Determination of the Ideal Specimen for the Detection of Aspergillus During Invasive Aspergillosis

Ref ID: 19476

Author:

B Greiderer-Kleinlercher1*, E Fréalle2, S Perkhofer3

Author address:

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

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose:
Invasive aspergillosis (IA) is increasingly recognised in immunocompromised patients and still
associated with a high mortality rate. The diagnosis of IA is still problematic. Often, different blood
specimens are used for PCR diagnosis including serum, plasma or EDTA blood. However, there is
no consensus on the blood sample type that should be used for the detection of Aspergillus spp.
The aim of this study was to systematically investigate the suitability of different blood specimens
for diagnostic analysis. Therefore, we spiked blood samples from healthy donors with Aspergillus
fumigatus conidia and hyphae at various concentrations. Then we examined fungal growth in the
different samples by solid- and fluid culture, fluorescence- and light-microscopy.
Methods:
Native, EDTA and citrated blood samples were taken from healthy volunteers. The samples were
spiked with A. fumigatus (102, 103, 104, 105 and 106 conidia/ml), gently mixed and incubated for
30 min at 37°C followed by selective centrifugation to gain different blood compartments namely
EDTA whole blood, citrated blood pellet, platelet rich plasma (PRP), serum, serum blood, solid and
lysed clot.
Same experiments were performed with hyphae. Lysis of the clot was performed with tissue lysis
buffer and proteinase K incubation for 60 min at 55°C. Then 10 μl and 100 μl of each specimen were
plated on solid sabouraud agar and poured into liquid Sabouraud and incubated for 24h, 48h and 72h
at 37°C. Fungal growth was determined by counting colony forming units (cfu) and monitoring the
confluence.
Untreated A. fumigatus served as positive control. Additionally, calcofluor-white fluorescenceand
light- microscopy out of the specimens were performed to detect conidia or hyphae in the
compartments.
Results:
Solid growth culure experiments at 106 cfu/ml showed fungal growth in EDTA (42%), PRP (30%),
citrated blood (50%) and the clot (50%) after 24h of incubation, whereas serum samples only showed
7% growth. After 72h all specimens except serum (40%) showed 100% fungal growth.
At 102 cfu/ml no growth for serum and PRP could be detected after 72h, whereas the clot (78%),
EDTA (60%), citrate blood (75%) and serum blood (49%) showed growth. Same results were found
with hyphae. With light and fluorescence microscopy more conidial or hyphal structures could be
found in the clot in comparison to all other specimens.
Conclusion:
In routine fungal diagnostic different blood specimens are used for Aspergillus identification. At low
fungal concentration we found strongest fungal growth in the clot and citrated blood and no growth
in serum samples. That absence or low detection of conidia or hyphae in serum is consistent with the
pelleting of fungal elements probably resulting from centrifugation. The increased incidence of fungal conidia and hyphal structures found by microscopy and growth
experiments might be an indication that highest fungal load is found in the clot also at low fungal
concentrations. A reason for this finding could be that platelets interact with Aspergillus species by
adhering and covering conidia. These finding has to be proven with real-time PCR and might help to gain better results and improve standardization for the diagnosis of IA.

Abstract Number: 4

Conference Year: 2014

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

New link: NULL

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