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

Abstract

e Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by highresolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/ HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes.

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

“…in serum samples, can guide treatment effectively and improve patients' outcomes. Furthermore, other real-time assays detect either a single Mucorales genus (Bernal-Martínez et al, 2013;Lengerova et al, 2014) or only groups of Mucorales pathogens, missing other clinically relevant species (Millon et al, 2013;Hata et al, 2008). In detail, Voigt et al (1999) used taxon-specific primer pairs in an end point PCR which would require performing 13 different assays.…”

“…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.…”

Diagnosis of invasive fungal diseases in haematology and oncology: 2018 update of the recommendations of the infectious diseases working party of the German society for hematology and medical oncology (AGIHO)

“…Detection of Mucor ales remains challenging, but recently PCR methods have been successfully established for detection in BAL or serum . For detection of fungi (moulds and yeasts) in tissue samples, in‐house methods and commercial molecular methods have been found to be useful .…”

Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer?

“…45,56 A semi-nested PCR targeting 18S rDNA and sequencing was used in two studies to identify the genera of Mucorales DNA in formalin fixed, paraffin embedded tissue. Conventional and real-time PCR techniques have been reported with most studies targeting the multi-copy 18S and 28S rDNA genes or ITS region, followed by sequencing.…”