Application Of Flow Cytometry To Test The Susceptibilities Of Mycobacterium Tuberculosis To Isoniazid By Flow Cytometry

Objective To develop a rapid method for testing susceptibility to isoniazid ofmycobacterium tuberculosis （MTb）by flow cytometry.Mehtods1. The optimal time and the concentration of FDA to identify activity ofmycobacterium tuberculosis were selected （preliminary experiment）.Based on thecharacteristics of viable MTb hydrolyzing flourescein diacetate(FDA),we used the FDA asfluorescent dyes in flow cytometry and tried to find the optimal time and the concentrationof FDA to identify activity of MTb: We select the optimal time and concentration of flowcytometry in detecting MTb activity through the observation of mean fluorescence intensity(MFI) under different proportion (0,25%,50%,75%,100%) of viable MTb at the sameconcentration of FDA、different time (10min,20min,30min,40min,50min,60min,70min,80min) and the same time (60min)、different concentration of FDA (100,200,250,300,400,500,600,700,800,900,1000,1100ng/ml).2. Susceptibility to isoniazid of mycobacteriumtuberculosis was detected. we detected the sensitivity to INH of10clinical isolated MTbstains by flow cytometry, critical concentration of INH to idenyify resistant strains andsensitive strains was preliminarily predicated according to the MFI ratio, which is the MFIdetected by the bacterial cultures at different concentrations of drug (5ug/ml,1ug/ml,0.2ug/ml,0.1ug/ml,0.05ug/ml) in24,48,72h versus to MFI detected by the positive control(no drug exposure). Furtherly, INH critical concentration inａnarrower scope of drugconcentration (0.1ug/ml,0.075ug/ml,0.05ug/ml,0.025ug/ml) was determined bydetecting the ratio of MFI from20cases of clinical isolates. In addition, comparison analysisof test results of susceptibility of30strains of clinical TB isolates between flow cytometricand proportional method were performed.Results1.After the100%pure viable MTb was stained for50minutes by FDA, theMFI rate of them rise quickly and stably.We futhermore observe the effect of FDA todifferent proportion (0,25%,50%,75%,100%) of viable MTb after different period(30,45,60min),the result turned out to be that the MFI value is more stable in the60min group.2.The MFI rate of the100%pure viable MTb stained by FDA rised up morerapidly and stably when the concentration of FDA is300ng/ml. Then we observed thedifferent rate of MFI after stained60min by three different concentration of FDA(200、250、300ng/ml),and MFI can distinguish the maximum life of MTb in the250ng/ml FDA group.3.We use the sensitive index (i.e. the ratio of the fluorescence intensity of drug-containing tubeto that of drug-free tube)≥0.8and INH critical concentration is0.05ug/ml as thejudgment standard, and30cases of clinical TB strains detected with the method of flowcytometry, the results of26strains were accordant to the ratio method, and the consistencyrate turned out to be86.7%.No significant difference was revealed between these twomethods.(P>0.05).Conclusion The optimal concentration and action time of FDA are250ng/ml and60minute to identify the activity of MTb by flow cytometric. Preliminary confirmed that flowcytometry susceptibility testing of MTb to isoniazid is a convenient,rapid and accuratemethod, but further verified the method need to expanding sample size due to small samplesin this study.