Mechanism of inhibition of biofilms in Candida albicans by phenyl aldehydes and propanoids

Abstract

Background: Quantitative cryptococcal cultures provide a measure of disease severity in cryptococcal meningitis, and the rate of fungal clearance by quantitative culture has become a widely accepted surrogate outcome measure in phase II clinical trials. Various quantitative methodologies have been used to quantify CSF fungal burden; however, the reproducibility of between techniques is unknown.Methods & Materials: 213 CSF samples were prospectively collected from 70 individuals with cryptococcal meningitis at Mulago Hospital in Kampala, Uganda during Sept-Nov 2013. Each sample was simultaneously cultured by three different quantitative culturing techniques: 1) "standard" 100mcL input volume of CSF with an additional five 1:10 serial dilutions; 2) a AIDS Clinical Trials Group (ACTG) method using various input volumes (1000,100,10mcL) and two 1:100 dilutions with (100,10 mcL input volume) per dilution; 3) 10mcL calibrated plastic loop of undiluted and 1:100 diluted CSF. Colony forming units (CFU)/mL were quantified on the tenth day of culture. In addition, CSF at time of diagnosis was analyzed by automated cell counter and cryptococcal antigen (CRAG) lateral flow assay (LFA) titers.Results: Mean log10 transformed CFU counts suggested no significant differences between the standard method and either of the two alternative methods by paired t-test (difference=+0.036 ACTG,p=.690;-0.053 log10CFU/mL loop, p=.671), although the ACTG and loop methods differed significantly (difference=+0.55,p=.001). Correlation between tests was high at r=0.82, 0.85, and 0.83 for the standard-ACTG, standard-loop, and ACTG-loop methods, respectively. A weighted kappa statistic allowing for 1 log10 difference between methods showed moderate agreement between all tests, with k=0.50, 0.57, and 0.45 for the standard-ACTG, standard-loop, and ACTG-loop methods, respectively. No significant relationships were identified between culture methods and automated cell counts (diff=-1.13,p<0.001 vs. standard, R2=0.49). Regression analysis showed a significant association between being in the highest tertile of LFA titers and higher CFUs by the standard method (p=.042) Conclusion: Overall, the three methods of quantitative culture produced highly comparable but not identical results. There were significant differences between ACTG and loop techniques. The choice of quantitative method should be made based on lab-specific capacity and reproducibility of each technique.