gms | German Medical Science

Invasive Aspergillosis (IA) is a life threatening infection, which particularly affects immunocompromized patients with hematologic malignancies, those receiving solid organ transplants or undergoing hematopoietic stem cell transplantation. A delayed or inappropriate treatment of this disease is associated with an inverse outcome of IA. Thus, early and reliable diagnosis is a key element for efficient patient management.

Recent studies suggested that combining Aspergillus circulating DNA detection by PCR in serum to Galactomannan (GM) assay as screening methods is an effective strategy for monitoring patients at risk. This approach involves a specimen easy-to-treat and results in early IA detection, recovery of underdiagnosed cases, reduced use of antifungals and consistent clinical performance scores. However, lack of standardization has hampered inclusion of PCR results into the EORTC/MSG criteria for patient classification. The European Aspergillus PCR Initiative (EAPCRI) has pointed out sample preparation and false positivity among the main limiting factors towards standardization.

The present work highlights an effort towards standardizing free fungal genomic DNA (gDNA) detection in serum specimens. Aspergillus fumigatus is the first targeted species as it causes the majority of reported IA cases. The bioMérieux easyMAG® system enabled optimizing a modular semi- automated procedure that includes sample preparation (pretreatment/lysis and fungal gDNA extraction/purification) from spiked healthy donor serum and subsequent detection of fungal DNA using Real Time PCR and a single copy gene as a target. Analytical performance studies showed an apparent Limit of Detection of 10<LoD<5 genomes/ml serum, no cross-reactivity but against Neosartorya fischeri gDNA, 0% false positivity and no interference by human DNA and/or serum components. On the top of that, an internal control has been integrated in the prototype in a duplex PCR setting.

The built prototype includes an internal control and fills the main required sensitivity and specificity standards set by EAPRCI (designated threshold of 10 genomes/mL serum, no false positives). Cross-reactivity with N. fischeri is not considered as a major issue as the corresponding IA reported cases are rare. Focus is now on clinical validity evaluation of this tool, i.e., clinical sample testing from retrospective collections.

Note: The authors M. Lackner, J. Springer, C. Lass-Flörl, J. Löffler, H. Einsele, J. Yugueros-Marcos, and A. Apostolaki belong to the aspBIOmics consortium funded by the ERA-NET Pathogenomics Action.