BACKGROUND: The diagnosis of invasive aspergillosis (IA) remains challenging and frequently is not made until after death. Histopathological examination remains central to confirmation of diagnosis but often requires invasive procedures to obtain tissue for the examination. Detection of aspergillus DNA by quantitative PCR (qPCR) offers the potential for earlier diagnosis due to higher sensitivity, but PCR in clinical use is poorly reproducible, with different centres reporting variable results and often using different extraction and analytical methods. AIMS: To optimise the performance of aspergillus PCR as a diagnostic modality. METHODS: A rat inhalation model of invasive aspergillosis was used to optimise the methodology of diagnostic aspergillus PCR. Infected animals were terminally bled at 4 days post-infection; samples of EDTA blood, serum and the residual clot were pooled for subsequent analysis. DNA was extracted from each fraction using a variety of methods and an optimised qPCR reaction using an Aspergillus fumigatus primer set performed. RESULTS: Significantly more aspergillus DNA was detected from the clot than EDTA and serum samples. Enzymatic and mechanical pretreatment reduced the yield of fungal DNA. There was some evidence that the average Ct values were greater for the EZ1 BioRobot than the MagNA Pure automated extractor, but this did not reach statistical significance at the 5% level (p = 0.078). CONCLUSIONS: Automated extraction from the clot present in a blood sample will increase DNA yield and improve the diagnostic sensitivity of the test.
BACKGROUND: The diagnosis of invasive aspergillosis (IA) remains challenging and frequently is not made until after death. Histopathological examination remains central to confirmation of diagnosis but often requires invasive procedures to obtain tissue for the examination. Detection of aspergillus DNA by quantitative PCR (qPCR) offers the potential for earlier diagnosis due to higher sensitivity, but PCR in clinical use is poorly reproducible, with different centres reporting variable results and often using different extraction and analytical methods. AIMS: To optimise the performance of aspergillus PCR as a diagnostic modality. METHODS: A rat inhalation model of invasive aspergillosis was used to optimise the methodology of diagnostic aspergillus PCR. Infected animals were terminally bled at 4 days post-infection; samples of EDTA blood, serum and the residual clot were pooled for subsequent analysis. DNA was extracted from each fraction using a variety of methods and an optimised qPCR reaction using an Aspergillus fumigatus primer set performed. RESULTS: Significantly more aspergillus DNA was detected from the clot than EDTA and serum samples. Enzymatic and mechanical pretreatment reduced the yield of fungal DNA. There was some evidence that the average Ct values were greater for the EZ1 BioRobot than the MagNA Pure automated extractor, but this did not reach statistical significance at the 5% level (p = 0.078). CONCLUSIONS: Automated extraction from the clot present in a blood sample will increase DNA yield and improve the diagnostic sensitivity of the test.
Authors: Jan Springer; Hannes Schloßnagel; Werner Heinz; Thomas Doedt; Rainer Soeller; Hermann Einsele; Juergen Loeffler Journal: J Clin Microbiol Date: 2012-05-16 Impact factor: 5.948
Authors: Stephen A Krawetz; Peter R Casson; Michael P Diamond; Heping Zhang; Richard S Legro; William D Schlaff; Christos Coutifaris; Robert G Brzyski; Gregory M Christman; Nanette Santoro; Esther Eisenberg Journal: Syst Biol Reprod Med Date: 2011-09-07 Impact factor: 3.061
Authors: Juergen Loeffler; Carlo Mengoli; Jan Springer; Stéphane Bretagne; Manuel Cuenca-Estrella; Lena Klingspor; Katrien Lagrou; Willem J G Melchers; C Oliver Morton; Rosemary A Barnes; J Peter Donnelly; P Lewis White Journal: J Clin Microbiol Date: 2015-06-17 Impact factor: 5.948
Authors: P Lewis White; Rosemary A Barnes; Jan Springer; Lena Klingspor; Manuel Cuenca-Estrella; C Oliver Morton; Katrien Lagrou; Stéphane Bretagne; Willem J G Melchers; Carlo Mengoli; J Peter Donnelly; Werner J Heinz; Juergen Loeffler Journal: J Clin Microbiol Date: 2015-06-17 Impact factor: 5.948