Phenomenon of Dip Effect in Antifungal Susceptibility Testing of Candida tropicalis by using MIC Testing Strips against Echinocandins

Abstract

Minimum Inhibitory Concentration (MIC) of an antifungal is the minimum concentration that completely inhibits the visible growth of fungi under regulated, uniform and consistent in-vitro conditions. Reporting of antifungal susceptibility testing in Clinical Microbiology laboratories through standardised broth dilution methods by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility testing (EUCAST) can produce reproducibility within ± one to two-fold dilution, but are labour intensive and resource demanding procedures, requiring accurate training of the concerned personnel for performing and reading of the results. Hence, it becomes difficult to perform them regularly in high throughput laboratories and also in absence of enough trained manpower [1,2]. In such settings the Etest concentration gradient MIC Testing Strips (MTS) provide a valuable and convenient alternative solution, combining the principles of agar based diffusion methods and dilution methods. MTS comes as a ready to use impermeable plastic or paper reagent strips having embedded continual concentrations of antifungal in a predetermined gradient manner, covering a continuous concentration range generally across 15 two-fold dilutions. After application of MTS onto an inoculated agar surface, there is diffusion of preformed antifungal agent into the agar for over an hour. There is a formation of ellipse shaped area of inhibition which is symmetrical and has central part along the strip after 16-20 hours of incubation. The point of intersection of edge of ellipse with the strip is read as MIC in terms of μg/mL. Although convenient to use, it is not a reference method and still lacks the objectivity of reading and interpretation. One of the inhibition patterns posing as a reading challenge and leading to variability in reading is the “dip” effect, corresponding to a narrow inhibition zone at sub-MIC values i.e. ellipse is below the strip (does not intersect the strip). Although manufacturer’s instructions suggest to read below the dip and report the MIC less than the lowest value on the MIC scale, such phenomenon have been scarcely reported in the published literature [3,4].

Keywords

• anidulafungin
• caspofungin
• fungal growth
• isolate
• micafungin
• minimum inhibitory concentration
• yeast