In Vitro Study On The Combined Antifungal Effects Of Cyclosporine A With Azoles By Different Approaches

OBJECTIVE:To investigate the in vitro combined antifungal effects of cyclosporine A with three azoles on Candida albicans isolates sensitive or resistant to azoles by using various methods such as checkerboard method and time-kill curves,and to compare the agreement of these methods.METHODS:By using checkerboard method,the in vitro antifungal effects of the non-antifungal immunodepressant cyclosporine A(CsA) combined with fluconazole (FLC),itraconazole(ITR) and voriconazole(VRC) for 48h against Candida albicans isolates sensitive or resistant to azoles were assessed.The fungal growth was quantified by using a visual observation method and a spectrophotometric method.The results of the former were analyzed by the approache of FICI(Fractional inhibitory concentration index) and the latter were analyzed by FICI,AE(Difference between the predicted and measured fungal growth percentages)and RSA(Response Surface-modeling Approach).The coincidence between various methods were evaluated.By using time-kill curves,the dynamic antifungal effects of drug combinations within 48h were assessed,the results were determined by using both colony counting and the colorimetric method utilizing XTT.RESULTS:Checkerboard method was used to determine the antifungal effects of CsA and three azoles when used in combination.For the azole-sensitive isolates,the medians of FICI-1(based on the results of visual observation method)were 0.50,0.50,1.00,and the medians of FICI-2(based on the results of spectrophotometric method)were 1.00,0.50,1.00,respectively;all the medians ofαcalculated by RSA method were 0;the medians ofΣSYN andΣANT calculated byΔE method were 75.6%,-86%,101%,-51%, 77.9%,-31.5%,respectively.On the whole,for azole-sensitive Candida albicans isolates, CsA showed no or weak sensitization to azoles,but it could eliminate the "trailing" phenomenon.The results obtained from different methods were not the same absolutely. The rate of agreement between FICI-1 and FICI-2 was 86.7%,and those between FICI-2 and RSA,FICI-2 andΔE,RSA andΔE were 93.3%,73.3%,66.7%,respectively.For the azole-resistant isolates,when CsA was combined with FLC,ITR,VRC,the medians of FICI-1 and FICI-2 were all 0.00;all the medians ofαwere＞10~7;the medians ofΣSYN andΣANT were 1086.1%,-30.7%,1508.9%,-30.1%,712.1%,-40%, respectively.On the whole,for azole-resistant isolates,strong synergistic effects were observed whichever method was used.The coincidence was so good that all the rates of agreement between various methods were 100%.The antifungal effects of CsA and three azoles when used in combination were confirmed by time-kill curves.For the azole-sensitive isolate,compared with the azoles, the mean decrement of logarithm of colony counting obtained by XTT method for drug combinations after 48h incubation were 1.55,1.43,1.40,indicated indifferent effects(＜2).CsA showed no or weak sensitization to azoles.For the azole-resistant isolate, compared with the azoles,the mean decrement of logarithm of colony counting obtained by XTT method were all＞2 for drug combinations after 48 h incubation,indicated synergistic effects.CsA showed strong sensitization to azoles.Finally,we found that there was good coincidence between colony counting and XTT method,with r=0.9622.CONCLUSION:For azole-sensitive Candida albicans isolates,CsA for test showed no or weak sensitization to azoles,but it could eliminate the "trailing" phenomenon.For azole-resistant isolates,CsA showed strong sensitization to azoles.Good agreements were found between the results from different methods.The RSA method is not appropriate to evaluate the interactions between non-antifungal and antifungal agents.