PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue

Abstract

Background: Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities.

Aims: To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples.

Methods: DNA of 52 blinded samples from five different centres was extracted and used as a template in two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of zygomycetes.

Results: Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological diagnosis of zygomycosis or aspergillosis, respectively. Aspergillus fumigatus DNA was amplified from one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax embedding. In addition, eight samples from clinically suspected fungal infections (without histopathological proof) were examined. The two PCR assays detected a concomitant infection with Absidia corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another two cases.

Conclusions: The two seminested PCR assays described here can support a histopathological diagnosis of mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal treatment.

Figure 1

Alignment of 18S ribosomal RNA sequences of zygomycetes amplified by inner primer set ZM1 and ZM3 based on a program according to Corpet (http://prodes.toulouse.inra.fr/multalin/multialin.html). The complimentary primer regions are indicated by black bars. Black letters indicate low or missing consensus in contrast to grey letters, which indicate high consensus at a given nucleotide position. Species and GenBank accession numbers used from top to bottom: Absidia corymbifera (AF113407), Rhizomucor pusillus (AF113434), Cokeromyces recurvatus (AF113416), Apophysomyces elegans (AF113412), Saksenaea vasiformis (AF113442), Mucor hiemalis (AF113428), Rhizopus oryzae, now named R arrhizus (AF113440), Cunninghamella bertholletiae (AF113421), and Syncephalastrum racemosum (X89437).