Objectives: Current detection and susceptibility testing of moulds have insufficient sensitivity, require up to 72 h of incubation and carries considerable risk of cross-contamination. We evaluated a closed-system method for real-time detection of growth-related heat production of Aspergillus fumigatus. Measurements were performed with liposomal amphotericin B (AMB) (AmBisome) to evaluate the potential of microcalorimetry for rapid, accurate and easy-to-perform susceptibility testing of moulds. Methods: A. fumigatus strain ATCC 204305 was used. Microcalorimetry was performed in duplicate by adding 0.05 ml of A. fumigatus suspension containing 104 cells (determined in a haematocytometer by microscopy) in 2.95 ml Sabouraud Dextrose Broth containing serial 2-fold dilutions of AMB. Heat production was measured at 37Â°C under static conditions and detection time (in h), peak heat flow (in microwatt) and total heat produced (in Joules) were determined. The lower detection limit of heat production was defined at 20 microW. The minimal heat inhibition concentration (MHIC) was defined as the lowest antifungal concentration inhibiting heat production during 24 h of incubation. Results: Heat production of A. fumigatus without AMB (positive control) was detected after 11.3 h, with a peak of 220 microW and a total heat of 5 J. Growth media with AMB but without A. fumigatus (negative control) showed no detectable heat production. With increasing AMB concentration, the detection time was proportionally delayed (Figure) by 3.9 h (at 0.5 ug/ml), 8.8 h (at 1 ug/ml), 11.8 h (at 2 ug/ml), 16.8 h (at 4 ug/ml), 20.8 h (at 8 ug/ml), 28.2 h (at 16 ug/ml), 35.7 h (at 32 ug/ml) and 39.2 h (at 64 ug/ml). The MHIC of AMB was 4 ug/ml. The peak of heat flow gradually decreased from 220 microW (without AMB) to 120 microW (with 64 ug/ml AMB), while the total heat produced remained similar at about 5 J. Conclusion: Microcalorimetry allowed evaluation of activity of liposomal AMB against A. fumigatus by inhibiting its growth-related heat production. This method has the potential for rapid (12-24 h), accurate and real-time susceptibility testing of various moulds and for evaluation of new antifungals. In further steps, systematic optimization and standardization of calorimetric conditions are needed.
Full conference title:
20th European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 20th (2010)