Study On Early Diagnosis And Clinical Application For Invasive Aspergillosis

Background and objectiveAspergillus is airborne in all environments, both inside and outside the hospital. It is the main pathogen of causing invasive aspergillosis (IA) in immunocompromised patients. IA occurs mainly in immunosuppressed hosts, such as acute leukemia, bone marrow stem cell transplantation, solid organ transplantation, chemotherapy in patients with malignant tumors. The epidemiology of patients showed that the incidence of IA has increased considerably in recent years. The incidence of which exceeded candidiasis in the autopsy cases. The mortality rates are very high which range from 56.0% to 88.1%. Clinical signs and symptoms of IA are non-specific. The traditional culture and histopathological methods have a limited role in the diagnosis of IA. Therefore, how to improve the diagnosis of IA has become a hot problem of common concern to medical workers at home and abroad. At present, serological methods and molecular biology methods are the most promising, referred to as non-culture diagnosis. In order to improve the level of the early detection and further evaluate the value of non-cultured diagnosis, this subject studied the clinical value of three diagnostic methods in early diagnosis and provided the basis for rational anti-fungal treatment, including serum galactomannan antigen (GM) detection , plasma (1,3) -β-D glucan (BG) detection and the DNA determination by the SYBR GreenΙreal-time quantitative PCR.Methods and resultsMethods and results of GM detection are as follows:①1236 of systematically collected serum samples obtained from 291 patients at high-risk of IA were detected by double sandwich enzyme-linked immunoabsorbent assay. According to retrospective analysis standards,291 patients were divided into 3 groups, including 60 confirmed IA group, 64 suspicious IA group and 167 Non-invasive Aspergillus infection group.②The distribution of risk factors showed no significant difference,including neutropenic time, the use of potent immunosuppressive agents and graft-versus-host disease.③Compared with different cutoff value for a positive (cutoff of 1.5, cutoff of 1.0, cutoff of 0.5), we can achieved the most optimal results using cut-off of 0.5 for GM detection. According to results of GM detection of confirm IA and non-invasive Aspergillus infection group , the sensitivity , specificity, positive predictive value(PPV), negative predictive value(NPV) of GM (cut-off of 0.5) were 91.7%, 92.2%, 80.9%, 96.9%, respectively.④Compared with conventional detection methods, GM detection preceded the development of characteristic findings on sputum cultures and imaging diagnosis about 2～8 days.⑤The mortality can be reduced to 13.5% when the preemptive antifungal therapy of voriconazole or itraconazole were administrated according to positive results of GM(cutoff 0.5) and the over-treatment and drug toxicity can be avoided.⑥Serum concentrations of GM declined when anti-fungal treatment was effective for IA, Otherwise the concentrations of GM were increased. Quantitive results of the serum-based GM antigen can represent the status of IA and may correlate the concentration in serum with treatment efficacy.Methods and results of BG detection are as follows:①Using a cutoff of 20pg/ml, the plasma BG concentration of 291 high-risk patients were detected by MB-80 microorganism rapid detection system. All patients were divided into 3 groups, including confirmed IA group, confirmed IFI group and excluded the possibility of IFI group. The BG detection for diagnosis of IFI with plasma has shown sensitivity, specificity, PPV and NPV values of 85.6%, 75.0%, 73.5%, 86.5%, respectively. The BG detection for diagnosis of IA with plasma has shown sensitivity, specificity, PPV and NPV values of 80.0%, 75.0%, 61.5%, 88.2%, respectively.②Using cutoff of 20 pg/ml for BG detection and cutoff of 0.5 for GM detection, the results of positive with BG or GM in patients with IA reaches 96.7%, while the results of positive with BG and GM in the same patients with IA are 66.7% in 60 confirmed IA patients and 120 exclude IFI patients. BG- negative or GM-negative amounted to 100%, while BG and GM were both negative to 70.8%. The combined analysis of BG and GM can improve the sensitivity of diagnosis of IA, while BG and GM were negative to exclude IA.③Use of mannan-peptide will cause abnormally high level of BG ,but will not affect the results of GM detection.Method and results of PCR detection are as follow:①Search the related fungus, the bacterium and humanity's 18S rRNA, 5.8S rRNA, 28S rRNA and ITS I, ITS11 gene sequences in the NCBI Genebank database. Through the compare of the Vector NTI 8 software for multi-sequences alignment, the target gene in Aspergillus rDNA transcribed spacers ITS I designed primers, specific amplification can produce a variety of Aspergillus strains specially.②We have improved and simplified the techniques of fungal DNA extraction to make them more suitable for use in the routine laboratory. It was easy to extract DNA from mycelial fungus cell. This method can obtain a good result by real-time PCR using the designed primers.③Take the Aspergillus fumigatus as the research object, the method for rapid detection of Aspergillus technology platform was developed using quantitative PCR . With this method,Aspergillus fumigatus were detected, the result has proven to be very good to distinguish between the different DNA density, the sensitivity of detection is 10fg/ml.④We have developed the extraction method of DNA from fungi, quantitative PCR procedure and the analysis program of results in the routine laboratory. Furthermore,the quantitative PCR method for monitoring Aspergillus in serum has been shown to be a useful tool for the early diagnosis of IA.ConclusionAccording to the results of GM detection, we got four conclusions.①The cutoff 0.5 of serum-based GM ELISA test result is the most optimal cutoff value for early diagnosis.②The detection of GM in patients at high risk for invasive aspergillosis has shown that the Aspergillus test is high specific and sensitive. The GM assay is a rapid, reliable method for early diagnosis and treatment of IA.③A positive result may help in choosing the most effective antifungal treatment. Negative results might provide a basis for further diagnostic test or for selecting the appropriate empirical therapy against moulds.④Monitoring of GM in serum has recently been shown to be a useful tool for predicting the therapeutic outcome of patients IA. GM assay is a good indicator for preemptive antifungal therapy in patients with haematological disease and malignant tumors.According to the results of BG detection, we got two conclusions.①As a new diagnosis method, BG detection has reasonable sensitivity and specificity. It is a reasonable approach to the clinical application of this assays would be serial screening of patients at high risk of IA.②The combined analysis of BG and GM can improve the sensitivity of diagnosis of IA, while BG and GM were negative to exclude IA.According to PCR results, we got two conclusions.①The method of Aspergillus DNA extraction was suitable for quantitative PCR detection. The detection of IA was successfully achieved by SYBR GreenⅠfluorescence quantitative PCR.②This method was useful for screening for Aspergillus spp. in patients with risk factors without antifungal treatment.