Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group

Abstract

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77–100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Microbiological and Molecular Diagnosis of Mucormycosis: From Old to New

“…Within the laboratories using the EPA assay, the only technical parameter, which demonstrated a significant impact on the result, was the qPCR platform, with Rotor-Gene achieving the lowest Cq values. Overall, the study demonstrated very good concordance in results throughout the laboratories and methods used [ 19 ].…”

“…Despite these difficulties, the Fungal PCR Initiative (FPCRI, www.fpcri.eu), working group of the ISHAM, aims to include PCR diagnostics in the EORTC/MSG criteria for fungal infections, which they achieved for Aspergillus PCR; Candida, Mucorales, and Pneumocystis PCR must follow. In this context, an inter-laboratory study on two simulated serum panels (spiked with Mucorales DNA) was performed in 2017-2018, with 23 European laboratories participating [19]. The study evaluated the reproducibility of different DNA extraction and qPCR methods.…”

“…Further, Scharf et al (2020) observed that for Aspergillus , bead beating could significantly increase the amount of DNA extracted from serum and respiratory samples [ 18 ]. However, an inter-laboratory study displayed no significant effect of the method of extraction of Mucorales DNA from the serum on the diagnostic sensitivity [ 19 ].…”

Diagnosis of Breakthrough Fungal Infections in the Clinical Mycology Laboratory: An ECMM Consensus Statement

“…Aspergillus PCR has demonstrated high sensitivity and high negative predictive value in severely immunocompromised patients in settings where antifungal prophylaxis is not used [78][79][80][81]. PCR is also an important diagnostic test for mucormycosis [82,83]. However, the performance of PCR from blood is impacted significantly in patients receiving mold-active antifungal agents as shown in a recent review [8,55,81,84].…”

Serial Detection of Circulating Mucorales DNA in Invasive Mucormycosis: A Retrospective Multicenter Evaluation

“…The exact primer sequences are kept confidential by the manufacturer. Recently, the Fungal PCR Initiative (FPCRI) and the ModiMucor study group compared the performance of these assays using spiked serum in a ring test, and found the MucorGenius ® and Millon assays to have the highest positivity rates with the lowest Cq values [19].…”