Detection of a Molecular Biomarker for Zygomycetes by Quantitative PCR Assays of Plasma, Bronchoalveolar Lavage, and Lung Tissue in a Rabbit Model of Experimental Pulmonary Zygomycosis

Abstract

We developed two real-time quantitative PCR (qPCR) assays, targeting the 28S rRNA gene, for the diagnosis of zygomycosis caused by the most common, clinically significant Zygomycetes. The amplicons of the first qPCR assay (qPCR-1) from Rhizopus, Mucor, and Rhizomucor species were distinguished through melt curve analysis. The second qPCR assay (qPCR-2) detected Cunninghamella species using a different primer/probe set. For both assays, the analytic sensitivity for the detection of hyphal elements from germinating sporangiospores in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates from rabbits was 1 to 10 sporangiospores/ml. Four unique and clinically applicable models of invasive pulmonary zygomycosis served as surrogates of human infections, facilitating the validation of these assays for potential diagnostic utility. For qPCR-1, 5 of 98 infarcted lung specimens were positive by qPCR and negative by quantitative culture (qCx). None were qCx positive only. Among 23 BAL fluid samples, all were positive by qPCR, while 22 were positive by qCx. qPCR-1 detected Rhizopus and Mucor DNA in 20 (39%) of 51 serial plasma samples as early as day 1 postinoculation. Similar properties were observed for qPCR-2, which showed greater sensitivity than qCx for BAL fluid (100% versus 67%; P ‫؍‬ 0.04; n ‫؍‬ 15). The assay detected Cunninghamella DNA in 18 (58%) of 31 serial plasma samples as early as day 1 postinoculation. These qPCR assays are sensitive and specific for the detection of Rhizopus, Mucor, Rhizomucor, and Cunninghamella species and can be used for the study and detection of infections caused by these life-threatening pathogens.

Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species

“…There are reports using different sources for detecting Mucorales DNA from fresh or formalin-fixed paraffin-embedded tissue (FF-PET), bronchoalveolar lavages or serum samples (Bialek et al, 2005;Rickerts et al, 2006;Kasai et al, 2008;Millon et al, 2013). Also, different methods to detect Mucorales by PCR have been described, including conventional, semi-nested and real-time (DNA intercalating dyes, molecular-beacon or hydrolysis probe-based) PCR (Millon et al, 2013;Bialek et al, 2005;Voigt et al, 1999;Hata et al, 2008;Bernal-Martínez et al, 2013;Hrncirova et al, 2010;Lengerova et al, 2014); each suffers from different disadvantages such as high turnaround time, vulnerability to contamination or limited detection of selected species or genera.…”

Rapid Detection and Identification of Mucormycetes in Bronchoalveolar Lavage Samples from Immunocompromised Patients with Pulmonary Infiltrates by Use of High-Resolution Melt Analysis

“…Until now, only a limited number of mucormycete-specific PCR methods have been published. Moreover, only some of them allow the quantification of the fungal DNA load in samples (6)(7)(8)(9)(10), which can be important to differentiate between a real infection and contamination/colonization of the sample/patient.…”

Molecular Methods To Improve Diagnosis and Identification of Mucormycosis

“…Detection of fungal DNA in tissue samples by PCR is a novel non-culture-based method that may allow improved diagnosis of mucormycosis (2,4,7,9,10). In particular, PCR with sequencing of the 18S ribosomal DNA of Mucorales in order to diagnose mucormycosis and identify the infecting species in paraffin-embedded tissue samples in clinical cases of invasive fungal infection has been described (2, 9, 10).…”