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

Abstract

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 ؋ 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؇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.Interest in antifungal susceptibility testing has increased in recent years, as witnessed by the growing number of published articles in this field. Most investigations have focused on tests with fluconazole against pathogenic yeasts, particularly since reports have appeared suggesting that Candida isolates with reduced fluconazole susceptibilities are being encountered clinically after long-term fluconazole treatment (4,5,19,24,27,31,32,34,35,41). Current methods for antifungal susceptibility testing need to conform to three considerations, which overlap mutually. They should give results that correspond to the clinical outcome of antifungal treatment, that show some correlation to those ...

Bloodstream yeast infections: a 15-month survey

“…As official CLSI interpretative breakpoints are not available for amphotericin B, the breakpoints recommended by Pfaller & Diekema were used [2]. The amount of glucose in the RPMI medium was doubled to 2% to support optimal growth of isolates [7].…”

“…As official CLSI interpretative breakpoints are not available for amphotericin B, the breakpoints recommended by Pfaller & Diekema were used [2]. The amount of glucose in the RPMI medium was doubled to 2% to support optimal growth of isolates [7].A total of 412 yeast isolates was collected from 402 patients ; 10 patients had a double yeast infection. As found in the 2002 survey [3], the majority of patients (54 .…”

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

“…Some of these works include spectrophotometric determination of endpoints, higher-inoculum utilization, and glucose supplementation (2,5,12,13,17). The utility of these modifications remains to be established because of a lack of agreement between study findings.…”

Estudo comparativo entre as metodologias preconizadas pelo CLSI e pelo EUCAST para avaliação da atividade antifúngica

“…14,33 Apesar de não ter sido notado neste trabalho, o crescimento trailing é de grande relevância para a determinação da CIM, onde sua interpretação subjetiva deve ser considerada, haja vista que, mesmo com o uso de um agente revelador como a resazurina, as nuances de cores entre o azul e o vermelho podem ocasionar dúvidas na determinação da CIM. Assim, o EUCAST considera como CIM o orifício onde haja um crescimento de inóculo, determinado pela sua absorvância, de 50% ou menos em relação ao controle positivo.…”

“…Estudos têm confirmado que a modificação do documento CLSI M27-A2 9 com a suplementação do meio RPMI 1640 com 2% de glicose no meio de cultura 8 tornou a metodologia mais vantajosa, por reduzir o tempo de incubação necessário (24 h) para se obter um crescimento suficiente para a determinação dos valores de CIM. [10][11][12][13][14] Nas últimas décadas, a busca por novos agentes antimicrobianos, principalmente de fontes naturais, vem ganhando destaque. Neste sentido, os vegetais constituem uma enorme e importante fonte de produtos naturais que são biologicamente ativos, sendo que muitos destes podem ser utilizados como modelos para a síntese de inúmeros fármacos.…”