A novel gradient technique for combination testing: configuration and applications

Abstract number: 1134_04_229

Bolmström A., Engelhardt A., Karlsson Å., Vidh P.

Objectives:  

Combination therapy regimens of serious infections are seldom guided by in vitro testing due to the lack of simple yet reliable quantitative technique. Currently used methods such as checkerboard titrations (CT) are non-standardised and imprecise while kill curves are cumbersome to set up and interpretations remain arbitrary. A novel gradient technique, Xact™ (AB BIODISK) for quantification of drug interactions is described using a trim/sulpha (TR+SU) synergy model and other drug combinations/applications are also addressed.

Method:  

Xact consists of a 50 mm or 75 mm square carrier with a gradient quadrant of two or more test drugs in perpendicular alignment that gives a predefined concentration pattern with 225 or 484 unique drug ratios. Xact gradients for TR + SU across 0.016–256 and 0.001–2048 mg/mL were compared to CT using 2 E. coli, 1 E. cloacae, 1 S. aureus and 1 S. epidermidis tested in 10 replicates. The carrier was placed onto an inoculated agar surface and left for 1 hour to transfer the gradient imprint to the agar after which the carrier was removed. After overnight incubation, FICIs (Fractional Inhibitory Concentration Indices) were read where the inhibition isobologram intersected the predefined FICI grid. Xact was used with other drug/organism models to evaluate potential applications for yeast, rapid growing Mycobacteria (RGM) and for bactericidal interactions by relica-plating of the inhibition isobolograms.

Results:  

Maximum synergy was seen at MIC:MIC ratios of TR+SU for all strains with excellent reproducibility of FICIs (n = 50, SD. of <25% of the mean FICI value at 95% CI. ) and good agreement with CT. Models for yeast and RGM showed various interactions with the antifungal and antimycobacterial combinations studied. Xact could be used to demonstrate bactericidal interactions using the replica plating method.

Conclusion:  

The novel gradient technique was equivalent to CT as assessed with a TR + SU synergy model. It is simple to use to quantify interactions over a wide range of drug ratios. It could also be used for yeast and RGM testing and was able to demonstrate bactericidal interactions. Xact deserves further investigation as a potential tool for combination testing.