Don’t throw your blood clot’s away: Use of Blood Clot may improve sensitivity of PCR Diagnosis in Invasive Aspergillosis

Abstract

Aims: The diagnosis of invasive aspergillosis (IA) remains challenging and frequently is not made until after death. Histopathological examination remains central to conformation 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. We aimed to optimise the performance of aspergillus PCR as a diagnostic modality.

Methods: We have used a rat inhalation model of invasive aspergillosis to optimise the methodology of diagnostic aspergillus PCR. Infected animals were terminally bled at 4 days post infection and the 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 A. fumigatus primer set performed.

Results: Significantly more aspergillus DNA was detected from the clot than EDTA and serum samples. Enzymatic and mechanical pre-treatment reduce 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.