Early Laboratory Diagnosis Of Invasive Aspergillus Fumigatus Infection

The morbidityand mortality of Invasive fungal infections(IFI) has been increasing global in recent years,particularly in immunocompromised patients which were treated with chemotherapy, or those patients receiving high-dose corticosteroids or antibiotics.The diagnosis of IFI is difficult due to the lack of specific clinical features and to the low sensitivity of blood culture for isolation of fungus, especially for invasive aspergillosis(IA).Therefore,it is needed to develope non-culture methods with high sensitivity and specificity to improve etiologic diagnosis for invasive fungal infections.ObjectiveTo inspect nested PCR and serum galactomannan detection for the early diagnosis of invasive aspergillosis through the animal model of invasive aspergillosis,screening for the dominant antigen of Aspergillus fumigatus that were released during infection for the diagnosis of IA.MethodsSix cell lysis methods including freezing-thawing in liquid nitrogen or in -70℃,enzyme digestion,sonication,chaotropic salts and urea buffer were investigated.Each lysis procedure was followed DNA purification using the Qiagen kit.The efficiency was estimated by quantitation and PCR.Compared with the results of nested PCR, blood culture and the imaging technique for the early diagnosis of IA in the infected model;evaluated the value of nested PCR and GM detection for the diagnosis of IA in clinical samples.Mycelium protein and secreted protein of Aspergillus fumigatus were extracted for screening for Aspergillus fumigatus immunoreactivity antigens with the specific antibodies that were produced in the blood of the animal model using immunoblot assay.Results(1) All of the six extraction methods,enzyme digestion combined with the kit resulted in high yield.The detection limit of Candida albicans,Aspergillus fumigatus and Cryptococcus neoformans in simulated blood samples with universal primer PCR was 10~2,10~3 and 10~3 spores per reaction,respectively.(2) With a good sensitivity,nested PCR could give a positive result in the early of IA(two days after infection),which was superior to the culture and the imaging technique in the diagnosis for the model;on the clinical samples detection,the sensitivity of nested PCR and GM detection were 69%and 62%,with the specificity of 100%and 95%, respectively.(3) The secreted protein of Aspergillus fumigatus had the higher power to stimulate the body to response than the mycelium protein, and the strong immunoreactivity protein belonged to 10-30 kDa. ConclusionsWe have studied out a simple DNA extraction method for fungal, that is enzyme digestion combined with the Qiagen kit,nested PCR has a better application for the diagnosis of IA;we have got the dominant antigen of Aspergillus fumigatus.