Incidence of Cyp51 A key mutations in Aspergillus fumigatus-a study on primary clinical samples of immunocompromised patients in the period of 1995-2013.

Jörg Steinmann,Ilse D. Jacobsen,Mark Reinwald,Oliver A. Cornely,Thomas Miethke,Martin Hoenigl,Axel Hamprecht,Melchior Lauten,Natalia Merker,Peter-Michael Rath,Silke Will,Dieter Buchheidt,Patricia Postina,Birgit Spiess,Cornelia Lass-Flörl,Wolf-Karsten Hofmann

doi:10.1371/journal.pone.0103113

Abstract

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.

Highlights

Aspergillus species cause invasive aspergillosis (IA), a lifethreatening infection in immunocompromised patients
Two established [17], one improved and a novel polymerase chain reaction (PCR) assay were used for the detection of the most common azole resistance mediating alterations in the A. fumigatus cyp51 A gene directly from clinical samples stored in our Aspergillus DNA collection
DNA sequence analysis of the A. fumigatus wildtype PCR amplicons confirmed the wildtype sequence of the cyp51 A gene, showing that A. fumigatus cyp51 A DNA fragments were amplified by the use of the particular PCR assays

Summary

Introduction

Aspergillus species cause invasive aspergillosis (IA), a lifethreatening infection in immunocompromised patients. As triazole antifungal drugs (itraconazole, voriconazole, posaconazole) represent prophylaxis or standard first-line therapy against IA, emerging of resistance is of clinical concern. Infections due to azole resistant Aspergillus fumigatus (A. fumigatus) have increased over recent years [1,2,3], and are associated with markedly higher case fatality rates in particular among patients with azole resistant IA [4]. To date target-site modification is the main observed mechanism conferring azole drug resistance. The most important target, the 14a-sterol demethylase, is encoded by the cyp A gene of A. fumigatus. It has been reported that mutational hotspots

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Conclusion