Study On The Antifungal Activity Of Amphotericin B And L-AmB In Vitro And The Detection Of The Minimum Inhibitory Concentration For L-AmB By Flow Cytometry

Objective: (1)To evaluate the difference and correlatability of the minimum inhibitory concentrations (MICs) between amphotericin B and L-AmB against 21 candida species. (2) To develop a fast method by flow cytometer for determining the minimum inhibitory concentrations(MICs)of L-AmB against candida isolates and then to evaluate applicability of this method by comparing result of the MICs,being double-blindly determinated , for L-AmB against 21 strains of Candida species with the recommendatory broth macrodilution-based method of the National Committee for Clinical Laboratory Standards (NCClS), USA. Methods : ( 1 ) The MICs of amphotericin B and L-AmB against 20 clinical isolates candida species and one quality control strain were determined in vitro by a broth macrodilution-based method in accord with the recommendations of NCClS . The difference of MICs between amphotericin B and L-AmB was evaluated and the regression analysis was demonstrated by SPSS10.0 of statistic software.(2)Strains of candida was tested for susceptibility to L-AmB by flow cytometry using propidium iodide (PI) as indicator of viability. On base of judgement level setted , MICs was defined according to the increasing degrees of mean fluorescence intensity(MFI)detected by flow cytometer for yeast suspension between the drug-treated and the growth control. After a series of preliminary tests, the optimal concentration of PI in staining solution, the suitable procedure for yeast suspension being labeled by PI ,the appropriate incubation time for sampe and the suitable judgement level of MIC were screened and selected and then, a method for measuring MICs by FCM had being established . Finally, we used this method to determine MICs of L-AmB against 20 clinical isolates strain of candida species and one quality control strain . The result of MICs for L-AmB by this method was compared with by a broth macrodilution-based method in accord with the recommendations of NCClS M27-A2. Results:(1)The result of MICs for L-AmB was 52.4%(+1 dilution)and 23.8%( +2 dilutions ) in agreement with AmB and mostly was higher , averagely ,with 0.82 dilution;Nonparametion correlation analysis with SPSS10.0 demonstrated that the data of MICs for L-AmB and AmB were correlated (r=0.643,p<0.01). (2)With the final concentration of 1.25mg/L PI ,increasing value of MFI detected by flow cytometer for yeast suspension between the dead control and the growth control was most obviously different from others and the cells often disintegrate into debris as the concentration of PI increased from 10mg/L to 100mg/L, which was show in forward scatter/side scatter( FS/SS) figure;between the drug-treated tube and the growth control tube , increasing degrees of mean fluorescence intensity(MFI)for yeast suspension containing L-Amb was 12.5%～94.6% and was -3.8 %～12.5% for the yeast suspension in which L-Amb being eliminated by washing , optimal staining being seen for yeast suspension with L-Amb being eliminated.;Between the drug-treated and the growth control, increasing degrees of mean fluorescence intensity(MFI)for yeast suspension incubating 1 hour, 2 hours, 3 hours and 4 hours were -5.2%～19.2%,-3.6 %～21.4 % ,7.0 %～59.7 % and 12.5 %～87.5 % , respectively,three hours incubation being appropriate for sample; The MICs, according to judgement level by 50% increase compared to growth control tube, were approximately comparable to the result of the reference macrodilution method developed by NCCLS on Antifungal Susceptibility Tests(M27-A2). For the 20 clinical isolates strain of candida species and one quality control strain tested with L-Amb, 23.8%(5/21)of the flow cytometry results were 100% agreenment with NCCLS M27-A2 method, 61.9%(13/21)of the results were within 1 dilutions, 85.7%(18/21)of the results were within 2 dilutions.and 95.2%(20/21)of the results were within 3dilutions. Conclusion:(1)The MICs of L-AmB against candida species in vitro was correlated with AmB and we could reason out the susceptibility breakpoint concentration of L-AmB from the breakpoint of AmB;On basis of the result obtained in our tests, we could estimate the breakpoint concentration of susceptibility for L-Amb against candida species would be 2μg/mL or so . But,after a great deal of strains tested and the results combined with clinical outcome, we could draw a conclusion about the exact susceptibility breakpoint concentration for L-AmB against candida species .(2) In this study with a series of preliminary tests , experimental condition ,influencing factor and judgement level being optimally selected , the method of FCM for detecting the MICs of L-Amb against candida species was established successfully . 95.2%(20/21)of the flow cytometry results were within 3 dilutions with NCCLS M27-A2. But flow cytometry antifungal susceptibility test (FCAST) was more rapid , sensitive, accurate and objective than others . Primitively ,we found that PI with the concentration from 10mg/L to 100mg/L often disintegrated yeast cells into debris and for optimal staining effect with PI , L-Amb in yeast suspension should being eliminated by washing,which we insisted on .