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

Abstract

PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n517). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n510). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.

Diagnosis of invasive mold diseases in patients with hematological malignancies using Aspergillus, Mucorales, and panfungal PCR in BAL

“…In our cohort, the specificity of Aspergillus and panfungal PCR was similar or better to previous reports with 36%‐94% and 67%‐86% in BAL‐fluid, respectively, while their sensitivities were much lower . There are several potential explanations for the poor sensitivities of our PCR in the BAL‐fluid.…”

“…We found that the In our cohort, the specificity of Aspergillus and panfungal PCR was similar or better to previous reports with 36%-94% [19][20][21] and were much lower. 5,7,9,20,21,23,24 There are several potential explanations for the poor sensitivities of our PCR in the BAL-fluid. First, all hematological patients at risk for fungal infection also comprising those without specific infiltrates were included in this study, which could have led to a negative selection of patients without suspected IMD.…”

“…DNA extraction from 200 μL BAL specimens was performed with the commercial available EZ1 DNA Tissue kit (Qiagen ® , Hilden, Germany) according the manufacturer's instructions with an additional heating step of 95°C for 12 minutes as analyzed in former studies before purification with EZ1 Advanced apparatus . Briefly, 200 μL BAL‐fluid was centrifuged at 12 000 g during 5 minutes.…”

“…The final mix containing 30 μL volume consists of each 900 nM primer Asp‐1 and Asp‐2, 200 nmol/L of the hydrolysis probe p‐panAsp, 15 μL GenExpression mastermix, 3 μL Exo IPC Mix, 0.6 μL Exo IPC DNA (Internal positive control) (Applied Biosystems, Foster City, CA, USA), nuclease‐free water (Qiagen) and 5 μL extracted DNA. The Mucorales real‐time PCR was performed as recently described, targeting parts of the 18S and 28S ribosomal fungal DNA, using in each case 2 primers and 1 probe as well as the internal control system used in the Aspergillus PCR. For Aspergillus and Mucorales , real‐time PCR tests a total of 45 cycles were performed.…”

“…These molds are increasing and have important implications regarding treatment and mortality . We recently developed an in‐house panfungal PCR and a real‐time Mucorales PCR achieving sensitivities of 69 to 100% and 86 to 90% and specificities of 63% and 100% in biopsies, respectively . Because of these encouraging results, we evaluated the performance of these in‐house PCRs additional to Aspergillus PCR in 167 hematologic patients at risk for IMD undergoing BAL.…”