Molecular Detection and Identification of Zygomycetes Species from Paraffin-Embedded Tissues in a Murine Model of Disseminated Zygomycosis: a Collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) Evaluation

Abstract

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

“…Four studies reported the development of PCR assays to diagnose the pathogens causing this disease (229)(230)(231)(232). An early study focused on the identification of Rhizopus spp.…”

Diagnosis and treatment of mucormycosis in patients with hematological malignancies: guidelines from the 3rd European Conference on Infections in Leukemia (ECIL 3)

“…Fresh or frozen samples are preferred; however, based on recent inter-laboratory experimental and clinical data, formalin-fixed paraffin-embedded tissues may also be used. 26,27 Molecular identification of agents of mucormycosis can help to confirm diagnosis and identify the fungus to the genus and species level. Different techniques have been reported: DNA probes targeting 18S subunit, ITS1 sequencing after polymerase chain reaction (PCR) with pan-fungal primers, 18S-targeted semi-nested PCR and real-time PCR targeting cytochrome b gene.…”

Identification of fungal pathogens in a patient with acute myelogenic leukemia using a pathogen detection array technology

“…5,8,9,15,16 Although these molecular methods could detect species of a particular zygomycetes genus, they are limited by their detection range of pathogenic fungi, which is an important limiting factor in the context of diagnosis of invasive fungal infections. Further, the sensitivity of detection of fungal species by PCR or sequencing is very low in formalin fixed tissues compared to fresh tissues, 16 thus making diagnosis from fixed tissue section unreliable. More recently, to specifically identify species of fungi/zygomycetes, a combined approach of both morphologic and molecular testing methods have been explored when a histological diagnosis is not always possible.…”