Introduction
Mucormycosis is a rare invasive fungal infection causes by Mucorales species and generally affects severely immunocompromised individuals. Mucormycosis is associated with a high mortality rate and prevalence is underestimated due to the limited diagnostic options. Correct and fast identification of mucorales infection is essential to start  appropriate antifungal therapy.
Due to this limitation in current diagnostics, PathoNostics developed a real-time PCR for the detection of the clinical most relevant Mucorales species. Several parameters were determined including the analytical specificity, inclusivity and limit of detection (LoD). The clinical sensitivity was examined on a ring trial and a set of clinical samples.

Material and methods
The real-time PCR assay (MucorGenius, PathoNostics) is designed to detect the 28S multi-copy gene in the most prevalent etiological agents of Mucormycosis including Rhizopus spp., Mucor spp., Rhizomucor spp., Lichtheimia spp. and Cunninghamella spp. The real-time PCR assay consists of a set op primers and probes enabling detection of Mucorales species together with an internal control. 
The analytical specificity and inclusivity was determined by testing several Mucorales strains, other fungi, yeast and bacteria strains which may be present in the respiratory tract. In addition,  the LoD was determined for all Mucorales species by testing dilution series of genomic DNA. Quantification was enabled by droplet digital PCR (ddPCR).
Clinical sensitivity was determined using eleven Mucormycosis-suspected BAL samples which were tested by culture and in-house PCR. Finally the MucorGenius was also evaluated on the Mucorales panel (6 samples) obtained from the Fungal PCR Initiative (FPCRI).   

Results
The MucoGenius PCR was able to detect Rhizopus oryzae, Mucor racemosus, Rhizomucor pusillis, Lichtheimia corymbifera and Cunninghamella bertholletiae with a LoD in the range of 2-4 for all strains. No cross-reactivity was observed with other fungi, yeast or bacterial respiratory strains. 
From the eleven BAL samples, eight were positive for Mucorales species with the MucoGenius PCR whereas culture and the in-house PCR assay only resulted in 5 positive samples. 
Out of six FPCRI samples, 5 were correctly identified as Mucorales species with the MucorGenius PCR. This was in agreement with the FPCRI data. 

Conclusions 
The MucorGenius real-time PCR from PathoNostics can detect the most clinically relevant Mucorales species. According to the initial clinical evaluation, the new MucorGenius PCR showed good performance on a small set of clinical samples and had a 100% score on the FPCRI panel.

2 / Development of the first commercial real-time PCR assay for the detection of Mucorales species