Purpose: A significant proportion of patients with chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA) who attend the National Aspergillosis Centre (Manchester, UK) harbour triazole-resistant Aspergillus fumigatus, mainly attributable to acquired polymorphisms in the target site, cyp51A. High volume culture (HVC) developed by our laboratory to improve the growth of fungal isolates from respiratory samples allows identification and EUCAST susceptibility testing although culture positivity remains low (25-42%) even when elevated qPCR for Aspergillus spp. is demonstrated. We developed an assay for patient respiratory samples to elucidate whether we could detect polymorphisms in cyp51A in clinically unresponsive patients on long-term azole therapy at the first signs of therapeutic failure.
Methods: Patients were identified at weekly medical multidisciplinary team meetings as potentially failing therapy when positive Aspergillus spp. qPCR but negative HVC results were obtained in the context of adequate antifungal drug levels. Their sputum or bronchopulmonary lavage (BAL) samples, already extracted and assayed for the presence of Aspergillus spp. rDNA as per routine practice at the Mycology Reference Centre Manchester (MRCM), underwent nested PCR amplification of cyp51A, yielding biotinylated products for subsequent pyrosequencing using the Qiagen Advanced PyroMark system. Positions at gene locations G54, L98, Y121, P216-F219-M220 and T289 were assayed as well as possible insertions in the promoter region of cyp51A and results were confirmed by Sanger sequencing.
Results: Of the one hundred fourteen patient samples analysed since March 2017, 29 were reported with no polymorphisms, 20 with cyp51A polymorphisms, 38 are in progress, 16 were processed only by Sanger sequencing and 11 failed to be amplified. Of the polymorphisms reported, 2 were TR34 and/or L98H, 5 were G54 (E, R or V) and 13 were Y121F/T289A polymorphisms but the latter without the accompanying TR46 insertion. When HVC was available (for validation purposes), pyrosequencing results were in agreement with the susceptibilities derived by EUCAST broth micro-dilution testing only half the time, possibly due to amplification insensitivity or PCR inhibition but also when mixed species and species other than A. fumigatus were prominent in patient samples or resistance was due to non-cyp51A polymorphisms. Sanger and pyrosequencing results were generally concordant, although pyrosequencing could discriminate subtleties in mixed samples while Sanger offered whole gene coverage.
Conclusion: Pyrosequencing can identify A. fumigatus cyp51A polymorphisms in patient samples at the earliest signs of treatment failure when culture fails and susceptibility testing is not possible. The impact of therapeutic management in light of resistance profiling is now being investigated in the context of antifungal drug stewardship and patient benefit. Having overcome considerable technical difficulties, this assay will be made available to all, including new, patients attending the National Aspergillosis Centre not only those failing triazole therapy.
Full conference title:
- AAA 8th (2018)