Bactericidal testing historically has exhibited variable reproducibility, even when prior standardized methods were employed. Several modifications to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method are proposed to improve reproducibility. Recommended changes from the approved NCCLS guidelines (M21-A and M26-A) include omitting serum supplementation of Mueller-Hinton broth, incubating tubes at 35°C for 24 h with no agitation until they are sampled, running all tests in duplicate with six dilutions instead of nine, reincubating the test for an additional 24 h to resolve discrepant bactericidal activity test results, using a single 0.1-ml sample from each clear tube for subculture, and adopting an alternate method for calculating endpoint determination. In order to test these recommendations in a clinical laboratory setting, we used the modified methodology on 224 separate tests for bactericidal activity. There were 102 serum bactericidal titer (SBT) and 122 minimum bactericidal concentration (MBC) assays performed. By defining reproducibility as agreement between duplicate tests ؎ 1 dilution, we found 207 of 224 tests (92%) were reproducible at the 24-h subculture point (94% for the SBT assay and 91% for the MBC assay). When the 17 assays with discrepant results were incubated an additional 24 h for a second subculture, only 1 of 224 tests (0.4%) remained discrepant. The method used is practical for a clinical laboratory that chooses to perform bactericidal activity testing and assures a high level of reproducibility between duplicate assays. The total cost of a test was approximately $25.00.
Disk diffusion and MIC quality control (QC) ranges were determined for nafithromycin, a new lactone-ketolide, following the completion of a nine-laboratory, Clinical and Laboratory Standards Institute (CLSI) document M23-defined tier 2 study. Five QC strains consistent with the spectrum of activity of nafithromycin were tested: ATCC 25923 (disk only), ATCC 29213 (broth only), ATCC 29212 (broth only), ATCC 49619 (disk and broth), and ATCC 49247 (disk and broth). Nafithromycin disk diffusion QC ranges were determined to be 25 to 31 mm for ATCC 25923, 25 to 31 mm for ATCC 49619, and 16 to 20 mm for ATCC 49247. Nafithromycin MIC QC ranges were determined to be 0.06 to 0.25 μg/ml for ATCC 29213, 0.016 to 0.12 μg/ml for ATCC 29212, 0.008 to 0.03 μg/ml for ATCC 49619, and 2 to 8 μg/ml for ATCC 49247. All disk diffusion and MIC QC ranges established in this study were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing at their June 2015 meeting and were initially reported in the 2017 M100S document. The QC ranges established in this study should be used for determining the activity of nafithromycin in phase 2 and phase 3 human clinical trials and subsequently for testing patient isolates and isolates in phase 4 surveillance studies.
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