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D Miller, F Marangon, A Romano, E Alfonso, S Gonzalez; Evaluation of an Agar Well Diffusion Assay to Validate and Correlate Invitro Efficacy of Topical Antibacterial and Antifungal Preparations with Conventional Susceptibility Techniques . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1608.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: : Laboratory investigation of the potency of commercial and fortified preparations of topical antibacterials and antifungals against common ocular pathogens are rarely performed. Efficacy is usually inferred from results of conventional disk diffusion or MICs tests. We investigate the utility an agar well diffusion assay (AWDA) to validate and compare invitro activity of topical antimicrobials against standard laboratory techniques. In addition, a wet disc method (WDM) was used to mimic and predict efficacy of the delivered concentration and volume of the antimicrobials during topical administration. Methods: Select ocular pathogens, Staphylococcus. aureus (10), Pseudomonas aeruginosa (10), Candida albicans (2), Aspergillus species 2) , Fusarium species (3), Curvularia (1) and Candida parapsilosis (2) were grown and maintained in log phase. A 0.5 McFarland standard suspension of each isolate was used as inoculum. Ocular preparations included: Tobradex, tobramycin (14 mg), gentamicin (3%), ciloxin (3%), ocuflox (3%) , quixin (5%), amphotericin B (1.5 mg) , Amphotericin B (2 mg) and Natamycin (5 mg). AWDA, An 8 mm biopsy bore was used to «punch out» , 6-8 equally spaced wells on Mueller Hinton (bacterial) and RPMI (fungi) agars prior to inoculation. 200 ul of test drug was then delivered to the well. WDA: After inoculation with the standard inoculum (bacterial only) sterile, 6mm , disks impregnated with 100 ul of commercial or fortified ocular drops were placed on the agar. Plates were then incubated for 18-24 hours. Results: Agreement between the Agar Well Diffusion and or the Wet Diffusion methods and conventional techniques was 90%. Correlation was both drug and organism dependent. There was 100% agreement for the P. aeruginosa isolates for all methods and all antibiotics. Discordant results were found for S. aureus methicillin resistant isolates and the quinolones- conventional (77%) vs AWD and or WD (both 100%) and aminoglycosides (85% vs 100%). Zones of inhibition were highest for the AWD followed by WD and least with the standard Kirby Bauer disk method. Measured difference ranged from 12 mm up to 28 mm for AWD and 5 mm to 16 mm for the WD assay. Correlation between the AWDA and E test for amphotericin B was 90%. Agreement of natamycin and amphotericin B was 100%. Conclusion: The Agar well diffusion assay and the wet disk technique may be used to validate the invitro efficacy of ocular drops. Despite the concentration difference, conventional susceptibility techniques offer comparable qualitative results.
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