Research Of Nucleic Acid Sequence-based Amplification Assay For Detecting And Diagnosis Of Invasive Aspergillosis

Objective：Invasive aspergillosis (IA) is a life-threatening infection inimmunocompromised patients. Accurate diagnosis and treatment at an earlystage are often crucial for favorable outcomes. A nucleic acidsequence-based amplification (NASBA) assay was developed forfacilitating the clinical diagnosis of IA in the first place. Then real-timequantitative PCR (qPCR) and GM test were also included to find out theoptimal clinical laboratory diagnostic strategy of IA.Methods： Firstly, a nucleic acid sequence-based amplification(NASBA) coupled to electrophoresis assay was developed by usingAspergillus fumigates (CMCC A1a) to detect Aspergillus. Its sensitivity andspecificity were evaluated as well. Feasibility of NASBA-molecular beacon(MB) platform for quantification Aspergillus was then explored. Finally,blood samples from80patients at a high risk for IA were tested by NASBA,qPCR and GM test and their diagnostic parameters were calculated,respectively. Prospective comparison of three methods was performed to find out the optimal clinical laboratory diagnostic strategy of IAResults：The NASBA assay was developed successfully in the lab. Itsspecificity was excellent and its detection limit was less than1Cfu. Thereaction time (minute) when the fluorescence signal rises above the fixedthreshold was linear correlated with the logarithm of the spores amount (y=-10.7x+81.6, r=0.9889).The sensitivity of NASBA, qPCR and GM test was76.47%,67.65%and52.94%, respectively, while their specificity was80.43%,89.13%,80.43%, respectively. The efficiency of various combinations of tests wasalso evaluated. Perfect specificity (100%) and positive predictive value(100%) were achieved by combining NASBA and qPCR as a serial testing.A combination of NASBA and qPCR as a parallel testing was the mostsensitive (94.12%).Conclusions：NASBA-MB platform for quantification Aspergilluswas feasible. NASBA has the highest sensitivity, qPCR has the highestspecificity, and GM test is inferior to NASBA and qPCR in the diagnosis ofIA. Considering its advantages in sensitivity, simplicity and speed, NASBAappears to be a promising tool for routine diagnosis of IA. Combination ofthese assays should improve the diagnostic efficiency and the accuracy ofearly IA diagnosis.