Molecular Diagnosis of Invasive Aspergillosis and Detection of Azole Resistance by a Newly Commercialized PCR Kit

Abstract

is the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance in is worrisome. The aim of this study was to validate the new MycoGENIE real-time PCR kit and to evaluate its performance on clinical samples for the detection of and its azole resistance. This multiplex assay detects DNA from the species complex by targeting the multicopy 28S rRNA gene and specific TR and L98H mutations in the single-copy-number gene of The specificity of mutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinical isolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for the 28S rRNA gene and 6 copies for the gene harboring the TR and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection of DNA and azole resistance due to TR and L98H mutations in clinical samples.

PCR‐based detection of Aspergillus fumigatus and absence of azole resistance due to TR34/L98H in a french multicenter cohort of 137 patients with fungal rhinosinusitis

“…Samples negative for both A. fumigatus , TR 34 and L98H and also negative (or with a cycling threshold >35 cycles) for the internal control, were considered uninterpretable due to PCR inhibition. Performances of this PCR assay in terms of sensitivity/specificity (limits of detection of 6 and below 1 copy per genome for the TR 34 and L98H mutations and A. fumigatus respectively) had been previously confirmed both on clinical isolates and on a large collection of clinical samples . The following clinical and mycological data were collected: age, sex, clinical form (type and location) and antifungal therapy for FRS, species identification when available (for culture‐positive samples).…”

Evaluation of Two Commercial Real-Time PCR Kits for Aspergillus DNA Detection in Bronchoalveolar Lavage Fluid in Patients with Invasive Pulmonary Aspergillosis

“…21,32,33 The MycoGENIE A. fumigatus study showed 92.9% sensitivity (versus culture) and 90.1% specificity. 34 Variation in sensitivity may be explained by various PCR targets, including 28S rRNA (AsperGenius, MycoGENIE), 18S rRNA (MycAssay), and ITS1 (A. fumigatus Bio-Evolution), as well as by different methods of DNA extraction. Although Wheat et al 2 described equivalent performances for manual and automated DNA extraction processes, a better sensitivity was observed by others with automated extraction.…”

“…In our study, no mutations were detected, confirming the MICs previously determined by Etest and consistent with the first MycoGE-NIE A. fumigatus study. 34 Until now, only rare cases of azole-resistant strains have been isolated in Eastern France, allowing us to use the MIC determination to test sensitivity to many azoles or other drugs, such as amphotericin B.…”

Non-culture based assays for the detection of fungal pathogens

“…On the other hand, MycoGENIE 1 showed theSn of 92.9% and Sp of 92.9% for detection of Aspergillus DNA in respiratory samples, while in serum samples, the Sn and Sp were 100% and 84.6%, respectively. It is important to emphasize that all isolates harboring the TR34/L98H mutations were accurately detected [108].…”