Purpose: Invasive diseases by Aspergillus spp have been recorded as an important cause of morbidity and mortality in patients under immunosuppressive therapy. Early diagnosis is essential for successful intervention due to high severity, poor response to therapy and resistance to antifungal drugs in some cases. Diagnosis of proved aspergillosis is more frequently established by histopathology and/or culture from sterile sites due to the low sensitivity of the blood culture and false positive results of galactomannan ELISA ASSAY. In recent years, molecular methods represent an alternative for early diagnosis considering their high sensitivity and specificity. Here we describe the sensitivity of Real Time PCR to detect DNA from A. fumigatus and its specificity with other fungi, bacteria and viruses, and human DNA. Methods: Test of sensitivity: extraction of DNA from culture of A. fumigatus by QIAmp kit (Qiagen); test for specificity: extracting DNA from culture of A. flavus, A. niger, A. terreus, Candida sp (tropicalis, parapsilosis, glabrata, albicans, dubliniensis and krusei) and human DNA by QIAmp kit (Qiagen). Extraction of DNA from other fungi (H. capsulatum, C. neoformans var neoformans and var gatti, Scedosporium sp, Fusarium sp, P. brasiliensis, F. pedrosoi, N. asteroides and N. brasiliensis), bacteria (S. flexineri, S. aureus, S. enteritidis, E. coli, T. pallidum, B. garinii, B. burgdorferi, S. typhimurium, L. copenhageni and P. aeruginosa) and virus (CMV, HSV1 and 2) performed according to Sambrook et al, 1989. PCR primers were designed based on the rRNA gene sequence and the amplified products were identified by the Melting temperature and confirmed by agarose gel. Results: Real Time PCR sensitivity was 5x10-13 g of DNA, equivalent to 1 cell. There was no amplification of other products in addition to the specific DNA of other fungi, bacteria and viruses and human DNA. Conclusions: Real Time PCR showed to be sensitive and specific for the diagnosis suggesting the applicability in the early diagnosis.
Full conference title:
4th Advances Against Aspergillosis
- AAA 4th (2010)