Performance of Molecular Approaches for Aspergillus Detection and Azole Resistance Surveillance in Cystic Fibrosis

Abstract

Aspergillus fumigatus triazole resistance is an emerging concern for treating chronically infected/colonized patients. This study sought to evaluate the performance of PCR assays to detect Aspergillus fungi together with azole resistance in sputum samples from cystic fibrosis (CF) patients. In total, 119 sputum samples from 87 CF patients were prospectively processed for Aspergillus detection by means of mycological culture and four qPCR assays, 2 in-house methods and two commercial multiplex real-time PCR assays simultaneously detecting Aspergillus and the most relevant cyp51A gene mutations (MycoGENIE ® and AsperGenius ® ). Azole susceptibility of A. fumigatus isolates was assessed using Etest ® method and cyp51A gene mutation were characterized by sequencing. The overall rate of Aspergillus detection with the four qPCR assays ranged from 47.9 to 57.1%, contrasting with 42/119 (35.3%) positive cultures with A. fumigatus . The high sensitivity of PCR on sputum could then contribute to more effective grading of Aspergillus disease in CF patients. Five out of 41 isolated strains (12.2%) exhibited azole-resistant MIC patterns, three of which harbored cyp51A mutations and only 1/3 with the sequence TR 34 /L98H. Combined with culture, PCR assay achieved high sensitivity Aspergillus screening in CF samples. However, cyp51A targeting was only moderately effective for azole resistance monitoring, while Aspergillus resistance remains of great concern.

Use of Aspergillus fumigatus real-time PCR in bronchoalveolar lavage samples (BAL) for diagnosis of invasive aspergillosis, including azole-resistant cases, in high risk haematology patients: the need for a combined use with galactomannan

“…16 This observation is indirectly confirmed by cohorts reporting higher performance of PCR, both in BAL and serum, in those receiving mould active agents. [21][22][23] Also in our study, the rate of qPCR positivity in GM positive BAL samples was higher form patients receiving mould active drugs compared to those not treated. Better specification which portion of BAL fluid should be used might be warranted, since by testing supernatant, DNA still within the organism or phagocytic host cells might not be detected.…”

“…Although in case of fungi other than Aspergillus co-infection might be present, it is unlikely based on negative BAL GM. On the contrary, cases of cross-reactivity of the method with strains of A. flavus have already been described in the literature, 23 and such a false positive result might be particularly likely in case of high fungal burden, as demonstrated in our two cases. From the clinical point of view, a qPCR should detect all species of Aspergilllus, since species other than A. fumigatus might be more prevalent, particularity in some geographical zones, for example, A. flavus in Mediterranean.…”

A New Age in Molecular Diagnostics for Invasive Fungal Disease: Are We Ready?

“…The sensitivity and specificity of the MycoGENIE were 92.9 and 90.1%, respectively, using respiratory samples (n = 88) and 100 and 84.6%, respectively, with serum samples; this study detected no CYP51A mutants directly from specimen (Dannaoui et al, 2017). Another study compared the AsperGenius R and MycoGENIE assays for the detection of Aspergillus and resistance mutations in sputum specimens from cystic fibrosis patients (Guegan et al, 2018). Neither test detected any CYP51A resistance mutations, and the AsperGenius R had only limited success in amplifying the CYP51A gene targets, likely due to low fungal burden (Guegan et al, 2018).…”

“…Another study compared the AsperGenius R and MycoGENIE assays for the detection of Aspergillus and resistance mutations in sputum specimens from cystic fibrosis patients (Guegan et al, 2018). Neither test detected any CYP51A resistance mutations, and the AsperGenius R had only limited success in amplifying the CYP51A gene targets, likely due to low fungal burden (Guegan et al, 2018). While time efficient, these commercial assays are limited by the fact that CYP51A is a single copy gene, and that they detect only azole resistance involving the tandem repeat.…”

Surveillance for Azole-Resistant Aspergillus fumigatus in a Centralized Diagnostic Mycology Service, London, United Kingdom, 1998–2017

“…This significant variation in the proportion of resistance, suggested that patients with chronic airway diseases might be at higher risk of colonization and/or infection with azole-resistant A. fumigatus when compared to general or mixed patient cohorts. Similarly, prevalence of azole resistance in clinical A. fumigatus in French patient populations was dependent on underlying clinical conditions; 1.1% in hematological patients, 1.8% in unselected patients ( Alanio et al, 2011 ), and 8% to 12.2% in patients with cystic fibrosis ( Morio et al, 2012 ; Guegan et al, 2018 ). This finding supports the recommendations by van der Linden et al (2016) about the need to determine azole resistance frequency at the hospital level and within different patient groups.…”