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The JONES Group/JMI Laboratories, North Liberty, Iowa,1 Tufts University School of Medicine, Boston, Massachusetts2
Received 13 February 2003/ Returned for modification 14 May 2003/ Accepted 24 May 2003
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Appropriate disk diffusion and MIC quality control (QC) ranges for garenoxacin have been previously established (1). These QC ranges were determined for seven commonly used American Type Culture Collection (ATCC) control strains, which were tested over a 10-day period by more than seven laboratories using reference methods (5). With QC ranges established for garenoxacin, and as this compound nears completion of Phase III clinical trials and advances into clinical use, routine susceptibility testing using commercially prepared MIC products will be necessary. To validate the garenoxacin MIC results derived from commercially prepared products with extended shelf lives, a comparison (validation) study conforming to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines was performed (6).
In accordance with NCCLS M7-A6 methods (5) and established precedent (4), a minimum of 100 isolates per organism group listed in the M100-S13 document (7) tables for which garenoxacin has proven activity were tested. The spectrum of garenoxacin activity was determined for the following seven organism groups: Staphylococcus spp. (153 strains), Streptococcus spp. (141 strains; viridans group and ß-hemolytic streptococcal species), S. pneumoniae (167 strains), Enterococcus spp. (102 strains), Haemophilus influenzae (308 strains), Enterobacteriaceae (105 strains), and other gram-negative bacilli (102 strains). Garenoxacin susceptibility was determined by using both commercial dry-form (Sensititre/TREK Diagnostics, Cleveland, Ohio) and frozen broth microdilution panels. All frozen-form reference panels were processed using the manufacturer's package insert procedures (also produced by TREK Diagnostics) and were maintained at -70°C or below until used. All panels were inoculated with a final concentration of 5 x 105 CFU/ml. The panels were incubated in ambient air at 35°C and were interpreted at 16 to 20 h for gram-negative rapid-growing species or 20 to 24 h for gram-positive and fastidious organisms (5). Gatifloxacin was tested concurrently as an internal QC quinolone agent for this MIC validation study. The tested range for garenoxacin on both the reference and commercial dry-form panels was 0.008 to 16 µg/ml. The tested gatifloxacin concentration range on commercial dry-form panels was 0.03 to 4 µg/ml, and it was 0.004 to 8 µg/ml for frozen-form panels.
Commercial MIC panel reproducibility was assessed for garenoxacin by testing 14 strains. These strains included 10 commonly used ATCC strains: Escherichia coli ATCC 25922 and ATCC 35218, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853, S. aureus ATCC 29213 and ATCC 25923, Enterococcus faecalis ATCC 29212, H. influenzae ATCC 49247 and ATCC 49766, and S. pneumoniae ATCC 49619, as well as four clinical isolates. Susceptibility testing was performed three times daily for 3 days, generating a total of 126 determinations. Ciprofloxacin was utilized as the control antimicrobial agent for this portion of the study. The acceptable agreement definition for susceptibility test reproducibility was that 95% of MIC results be within ±1 log2 dilution step. All MIC results for QC strains were within published NCCLS M100-S13 ranges (7).
Table 1 summarizes the validation phase results for testing garenoxacin and gatifloxacin to compare reference NCCLS frozen panels (5) to commercial dry-form panels. Garenoxacin MIC results were slightly less variable than gatifloxacin test comparisons. All dry-form MIC results for garenoxacin were within ±1 log2 dilution. The gatifloxacin MICs for six strains were 2 log2 dilutions higher or lower than the reference method MIC.
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Garenoxacin MICs for the Enterobacteriaceae also demonstrated a slight shift towards a 1-log2-higher dilution for on-scale dry-form values than with frozen-form panels. Identical MICs occurred in 44.8% of tests with Enterobacteriaceae, while 50.7% of MICs were twofold higher. Among the nonfermentative gram-negative bacilli, identical MICs occurred for 53.4% of isolates, and 43.1% of tests demonstrated a twofold-higher MIC. All garenoxacin MICs for H. influenzae were within ±1 log2 dilution, with no significant shift or trend toward either higher or lower values. When all MIC results were analyzed for gatifloxacin, MICs for only 1.3% of the isolates were observed to be beyond the ±1-log2-dilution range.
Garenoxacin MIC results for the commercial dry-form panel reproducibility studies are listed in Table 2. Garenoxacin had a slightly greater same-day MIC reproducibility result, with 92.1% being identical versus 90.5% for the ciprofloxacin control. The MICs were identical for 90.5% of garenoxacin between-day replicates, compared to 88.9% for ciprofloxacin. There was no clear tendency for the MIC of either quinolone skewing towards higher or lower values or for unacceptable low levels of reproducibility. Ciprofloxacin MIC comparison results were on-scale for 8 of the 14 organisms tested, while garenoxacin demonstrated on-scale MIC results for 12 of the 14 strains tested. For both garenoxacin and ciprofloxacin, all replicate MICs were within ±1 log2 dilution for same-day and for between-day testing, meeting the definition of acceptable reproducibility.
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Garenoxacin, a novel des-fluoro(6) quinolone, has proven to be a potent antimicrobial agent in vitro, with a wide spectrum of activity, especially against gram-positive organisms and fastidious respiratory tract pathogens (2, 3, 8, 9; Takahata et al., 37th ICAAC). The study results reported here demonstrate that garenoxacin can be accurately and reproducibly tested following the Food and Drug Administration release of this product by using commercial dry-form (Sensititre, TREK Diagnostics) reagents and can be controlled by methods and QC ranges found in contemporary NCCLS tables (1, 5, 7).
ACKNOWLEDGMENTS |
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We also extend our thanks to K. Meyer, T. Anderegg, M. Beach, and D. Biedenbach for their technical support in the preparation of the manuscript and the execution of this comparative trial.
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