Development and evaluation of three independent real-time polymerase chain reaction (RT-PCR) assays for the detection and identification of Mucorales species

Ref ID: 19212

Author:

D. Goldenberger*, R. Frei, F. Schmitt, H. Einsele, J. Loeffler

Author address:

Basel, CH; Würzburg, DE

Full conference title:

23rd European Congress of Clinical Microbiology and
Infectious Diseases

Date: 27 April 2014

Abstract:

Objectives: Mucormycosis is an emerging invasive fungal infection in immunocompromised patients with aggressive course and high mortality rate. The infection is caused by a heterogeneous group of filamentous fungi classified in the order Mucorales with the principal organism Rhizopus arrhizus. Conventional diagnosis of mucormycosis often lacks sensitivity (culture) or specificity (histopathology) and phenotypic identification to the species level is time-consuming and needs a high level of laboratory skills. The use of molecular methods has the potential for a rapid and accurate detection and identification of Mucorales organisms.
Methods: We developed a molecular detection system for Mucorales consisting of 3 real-time PCR assays. Primers and probes were selected based on multiple sequence alignments of Mucorales and clinically relevant non-Mucorales fungal organisms. Two tests amplify parts of the 18S and 28S rRNA gene (fragment length approximately 180bp and 110bp, ), respectively) detecting Mucorales DNA within a few hours. The third assay amplifies the internal transcribed spacer 2 (ITS2) (approximately 450bp) and allows after direct sequencing fungal identification up to the species level. The 3 tests were applied to a panel of 21 Mucorales reference strains and to various non-Mucorales organisms evaluating sensitivity, specificity and efficiency of the PCR tests. The Mucorales-specific assays were also applied to clinical specimens containing etiologic Mucorales or non-Mucorales fungal organisms.
Results: Twenty of 21 reference strains were detected with the 3 Mucorales PCR tests. Among the 13 non-Mucorales fungal organisms such as different Aspergillus and Candida species, no amplification occurred in the 3 detection systems with the exception of Fusarium species in the ITS2 PCR. Based on 10-fold dilutions the lower detection limit of the 18S and the 28S rRNA PCR ranged from 1 to 10 genomes, and for the ITS2 PCR from 10 to approximately 500 genomes, depending on the fungal species. No contaminating DNA was detected in the reagents for PCR amplification or in the DNA extraction compounds performing repeated testing. Our detection system was applied to 11 clinical samples. Results of the Mucorales PCR assays were in high accordance to culture as well as panfungal PCR and sequencing.
Conclusions: There exists only few data on molecular diagnosis of Mucorales species. We provide a novel culture-independent detection algorithm for all clinically relevant Mucorales species. Its gradual use has the potential for a rapid, sensitive, and highly specific laboratory diagnosis of this devastating fungal infection.

Abstract Number: P1074

Conference Year: 2013

Link to conference website: http://registration.akm.ch/einsicht.php?XNABSTRACT_ID=162688&XNSPRACHE_ID=2&XNKONGRESS_ID=180&XNMASK

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