Objectives:
Mucormycosis are rare invasive fungal infections due to environmental molds from the order of Mucorales, whose incidence has increased during the last decade. These infections affect mainly immunosuppressed patients and are associated to a poor prognosis. The diagnosis, previously based solely on conventional mycological examination, has been improved by the detection of Mucorales circulating DNA with q-PCR. The yield of DNA extraction procedure remains a critical issue for Mucorales DNA detection in blood. Our objective was to compare currently available automated methods for DNA extraction from serum in patients with mucormycosis (MM).
Methods:
The Applied Biosystems® 7500 Real-Time PCR System, whose performance has been evaluated previously as similar to LightCycler® 480 Instrument, was used for DNA amplification. We compared three automated methods designed for circulating DNA extraction, i) MICROLAB STARlet instrument (Hamilton®) coupled with NucleoSpin® 96 DNA plasma kit (Macherey-Nagel), ii) AutoMag instrument coupled with MycoGENIE® Af DNA kit (Ademtech) and iii) MagNA Pure Compact Instrument coupled with Large Volume MagNA Pure Nucleic Acid Isolation kit (Roche).
Depending on the extraction method used, the volume of sample varies from 200 µL to 1 mL. The first method use silica membrane, whereas the two others use magnetic beads to extract DNA. Analysis was performed on both clinical (39 sera sequentially collected from 14 patients with MM) and spiked blood samples. Q-PCR was performed as previously reported by Millon et al. (1).
Results:
The yields of DNA extraction for all tested procedures were almost similar with a better limit of detection (LoD) for Roche and Hamilton/Macherey-Nagel methods (<1 fg/µl). The cost per DNA extraction test varies from 5€ (Ademtech) to 20€ (Roche), whereas the estimated time of extraction process was 1 to 4h with MICROLAB STARlet system. For clinical samples, q-PCR was positive in 8/14 patients with MM and for 7 of them q-PCR could have reduced the time to diagnosis of MM in comparison to mycological methods (until 17 days). All q-PCR positive results were concordant with mycological identification. Q-PCR also revealed a co-infection by 2 different Mucorales species. The sensitivity of q-PCR was 61.5% and most negative results were observed in patients who were tested after initiation of antifungal therapy and/or presented with cutaneous MM. The analytic performance of this method has been validated through external evaluation quality samples distributed by PHRC ModiMucor/FPCRI Muc Lab working group.
Conclusion:
In total, our study showed that MICROLAB STARlet system coupled with NucleoSpin® 96 DNA plasma kit (Macherey-Nagel) and AB7500 real-time PCR system represent a suitable method allowing to conciliate the technical, economic and clinical requirements for a reliable diagnosis of MM.

(1) Millon L. et al. Quantitative Polymerase Chain Reaction Detection of Circulating DNA in Serum for Early Diagnosis of Mucormycosis in Immunocompromised Patients. Clin Infect Dis. 2013

Evaluation of three automated methods for the extraction of circulating DNA: application to the diagnosis of mucormycosis by q-PCR