Investigation of primary clinical samples and azole-resistant isolates of immunocompromised patients for cyp51A mutations in Aspergillus fumigatus using established and a novel TR46 PCR assays

Spiess, B.(1)*; Postina, P.(1); Hamprecht, A.(2); Vehreschild, J.(3); Lass-Flörl, C.(4); Hönigl, M.(5); Rath, P.-.M.(6); Miethke, T.(7); Hofmann, W.-.K.(8); Buchheidt, D.(1);


Objectives. Increasing numbers of azole resistant Aspergillus isolates have recently been reported, but, in immunocompromised patients, the diagnosis of invasive aspergillosis (IA) is rarely based on positive culture yield. Therefore, we used an established and a novel nested TR46 PCR assays for the detection of azole resistance mediating cyp51A mutations in Aspergillus fumigatus directly from clinical samples and in azole resistant clinical A. fumigatus isolates. Methods. We established a nested PCR assay for the detection of the 46 bp tandem repeat (TR46) directly from clinical samples (BAL, tissue biopsies) as a marker of the TR46/Y121F/T289A genotype, which is known to cause pan-azole resistance. The assay was tested using DNA of a known TR46/Y121F/T289A positive, multi-azole resistant clinical isolate (A12519) of a hematological patient with IA. We furthermore investigated 30 clinical samples, the DNA of a clinical sample of a NHL patient with IA and two further multi-azole resistant clinical A. fumigatus isolates (A68, A71) of immunocompromised patients for the occurrence of TR46. Furthermore, we screened 181 clinical samples of our Aspergillus DNA sample collection (1995-2013) and the DNA of all isolates using our established TR34/M220/L98H cyp51A azole resistance PCR assays. Results. The detection threshold for the TR46 PCR assay was 6 pg of A. fumigatus DNA. Using this assay, 30 primary clinical samples and all isolates were investigated by PCR and DNA sequence analysis revealing the TR46 alteration in the clinical isolate (A12519) used as positive control; all other samples were TR46 negative. Screening of 181 primary clinical samples for the TR34/L98H/M220 alterations revealed a single L98H mutation in a lung tissue specimen of a COPD/AML patient, a L98H alteration in combination with the TR34 in a brain tissue sample of a patient with ALL and in the BAL sample of a patient with AML. Investigating a BAL sample of a CLL patient from 1998, we detected the so far unknown N90K mutation. No microbiological data of this patient is available, so the potential contribution on azole resistance of this novel mutation cannot be clarified. DNA sequence analysis revealed the L98H/TR34 cyp51A alteration combination in both isolates A68 and A71. Conclusions. In addition to our TR34/L98H/M220 PCR assays, we established a novel TR46 PCR assay to detect these cyp51A mutations directly from primary clinical samples. The mutations were also detected in multi-azole resistant clinical isolates showing the feasibility of the approach. We consider our assays of epidemiological and clinical relevance to detect azole resistance and to optimise antifungal therapy in hematological patients with IA. We are indepted to Prof. Dr. Uwe Gross, Göttingen, for providing a clinical sample and to Silke Will, Natalia Merker

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