The Study Of Laboratory Diagnosis For Invasive Aspergillosis In Patient With Hematologic Diseases And Malignant Tumour And Clinical Application

objectiveThe incidence of invasive aspergillosis(IA) has increased dramatically during the last decade. These infections are associated with high mortality, ranging from 50% to 90%, especially in hematological diseases and malignant tumor patients.The critical problem is the difficulty in making the early diagnosis. Unfortunately, delayed diagnosis and treatment for IA are associated with poor outcomes and high mortality regardless of therapeutic modalities used. Tissue biopsy or histopathological specimens which involves invasive procedures are still considered the gold standard for establishing the diagnosis. However, these procedures are rarely performed, especially in the setting of immunosuppression or patients with thrombocytopenia, where such invasive procedures can be life-threatening. There has been an increased search for better noninvasive diagnostic methods for IA. At present, the serum antigen detection and molecular detection is the focus of research。This subject studied the clinical value of (1,3)-β-D-glucan(BG) antigen detection,galactomannan(GM) antigen detection and combined detection in early diagnosis and anti-fungal effect in hematological diseases and malignant tumor patients. We expect to develop and evaluate a Taqman Real-Time fluorescence PCR diagnostic techniques for IA and preliminary study the clinical value of this method in early diagnosis of IA .MethodsGM and BG detection are as follows:The serum and plasma samples obtained from 226 patients were detected with platelia Aspergillus assay kit and MB-80 microorganism rapid detection system. According to the hematological disease/malignant tumor patients with invasive fungal infections (IFI) diagnostic criteria and treatment principles as the standard: The patients were divided into three groups,including diagnosis and clinical diagnosis of IA (IA group),clinical suspected IA group and non-invasive Aspergillus infection group.PCR detection method is as follows: we extracted bacterial and serum DNA in clinical specimens by automatic nucleic acid extraction system; According to the GenBank database, we used the Blast and DNASIS tools to search highly conservative sequence of Aspergillus. One pair of Real-Time PCR universal primers and fluorescent probes of Aspergillus were designed by Primer Express 2.0 and Primer Express software; The standards were established and used to establish standard curves. The Real-Time PCR method were used to detected the Aspergillus DNA in clinical patients.ResultsGM and the BG test results:⑴A ccording to results of GM antigen detection,the sensitivity and specificity of serum GM test were 84.4% and 90.1%. The mortality can be reduced to 13.7% when the clinical anti-Aspergillus treatment were administrated according to positive results of GM.⑵Based on ROC curve,we can achieved the optimal results using the positive value of 20pg/ml for plasma BG detection in invasive fungal infections. The BG detection for diagnosis of IFI has shown sensitivity and specificity were 88.3% and 86.8%. The BG detection for diagnosis of IA has shown sensitivity and specificity were 88.9% and 62.5 %. The comparison of plasma BG concentrations between Aspergillus infections and other fungal infections had no statistical significance(P>0.05).⑶The combined detection of BG and GM for diagnosis of IA has shown sensitivity and specificity were 82.2% and 99.3%.⑷Compared with conventional detection methods,the positive results in 27 patients were obtained using GM detection method which preceded the findings on imaging diagnosis and sputum cultures about 3～10 days. 25 patients'BG detection preceded the findings on imaging diagnosis and sputum cultures about 1～7 days.⑸the concentrations of GM antigen and BG antigen decreased when anti-fungal treatment was effective for IA and increased as the treatment ineffective.Real-Time PCR detection results:⑴D NA extraction from fungal suspensions and serum can obtain a good result by real-time PCR and general PCR and analysis.⑵The technological platform for rapid detection of Aspergillus was established using Real-Time PCR. With this method,different concentrations of DNA templates were amplified,the best linearity range was 107-103Copies/ml; the sensitivity of detection is 10copies/ml,⑶Real-Time PCR for clinical diagnosis of IA in patients has shown sensitivity and specificity were 86.7% and 85.5 %.ConclusionAccording to the above results, we got five conclusions:⑴The detection of GM antigen has shown high specific and sensitive. The GM assay has a guiding value in the early diagnosis of IA .⑵The positive value of 20pg/ml of plasma BG is the most optimal for IFI early diagnosis. BG detection has a good clinical diagnosis value of the IFI. BG assay has low specificity for clinical diagnosis IA.⑶The combined detection of BG and GM can reduce false-positive rate ,improve the specificity and clinical diagnostic accuracy in the diagnosis of early IA.⑷GM and BG assay is a good indicator for antifungal therapy in IA patients . Monitoring of GM and BG concentration has been shown to be a a clinical index for appraising the therapeutic efficacy and prognosis of IA patients.⑸The Real-Time quantitative PCR techniques for IA was preliminary established. This method was useful for screening Aspergillus spp. in patients with high risk IFI factors and it has a good early diagnosis value of IA in hematologic diseases and malignant tumour patients.