Comparison of Two Molecular Assays for Detection and Characterization of Aspergillus fumigatus Triazole Resistance and Cyp51A Mutations in Clinical Isolates and Primary Clinical Samples of Immunocompromised Patients

Abstract

In hematological patients, the incidence of invasive aspergillosis (IA) caused by azole resistant Aspergillus fumigatus (ARAf) is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR) assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the Aspergillus fumigatus (A. fumigatus) Cyp51A gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the Cyp51A alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL), 15 cerebrospinal fluid (CSF) samples) and ARAf isolates (n = 3) of immunocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of Aspergillus DNA and Cyp51A alterations. As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two A. fumigatus isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known Cyp51A alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML). The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61% in biopsies, 29% in CSF, 67% in BAL samples and 100% in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47% in biopsies, 42% in CSF, 59% in BAL samples and 100% in isolates. Altogether 17 Cyp51A alterations were detected using our ARAf PCRs plus DNA sequencing and therefrom 10 alterations also by the AsperGenius® system. The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant. The advantage of the AsperGenius® system is the time saving aspect. We consider non-culture based molecular detection of Aspergillus triazole resistance to be of high epidemiological and clinical relevance in patients with hematological malignancies.

Use of Aspergillus fumigatus real-time PCR in bronchoalveolar lavage samples (BAL) for diagnosis of invasive aspergillosis, including azole-resistant cases, in high risk haematology patients: the need for a combined use with galactomannan

“…16 This observation is indirectly confirmed by cohorts reporting higher performance of PCR, both in BAL and serum, in those receiving mould active agents. [21][22][23] Also in our study, the rate of qPCR positivity in GM positive BAL samples was higher form patients receiving mould active drugs compared to those not treated. Better specification which portion of BAL fluid should be used might be warranted, since by testing supernatant, DNA still within the organism or phagocytic host cells might not be detected.…”

Genotyping of Aspergillus fumigatus in Formalin-Fixed Paraffin-Embedded Tissues and Serum Samples From Patients With Invasive Aspergillosis

“…Amplification of resistance targets was not successful in 7 of 52 PCR reactions and these materials originated from three sera and a lung biopsy. The other six lung biopsies with A. fumigatus were successfully characterized, demonstrating a success rate of 86% for FFPE tissues, which is very similar to the performance of this assay in non-FFPE clinical materials (White et al, 2017 ; Buil et al, 2018 ; Postina et al, 2018 ).…”

A Novel Broad Allele-Specific TaqMan Real-Time PCR Method To Detect Triazole-Resistant Strains of Aspergillus fumigatus, Even with a Very Low Percentage of Triazole-Resistant Cells Mixed with Triazole-Susceptible Cells

“…and it only took about 6 h, which included about 2 h for DNA extraction, 3 h for allele-specific real-time PCR detection, and 1 h for data analysis. Our assay had favorable sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume) for all mutations tested compared to the sensitivity of currently established PCR assays detecting triazole resistance (600 fg of A. fumigatus DNA for the TR 34 mutation, 4 pg for the M220 mutation, and 300 fg for L98H mutation) (7,31). Moreover, we interpreted the sensitivity of our allele-specific real-time PCR method by using CFU counts, which are widely used in evaluating the number of viable cells of microbes (32).…”