Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes.

Rita Caramalho,Cornelia Lass-Flörl,Ana Alastruey-Izquierdo,Lisa Madl,Thomas Larentis,Ricardo Araujo,Cornelia Speth,Michaela Lackner,Johannes Pallua,Katharina Rosam,Günter Rambach

doi:10.3390/jof5040098

Abstract

Mucormycosis infections are infrequent yet aggressive and serious fungal infections. Early diagnosis of mucormycosis and its discrimination from other fungal infections is required for targeted treatment and more favorable patient outcomes. The majority of the molecular assays use 18 S rDNA. In the current study, we aimed to explore the potential of the mitochondrial rnl (encoding for large-subunit-ribosomal-RNA) gene as a novel molecular marker suitable for research and diagnostics. Rnl was evaluated as a marker for: (1) the Mucorales family, (2) species identification (Rhizopus arrhizus, R. microsporus, Mucor circinelloides, and Lichtheimia species complexes), (3) growth stage, and (4) quantification. Sensitivity, specificity, discriminatory power, the limit of detection (LoD), and cross-reactivity were evaluated. Assays were tested using pure cultures, spiked clinical samples, murine organs, and human paraffin-embedded-tissue (FFPE) samples. Mitochondrial markers were found to be superior to nuclear markers for degraded samples. Rnl outperformed the UMD universal® (Molyzm) marker in FFPE (71.5% positive samples versus 50%). Spiked blood samples highlighted the potential of rnl as a pan-Mucorales screening test. Fungal burden was reproducibly quantified in murine organs using standard curves. Identification of pure cultures gave a perfect (100%) correlation with the detected internal transcribed spacer (ITS) sequence. In conclusion, mitochondrial genes, such as rnl, provide an alternative to the nuclear 18 S rDNA genes and deserve further evaluation.

Highlights

The order Mucorales is assigned to the subphylum Mucoromycotina, one of the most ancient, early divergent groups of fungi [1]
The qPCR limit of detection (LoD) of the ID tool was tested using clinical sample mimics based on DNA samples from
Patients suffering from mucormycosis exhibit signs and symptoms similar to patients suffering from other invasive fungal infections, such as aspergillosis [26,27]

Summary

Introduction

The order Mucorales is assigned to the subphylum Mucoromycotina, one of the most ancient, early divergent groups of fungi [1]. Mucorales have been identified using primarily histological and standard mycological methods, as well as DNA sequencing of the internal transcribed spacer (ITS) region [6] These gold standard approaches are time-consuming and can be performed only for well-established infections. To test for potential application in routine diagnostics, the extraction method used instead was the MolYsisTM Basic kit (Molzym GmbH and Co. KG, Bremen, Germany) and the rnl real-time. Rnl Real-Time PCR-HRM Evaluation of Assay Performance in Clinical Sample Mimics. Aliquots of 200μL EDTA whole blood collected from healthy volunteer donors were inoculated with a ten-fold dilution series of 1 × 107 –1 × 102 R. arrhizus (KOG-D3). DNA extraction was performed for all samples, including non-spiked human blood as negative controls. The MolYsisTM Basic kit was used to recover the microbial lysate depleted of human DNA, followed by the DNeasy® Blood and Tissue Kit (QIAGEN GmbH)) according to the manufacturer’s instructions

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