gms | German Medical Science

Purpose: Invasive fungal infections (IFI) are life-threatening complications in immunocompromised patients. Due to improved diagnosis and treatment options, infections caused by Candida spp. and Aspergillus spp. are decreasing, while other rare fungal pathogens like Mucorales are on the rise. Mucormycosis is a very severe fungal infection and difficult to diagnose. Standard culture is insensitive and microscopy as well as radiological imaging unspecific. Most diagnostic procedures are invasive and thus cannot be used for screening. DNA detection from serum as described for Aspergillus or Mucorales (Yamakami et al., 1996; Millon et al., 2013) by polymerase chain reaction (PCR) is a rapid and very sensitive tool to identify pathogens and can improve diagnosis and treatment.

Methods: Forty-six serum specimens from 5 hematological patients were collected from 2008 to 2012 at Medical University Hospitals of Innsbruck and Wuerzburg. Sera were drawn twice weekly. According to EORTC/MSG criteria (DePauw et al., 2008), 3 patients were classified having proven/probable IFI either by histology or positive culture from bronchoalveolar lavage (BAL).

Sera were available up to 127 days before EORTC classification date. DNA from 1 ml of serum was manually extracted by using a commercially available kit (UltraSens Virus Kit, Qiagen). Extracts were analysed by real-time PCR assay (qPCR) amplifying the 18S rDNA region. This assay was modified using the primers published by Bialek et al. (2005) comprising in addition a Mucorales specific probe and degenerated nucleotides. A single round PCR cycling was used. Amplicons of positive PCR reactions were sequenced and aligned with reference sequences using BLAST analysis search.

Results: DNA extracts of serum samples were analysed by a Mucorales-specific qPCR assay within 4 hours. Four samples of the 3 probable/proven Mucorales IFI cases were positive. Sequencing revealed 2 different Mucorales strains (Lichtheimia sp., Rhizopus sp.) confirming pathogen identification by conventional methods. PCR from serum detected Mucorales DNA up to 3 days earlier than BAL or biopsy. Day “zero” was defined as time point of EORTC classification (positive BAL culture, biopsy). None of the unclassified control samples were positive.

Conclusion: Using qPCR allowed Mucorales specific detection of DNA in serum. This probe-based assay detecting a broad spectrum of Mucorales was used as an add-on tool to confirm mucormycosis in serum. Due to easy availability of serum even in severely ill patients, qPCR can be used as a screening tool in high-risk patients with suspected mould infection. The method used is faster and more sensitive than standard methods. It can help to distinguish between infections with Aspergillus and Mucorales, which is relevant for antimycotic regimens and patients’ outcome.