Development of novel PCR assays to detect azole resistance-mediating mutations of the Aspergillus fumigatus cyp51A gene in primary clinical samples from neutropenic patients

Abstract

The increasing incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised/hematological 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 a positive culture yield in this group of patients. We generated primers from the unique sequence of the Aspergillus fumigatus cyp51A gene to establish PCR assays with consecutive DNA sequence analysis to detect and identify the A. fumigatus cyp51A tandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially diluted A. fumigatus and human DNA, A. fumigatus cyp51A gene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg of A. fumigatus DNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons for A. fumigatus wild-type DNA confirmed the cyp51A wild-type sequence, and PCR products from one azole-resistant A. fumigatus isolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.

Figures

Fig 1

Partial promoter and coding sequence of the Aspergillus fumigatus cyp51A gene (GenBank accession number AF338659.1). Primer sequences are underlined and marked by bold letters. Orientation of the primers is indicated by arrows. The TR in the promoter region is marked by bold letters and horizontal lines. Cds, coding sequence; TR, tandem repeat.

Fig 2

Determination of the sensitivity of the L98H one-step PCR (A), the TR two-step PCR (B), and the M220 one-step PCR (C) assays by ethidium bromide agarose gel electrophoresis using serially diluted A. fumigatus wild-type DNA. (A) Lane 1, 9 pg; lane 2, 8 pg; lane 3, 7 pg; lane 4, 6 pg; lane 5, 5 pg; lane 6, 100 ng human DNA from a healthy person (negative reagent control); lane 7, 100 ng human DNA plus 50 pg A. fumigatus DNA (positive control); lane 8, sample from patient 1; lane 9, sample from patient 2. (B) Lane 1, 2 pg; lane 2, 1 pg; lane 3, 800 fg; lane 4, 600 fg; lane 5, 400 fg; lane 6, 200 fg; lane 7, 100 fg; lane 8, 50 fg; lane 9, 100 ng human DNA from a healthy person (negative reagent control); lane 10, 100 ng human DNA plus 50 pg A. fumigatus DNA (positive control); lane 11, sample from patient 1; lane 12, sample from patient 2. (C) Lane 1, 9 pg; lane 2, 8 pg; lane 3, 7 pg; lane 4, 6 pg; lane 5, 5 pg; lane 6, 4 pg; lane 7, 3 pg; lane 8, 100 ng human DNA from a healthy person (negative reagent control); lane 9, 100 ng human DNA plus 50 pg A. fumigatus DNA (positive control); lane 10, sample from patient 1; lane 11, sample from patient 2. The PCR amplicons of the patient samples could be used directly for DNA sequencing analysis. (D) Positive-control amplifications for the three different PCR assays (L98H, lanes 1 to 3; TR, lanes 5 to 7; M220, lanes 9 to 11) were performed using A. fumigatus wild-type DNA (50 pg) and DNA of two azole-resistant A. fumigatus isolates (50 pg) (strains F16216 and F14532) diluted in 100 ng human genomic DNA. A negative reagent control amplification without addition of DNA (lanes 4 and 8) resulted in no signals. The 34-bp TR in the promoter region of the cyp51A gene of strain F16216 was predicted by visualizing the longer PCR fragment (lane 6). Lane 1, A. fumigatus wild-type L98H; lane 2, strain F16216 L98H; lane 3, strain F14532 L98H; lane 4, mixed control; lane 5, A. fumigatus wild-type TR; lane 6, strain F16216 TR; lane 7, strain F14532 TR; lane 8, mixed control; lane 9, A. fumigatus wild-type M220; lane 10, strain F16216 M220; lane 11, strain F14532.