Development of Novel PCR Assays to Detect Azole Resistance Mediating Mutations of the Aspergillus fumigatus cyp51A Gene in Non Culture Based Clinical Samples from Neutropenic Patients

Ref ID: 18650

Author:

B. Spiess – Scientific Assistent1, W. Seifarth – Senior Scientist 1, N. Merker – Technical Assistent 1, S. J. Howard – Scientist 2, S. Epple – Technical Assistant 1, A. Dietz – Technical Assistant 3, W. K. Hofmann – Director 1, D. Buchheidt, MD (Doct

Author address:

13rd Dept of Int. Med., Mannheim, Germany, 2Sch. of Translational Med., The Univ. of Manchester, Manchester Academic Hlth. Sci. Ctr., Manchester, United Kingdom, 3Inst. of Med. Microbiol. and Hygiene, Mannheim, Germany.

Full conference title:

52nd Annual ICAAC

Date: 9 September 2014

Abstract:

Background: The incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised patients emphasizes the need to improve the detection of resistance mediating cyp51A gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on positive culture yield. Methods: We established PCR assays with consecutive sequence analysis to detect the A. fumigatus cyp51A TR (tandem repeat) mutation in the promoter region and two mutations leading to the L98H and M220I alterations in cyp51A coding region. After testing sensitivity and specificity, A. fumigatus cyp51A gene fragments of about 150 bp potentially carrying the mutations were amplified directly out of clinical samples (blood, BAL and tissue) and sequenced. Results: PCR assays are specific for A. fumigatus DNA and did not show crossreactivity with human genomic DNA. Detection limit for the TR assay is 1 pg, for the L98H assay 6 pg and for the M220I assay 4 pg of A. fumigatus DNA. Sequencing of the PCR amplicons for A. fumigatus wildtype DNA confirmed the cyp51A wildtype sequence. Sequencing of PCR DNA sequence analysis to rapidly detect azole resistance mediating mutations of the A. fumigatus cyp51A gene directly from clinical samples. products from one azole resistant A. fumigatus strain showed the L98H and TR mutations. Conclusions: We established three sensitive and specific PCR assays with consecutive sequencing. For epidemiological and clinical issues, we actually analyze clinical samples from our Aspergillus DNA bank positive in our diagnostic nested Aspergillus PCR assay. We consider our assay of high epidemiological and clinical relevance to detect azole resistance and to allow significantly quicker targeted antifungal therapy in patients with IA.

Abstract Number: M-1537

Conference Year: 2012

Link to conference website: NULL

New link: NULL

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