]) recommended in the National Committee for Clinical Laboratory Standards M27A standard were examined. Growth abilities and minimum inhibitory concentrations (MICs) in microplates incubated at 35°C for 48 h were determined. The results indicated that shaking and an inoculum size of 105 CFU/ml yielded optimal growth of this yeast. Compared to RPMI 1640, supplementation of RPMI 1640 with 2% glucose did not significantly improve growth of C. neoformans and resulted in an 8.7-h delay of exponential growth. Cryptococcal growth in RPMI 1640 at 24 h was notably better than that in RPMI–2% glucose, although by 48 h the growths were comparable. The MIC range of amphotericin B observed for the C. neoformans strains grown in RPMI 1640 with or without glucose was too narrow to allow the separation of susceptible and resistant strains based on clinical outcome. The widest ranges of MICs of flucytosine and fluconazole were obtained with BYNB. This work demonstrates the need for a new antifungal susceptibility test for C. neoformans.

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