RT-PCR DETECTION OF ASPERGILLUS FUMIGATUS FROM FROZEN WHOLE BLOOD IN A GUINEA PIG MODEL OF INVASIVE PULMONARY ASPERGILLOSIS (IPA)

M. Herrera1*, L. Najvar1, R. Bocanegra1, P. Donnelly2, J. Loeffler3, L. White4, B. Wickes1, W.Kirkpatrick1, N. Wiederhold1

Author address: 

1The University of Texas Health Science Center at San Antonio, USA 2Nijmegen Medical Centre, The Netherlands 3Wuerzburg University, Germany 4NPHS Microbiology Cardiff, UK

Abstract: 

Purpose: Early detection of clinical invasive aspergillosis can have a positive impact on patient outcome. Whole blood samples may be obtained from patients in a relatively unobtrusive manner, yet little is known about the utility of using frozen whole blood as a diagnostic sample in invasive aspergillosis. We examined the utility of PCR methodology to detect early signal of infection from Aspergillus fumigatus in whole blood from our guinea pig model of IPA Methods: Male Hartley guinea pigs received cortisone acetate and cyclophosphamide 2 days before and 3 days after infection for immunosuppression. The guinea pigs received an inhalational challenge of 4x108 colony forming units (CFU) Aspergillus fumigatus AF293 for 1 hour in an acrylic chamber. Whole blood samples were obtained from anaesthetized guinea pigs at 1 hour, 3 and 5 days post-infection. Blood was collected into vacutainer tubes containing EDTA and rocked for 5 minutes. Blood was pooled by day of infection, aliquoted, and frozen. DNA was extracted from 3 mL of blood. The EAPCRI protocol for whole blood extraction consisting of several RBC and WBC lysis steps was modified by adding a Dnase step before bead beating and an automated final step. RT-PCR was performed in ABI/PRISM7900 Sequence Detector System to detect binding to an 18S rDNA MGBFAM probe. (1,3)-β -D-glucan (BG) was assessed with the commercial Fungitell kit and galactomannan (GM) was measured using the Platelia Aspergillus kit. Lung Aspergillus burden was quantified by CFU. Results: PCR of the negative controls showed low level, non-specific detection of signal. CFU from lung samples at 1 hour and days 3 and 5 post-infection indicated consistent infection had occurred, which was substantiated by RT-PCR data. PCR of samples obtained at 1 hour and days 3 and 5 showed low levels of PCR product with Ct values of 35, 32 and 33 respectively from blood. BG and GM remained below the level of detection for signal from Aspergillus at these time points. Conclusions: RT-PCR of frozen blood samples from our guinea pig model showed rapid, specific detection of Aspergillus conidia at 1 hour, and detected Aspergillus DNA at later time points, even when other available methods failed to detect Aspergillus in whole blood from matched time points. PCR signal in each case may have been decreased by pooling. Effective extraction of DNA from whole blood, followed by RT-PCR may be a useful means of detecting IPA in its early stages.
2010

abstract No: 

144

Full conference title: 

4th Advances Against Aspergillosis
    • AAA 4th (2010)