Comparison of Two Molecular Assays for Detection and Characterization of Aspergillus fumigatus Triazole Resistance and Cyp51A Mutations in Clinical Isolates and Primary Clinical Samples of Immunocompromised Patients.

Patricia Postina,Dieter Buchheidt,Julian Skladny,Peter-Michael Rath,Jörg Steinmann,Thomas Miethke,Birgit Spiess,Anne Dietz,Tobias Boch,Natalia Merker,Wolf-Karsten Hofmann,Axel Hamprecht,Oliver Bader,Oliver A Cornely

doi:10.3389/fmicb.2018.00555

Abstract

In hematological patients, the incidence of invasive aspergillosis (IA) caused by azole resistant Aspergillus fumigatus (ARAf) is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR) assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the Aspergillus fumigatus (A. fumigatus) Cyp51A gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the Cyp51A alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL), 15 cerebrospinal fluid (CSF) samples) and ARAf isolates (n = 3) of immunocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of Aspergillus DNA and Cyp51A alterations. As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two A. fumigatus isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known Cyp51A alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML). The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61% in biopsies, 29% in CSF, 67% in BAL samples and 100% in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47% in biopsies, 42% in CSF, 59% in BAL samples and 100% in isolates. Altogether 17 Cyp51A alterations were detected using our ARAf PCRs plus DNA sequencing and therefrom 10 alterations also by the AsperGenius® system. The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant. The advantage of the AsperGenius® system is the time saving aspect. We consider non-culture based molecular detection of Aspergillus triazole resistance to be of high epidemiological and clinical relevance in patients with hematological malignancies.

Highlights

Aspergillus fumigatus (A. fumigatus) is one of the major live-threatening fungal pathogens (Brown et al, 2012)
Four established (Spiess et al, 2012, 2014) and two novel inhouse azole resistant Aspergillus fumigatus (ARAf) polymerase chain reactions (PCR) assays were compared to the commercially available AsperGenius R kit system concerning the detection of A. fumigatus DNA and Cyp51A key mutations directly from clinical samples of immunocompromised patients
Overall 74% (98/132) of our in-house ARAf PCRs showed positive signals accompanied by 67% (88/132) of successful sequencing, whereas 59% (52/88) of the AsperGenius R PCR assays were positive for the detection of A. fumigatus DNA from bronchoalveolar lavage (BAL) samples

Summary

Introduction

Aspergillus fumigatus (A. fumigatus) is one of the major live-threatening fungal pathogens (Brown et al, 2012). The situation is worsened by an increasing prevalence of triazole resistant Aspergillus infections (Steinmann et al, 2015; van der Linden et al, 2015; Verweij et al, 2016; Garcia-Rubio et al, 2017) which is associated with a much higher mortality rate (van der Linden et al, 2011; Steinmann et al, 2015; Verweij et al, 2015; Chong et al, 2016; Meis et al, 2016). About 50– 80% of triazole resistance in A. fumigatus is caused by mutations in the Cyp51A gene (Dudakova et al, 2017). In 2015 van der Linden et al described the TR46/Y121F/T289A mutation combination as the second most frequent resistance-mechanism causing high level triazole resistance (van der Linden et al, 2015; van Ingen et al, 2015)

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Conclusion