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Quality Control Limits for Posaconazole Disk Susceptibility Tests on Mueller-Hinton Agar with Glucose and Methylene Blue

Quality Control Limits for Posaconazole Disk Susceptibility Tests on Mueller-Hinton Agar with Glucose and Methylene Blue,10.1128/JCM.01732-06,Journal

Quality Control Limits for Posaconazole Disk Susceptibility Tests on Mueller-Hinton Agar with Glucose and Methylene Blue   (Citations: 10)
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An international collaborative study was performed in order to propose quality control limits for fluconazole disk diffusion susceptibility tests on Mueller-Hinton agar with 2% glucose and 0.5 g of methylene blue per ml. The supplements may be added before autoclaving the agar, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates generated data to propose zone size limits for tests of Candida parapsilosis ATCC 22019 (22 to 33 mm), C. tropicalis ATCC 750 (26 to 37 mm), and C. albicans ATCC 90028 (28 to 39 mm). C. krusei ATCC 6258 was not useful for this purpose. Immunocompromised patients are often colonized by or in- fected with fungi, especially Candida spp. Consequently, they frequently receive antifungal agents such as fluconazole throughout their period of hospitalization. Prolonged exposure to an azole can select strains with diminished susceptibilities (4), and consequently, increasing doses may be necessary. For that reason, clinical laboratories may be asked to monitor the susceptibility of isolates from such patients in order to deter- mine when strains with diminished susceptibility have been selected (3). For many laboratories, the disk diffusion proce- dure may be the most practical method for this purpose. A standardized method and well-defined quality control proce- dures are essential in order to determine when significant changes are being observed over an extended period of time. In 1996, we proposed a disk diffusion procedure that was performed on RPMI 1640 broth with 2% glucose and 1.5% agar (1). Subsequent studies were carried out to find an agar medium that should be readily available in most clinical labo- ratories. Mueller-Hinton (MH) agar could be used for this purpose, but it had to be supplemented with 2% glucose in order to increase the number of clinical isolates that would grow adequately. Unfortunately, the added glucose resulted in a significant amount of growth within zones of inhibition and zone edges that were not well defined. Addition of a low concentration of methylene blue (0.5 g/ml) made the zones of inhibition clearer and easier to measure precisely (2, 8). Plates with MH-glucose-methylene blue (GMB) agar can be prepared in advance and stored until needed, but the shelf life of such plates has not been determined. Prepared agar plates are practical for surveys, when the number of tests can be predicted in advance and prolonged storage of the plates is not necessary. An alternative approach is preferred for clinical laboratories where susceptibility tests are only sporadically needed. Fresh MH agar plates are commonly available in clin- ical laboratories, and they can be supplemented by flooding the surface with a GMB solution. Flooding procedure. A stock solution of methylene blue (5 mg/ml) was prepared and refrigerated at 2 to 8°C. To 100 ml of a 40% glucose solution, 200 l of the stock methylene blue was added to give 10 g of methylene blue per ml of 40% glucose (GMB solution). The GMB solution was dispensed into screw- cap tubes (3.5 ml for 150-mm-diameter plates or 1.5 ml for 100-mm-diameter plates), and that solution was then sterilized by autoclaving. The day before testing, refrigerated tubes with the GMB solution were allowed to warm to room temperature and at the same time MH agar plates were dried in a 35°C incubator with lids ajar until all of the surface moisture evap- orated (usually 1 to 2 h). The dried agar surfaces were then flooded with the GMB solution, and that liquid was allowed to adsorb overnight at room temperature on a flat surface. Ad- sorption was completed within a few hours when the MH agar plates had been dried by prolonged storage, but freshly pre- pared agar plates required a longer time. Assuming that the supplements are completely adsorbed and evenly dispersed throughout the 70 ml of agar in each 150-mm-diameter plate, the final concentrations of glucose and methylene blue should be 2% and 0.5 g/ml, respectively.
Journal: Journal of Clinical Microbiology - J CLIN MICROBIOL , vol. 45, no. 1, pp. 222-223, 2007
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