Digital Droplet PCR: a Promising Method for Rare Events Detection for Invasive Aspergillosis Diagnosis

Ref ID: 19599

Author:

B Greiderer-Kleinlercher1*, E Fréalle2, V Coiteux3, S Perkhofer4

Author address:

1Biomedical Science, University of Applied Sciences Tyrol, Innsbruck, Austria
2Parasitology-Mycology Laboratory, Lille University Hospital Center & Pasteur Institute of Lille, Lille
Cedex, France
3Haematology Service, Lille University Hospital Cen

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose:
Molecular diagnosis of invasive aspergillosis (IA) has still no contenting sensitivity for clinical
routine diagnosis despite vigorous efforts of standardisation. Real-time PCR is a fast method for
identifying fungal load in immunocompromised patients. However, the detection of small amounts
of fungal DNA in clinical samples makes the diagnosis difficult. These small amounts of fungal
DNA may be an explanation for the non-adequate diagnostic sensitivity and high amounts of human
genomic DNA and may lead to unspecific amplifications. Digital droplet PCR (ddPCR) could be
helpful to improve sensitivity and specificity of this molecular technique for diagnosis of IA. The
purpose of this study was to test clinical samples by use of ddPCR in comparison to real-time PCR
results.
Methods:
For detection limit of the real-time PCR fungal DNA was extracted from whole blood samples
from healthy volunteers spiked with A. fumigatus conidial concentrations ranging from 102 to
106 conidia/ml. Human whole blood samples without fungi served as a negative control. DNA of
serum samples from patients with probable IA (n=5) and serum samples from patients without IA
(n=5) were extracted and tested with ddPCR and real-time PCR. The ddPCR technique separates
the sample into a large number of droplets (20,000 droplets/20 μl of reaction volume) and a single
amplification is carried out in each droplet individually.
Results:
The absolute detection limit was 1 conidia/μl (Cq 37) for real-time PCR and 2 events for ddPCR.
The cut off level based on negative control results were Cq > 40 for real-time PCR and < 2 events for ddPCR. For real time PCR none of the positive serum samples was detected as positive sample (Cq > 40 for all samples) and one sample out of five negative IA patient samples was detected
positive (Cq 39) due to the detection limit. With ddPCR three out of the five positive serum samples
were detected as positive with 5.6, 2.9 and 4.4 events. All other samples including negative patient
serum samples were detected as negative (0 to 1.5 events).
Conclusion:
None of the clinical samples from patients with probable IA gave positive results by use of real time
PCR. One out of the five negative serum samples was detected positive. As fungal burden in the
bloodstream of patients is low, a high degree of analytical sensitivity and specificity is needed to
avoid false negative and false positive results.
With ddPCR three out of five serum samples of patients with probable IA were tested positive and all
negative samples were tested negative. One of the potential advantages of ddPCR versus real-time
PCR is the capability to obtain absolute quantification without external references and robustness to
variations in PCR efficiency leading to more precise results.
Due to the fact that serum samples from patients with probable IA were taken early in the course of
IA, the ddPCR showed excellent results at an early stage of disease. Using this new PCR technique
is an innovative approach and represents a good tool for detection of IA by enhancing diagnostic
capabilities.

Abstract Number: 124

Conference Year: 2014

Link to conference website: http://www.AAA2014.org

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