Comparison between two culture media for in vitro evaluation of antifungal susceptibility of the Sporothrix schenckii complex / Comparação entre dois meios de cultura para avaliação in vitro da suscetibilidade a antifúngicos do complexo Sporothrix schenckii

BACKGROUND: The standard methodology for determining the antifungal sensitivity against the Sporothrix schenckii complex recommends the use of the 1640 Roswell Park Memorial Institute culture medium (RPMI) buffered with morpholinepropanolsulfonic acid (MOPS). However, while this is a high-cost medium which requires a laborious implementation and sterilization by filtration, the Sabouraud dextrose broth is a low-cost medium, widely used in mycology, sterilized by autoclave. OBJECTIVE: To evaluate the performance of the Sabouraud dextrose broth culture medium as a substitute for the RPMI 1640-MOPS in determining the antifungal sensitivity of S. schenckii. METHODS: Forty-eight clinical isolates were evaluated against five antifungal agents: itraconazole, ketoconazole, fluconazole, amphotericin B and terbinafine, using the method of broth microdilution advocated by the M38-A2 protocol of the Clinical and Laboratory Standards Institute. RESULTS: There were no significant differences between the Minimum Inhibitory Concentrations obtained in the two culture media for all the antifungals, with the exception of the amphotericin B. Regarding this drug, the Minimum Inhibitory Concentration range obtained were wider for the Sabouraud dextrose broth than for the Roswell Park Memorial Institute morpholinepropanelsulfonic acid. CONCLUSIONS: The Sabouraud dextrose broth showed potential to be used in the in vitro evaluation of the S. schenckii complex antifungal activity.

FUNDAMENTOS: A metodologia padronizada para a determinação da sensibilidade aos antifúngicos frente ao complexo Sporothrix schenckii preconiza a utilização do meio de cultura Roswell Park Memorial Institute (RPMI) 1640 tamponado com ácido morfolinopropanosulfônico (MOPS). No entanto, este meio possui custo elevado, execução trabalhosa e esterilização por filtração. Já o caldo Sabouraud-dextrose é amplamente utilizado em micologia, de baixo custo e pode ser esterilizado por autoclavagem. OBJETIVO: Avaliar o desempenho do meio de cultura caldo Sabouraud-dextrose em substituição ao RPMI 1640-MOPS na determinação da sensibilidade de S. schenckii a antifúngicos. MÉTODO: Foram avaliados 48 isolados clínicos frente a cinco antifúngicos: itraconazol, cetoconazol, fluconazol, anfotericina B e terbinafina, utilizando a metodologia da microdiluição em caldo preconizada pelo protocolo M38-A2 do Clinical and Laboratory Standards Institute. RESULTADOS: Não houve diferenças significativas nas Concentrações Inibitórias Mínimas obtidas nos dois meios de cultura para todos os antifúngicos, com exceção da anfotericina B. Para este fármaco, foram obtidas faixas mais amplas de Concentrações Inibitórias Mínimas para caldo Sabouraud-dextrose do que para Roswell Park Memorial Institute-morfolinopropanosulfônico. CONCLUSÕES: O caldo Sabouraud-dextrose mostrou potencial para ser utilizado na avaliação in vitro da atividade antifúngica do complexo S. schenckii.

In-vitro susceptibility testing by agar dilution method to determine the minimum inhibitory concentrations of amphotericin B, fluconazole and ketoconazole against ocular fungal isolates.

PURPOSE: To standardize in-vitro antifungal susceptibility testing by agar dilution method to find out the minimum inhibitory concentration (MIC) of amphotericin B, fluconazole and ketoconazole on ocular fungal isolates. METHODS: A total of 180 ocular fungal isolates (130 filamentous fungi and 50 yeasts) were included. The antifungal drugs such as amphotericin B (0.0625-8 microg/mL), fluconazole (0.2-819.6 microg/mL) and ketoconazole (0.025-6.4 microg/mL) were incorporated in doubling dilutions in the yeast nitrogen base medium. The MIC was determined as the lowest concentration of the antifungal drug preventing growth of macroscopically visible colonies on drug containing plates when there was visible growth on the drug-free control plates. RESULTS: All 50 ocular isolates of yeast were susceptible to amphotericin B, while two (4%) and five (10%) strains were resistant to fluconazole and ketoconazole respectively. Of the 130 filamentous fungi tested, six (4.6%) were resistant to amphotericin B, 49 (37.7%) and 10 (7.6%) were resistant to fluconazole and ketoconazole respectively. Percentile 50 (MIC 50) and Percentile 90 (MIC 90) for all the three antifungal agents were calculated. Aspergillus niger, Aspergillus terreus and Candida krusei were found to be resistant to fluconazole and ketoconazole. CONCLUSION: This technique was found to be reliable, cost effective and easy to perform with consistent results.

Evaluation of rapid alternative methods for drug susceptibility testing in clinical isolates of Mycobacterium tuberculosis

A study was carried out to compare the performance of a commercial method (MGIT) and four inexpensive drug susceptibility methods: nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS) assay, MTT test, and broth microdilution method (BMM). A total of 64 clinical isolates of Mycobacterium tuberculosis were studied. The Lowenstein-Jensen proportion method (PM) was used as gold standard. MGIT, NRA, MODS, and MTT results were available on an average of less than 10 days, whereas BMM results could be reported in about 20 days. Most of the evaluated tests showed excellent performance for isoniazid and rifampicin, with sensitivity and specificity values > 90 percent. With most of the assays, sensitivity for ethambutol was low (62-87 percent) whereas for streptomycin, sensitivity values ranged from 84 to 100 percent; NRA-discrepancies were associated with cultures with a low proportion of EMB-resistant organisms while most discrepancies with quantitative tests (MMT and BMM) were seen with isolates whose minimal inhibitory concentrations fell close the cutoff. MGIT is reliable but still expensive. NRA is the most inexpensive and easiest method to perform without changing the organization of the routine PM laboratory performance. While MODS, MTT, and BMM, have the disadvantage from the point of view of biosafety, they offer the possibility of detecting partial resistant strains. This study shows a very good level of agreement of the four low-cost methods compared to the PM for rapid detection of isoniazid, rifampicin and streptomycin resistance (Kappa values > 0.8); more standardization is needed for ethambutol.

Antifungal susceptibility testing method for resource constrained laboratories.

PURPOSE: In resource-constrained laboratories of developing countries determination of antifungal susceptibility testing by NCCLS/CLSI method is not always feasible. We describe herein a simple yet comparable method for antifungal susceptibility testing. METHODS: Reference MICs of 72 fungal isolates including two quality control strains were determined by NCCLS/CLSI methods against fluconazole, itraconazole, voriconazole, amphotericin B and cancidas. Dermatophytes were also tested against terbinafine. Subsequently, on selection of optimum conditions, MIC was determined for all the fungal isolates by semisolid antifungal agar susceptibility method in Brain heart infusion broth supplemented with 0.5% agar (BHIA) without oil overlay and results were compared with those obtained by reference NCCLS/CLSI methods. RESULTS: Comparable results were obtained by NCCLS/CLSI and semisolid agar susceptibility (SAAS) methods against quality control strains. MICs for 72 isolates did not differ by more than one dilution for all drugs by SAAS. CONCLUSIONS: SAAS using BHIA without oil overlay provides a simple and reproducible method for obtaining MICs against yeast, filamentous fungi and dermatophytes in resource-constrained laboratories.

Consenso sobre las pruebas de sensibilidad a los antimicrobianos en Enterobacteriaceae / Consensus for antimicrobial susceptibility testing for Enterobacteriaceae

En este documento se elaboraron una serie de recomendaciones para el ensayo, lectura, interpretación e informe de las pruebas de sensibilidad a los antimicrobianos para las enterobacterias aisladas con mayor frecuencia de especímenes clínicos. Se adoptaron como base las recomendaciones del National Committee for Clinical Laboratory Standards (NCCLS) de los EEUU, los de la subcomisión de Antimicrobianos, de la Sociedad Argentina de Bacteriología Clínica (SADEBAC), división de la Asociación Argentina de Microbiología (AAM) y de un grupo de expertos invitados. En él se indican las resistencias naturales de los diferentes miembros que integran la familia Enterobacteriaceae y se analiza la actividad de las diferentes beta-lactamasas cromosómicas, propias de cada especie, sobre las penicilinas, cefalosporinas y carbapenemes. Se recomiendan los antimicrobianos que se deberían ensayar, ubicados estratégicamente, para detectar los mecanismos de resistencia más frecuentes y cuales se deberían informar de acuerdo a la especie aislada, el sitio de infección y el origen de la cepa (intra o extrahospitalario). Se detallan los métodos de "screening" y de confirmación fenotipíca para detectar beta-lactamasas de espectro extendido (BLEE) que son más adecuados a nuestra realidad. Por último, se mencionan patrones infrecuentes de sensibilidad/resistencia que deberían verificarse y los perfiles de sensibilidad que pueden hallarse en las distintas enterobacterias en relación con los probables mecanismos de resistencia. Se debe resaltar que el contenido de este documento debe ser considerado como recomendaciones realizadas por expertos argentinos basadas en una revisión de la literatura y datos personales.

Taking into account previous recommendations from the National Committee for Clinical Laboratory Standards (NCCLS), the Antimicrobial Committee, Sociedad Argentina de Bacteriología Clínica (SADEBAC), Asociación Argentina de Microbiología (AAM), and the experience from its members and some invited microbiologists, a consensus was obtained for antimicrobial susceptibility testing and interpretation in most frequent enterobacterial species isolated from clinical samples in our region. This document describes the natural antimicrobial resistance of some Enterobacteriaceae family members, including the resistance profiles due to their own chromosomal encoded beta-lactamases. A list of the antimicrobial agents that should be tested, their position on the agar plates, in order to detect the most frequent antimicrobial resistance mechanisms, and considerations on which antimicrobial agents should be reported regarding to the infection site and patient characteristics are included. Also, a description on appropriate phenotypic screening and confirmatory test for detection of prevalent extended spectrum beta-lactamases in our region are presented. Finally, a summary on frequent antimicrobial susceptibility profiles and their probably associated resistance mechanisms, and some infrequent antimicrobial resistance profiles that deserve confirmation are outlined.

Métodos automatizados: ventajas y desventajas / Automated methods: advantages and disadvantages

Los estudios de susceptibilidad constituyen una herramienta de gran utilidad en la terapia exitosa de un paciente infectado. Los sistemas automatizados de identificación y evaluación de susceptivilidad ofrecen como principal ventajas la disminución del tiempo requerido en la obtención de los resultados; sin embargo, son de mayor costo que los sistemas manuales convencionales. Estudios de costo/beneficio han mostrado que disponer de un resultado hasta 12 horas antes, reduce los costos en antimicrobianos, reduce los días de hospitalización en unidades de cuidados intensivos y reduce el costo y de imágenes. Sin embargo, en todos estos estudios se contaba con un sistema de informe de exámenes que permitía la obtención oportuna de los resultados y la disponibilidad de los médicos clínicos para realizar los cambios de terapia en cualquier momento del día. Estos son los aspectos más importantes a considerar al momento de decidir la adquisición de algunos de estos sistemas y que podrían justificar el alto costo de equipamiento y de los insumos