Rapid discrimination of Aspergillus fumigatus cyp51A resistance mutations by pyrosequencing

Ref ID: 19570

Author:

L Novak Frazer1*, R Masania2, DW Denning1,2, MD Richardson1,2

Author address:

1Manchester Academic Health Science Centre, University of Manchester, Manchester, UK
2Mycology Reference Centre, National Aspergillosis Centre, University Hospital of South Manchester,
Manchester, UK

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose:
A significant proportion of patients who attend the National Aspergillosis Centre suffer from azole
resistance in Aspergillus fumigatus attributable to mutations in the target site (cyp51A). Our challenge
is to develop an assay to elucidate whether the resistance observed clinically in patients, who are
unresponsive to long-term azole therapy and have been confirmed to be positive for Aspergillus
infection or colonisation by qPCR, is due to mutations in the cyp51A target.
Methods:
Primers were designed using Qiagen Assay Design software. DNA extracted from wild-type
A. fumigatus and isolates confirmed by Sanger sequencing to be carrying cyp51A mutations were
processed with Qiagen QIAamp DNA Mini Kits. Amplification of cyp51A for pyrosequencing was
carried out on an Applied Biosystems Veriti PCR. Pyrosequencing was carried out on a Qiagen
PyroMark Q24 platform. All pyrosequencing results were confirmed by Sanger sequencing.
Results:
We have developed a pyrosequencing assay which can detect all the mutations involved in azole
resistance that have been described to date (TR/L98, G54, M220, TR46/Y121/T289). The processing
time, from PCR to pyrosequencing result is 6h. We have processed WT and mutant isolates to
confirm the validity and reliability of our assay.
Conclusion:
We have shown that cyp51A mutations can be monitored quickly with this novel pyrosequencing assay.
We will also process Aspergillus PCR-positive patient sputum and BAL samples for the presence
of these mutations, with the intention of providing this assay as a routine service for future patients
undergoing azole therapy. The aim is to have the results available to clinicians within 24h of a positive
Aspergillus PCR. The assay is amenable to processing several patient samples simultaneously and
may be useful in quickly determining the genetic cause of azole therapy failure. The presence of WT
and mutant A. fumigatus cyp51A sequences can be detected simultaneously and the ratio quantified
as well. The advantage of using pyrosequencing is that this technique can be adapted easily to detect
new cyp51A gene mutations in A. fumigatus as they arise. In addition, mutations in other genes
involved in resistance to triazoles and other antifungal drugs can be monitored in A. fumigatus and
in other fungal species. It is envisaged that the results of this assay will have a positive impact on
patient treatment and antifungal stewardship.

Abstract Number: 96

Conference Year: 2014

Link to conference website: http://www.AAA2014.org

New link: NULL

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