Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species

Abstract

The incidence of azole resistance in species has increased over the past years, most importantly for. This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.

A New Age in Molecular Diagnostics for Invasive Fungal Disease: Are We Ready?

“…Other molecular beacon-based assays since including those employing TaqMan technology have shown similar good results, when validated against the gold standard of DNA sequencing. As such, the most commonly used is a conventional PCR amplifying the CYP51A gene and promoter regions with Sanger sequencing (Dudakova et al, 2017). Ahmad et al (2014) developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay for detection of TR 34 as well as the L98H substitution.…”

“…They combine high sensitivity with high specificity to detect the small quantities of Aspergillus DNA in biological samples. Most formats are PCR-based followed by sequence analysis to identify mutations (Dudakova et al, 2017). Using a nested PCR, in culture-negative PCR-positive samples, the TR 34 /L98H and M220 mutations were detected in 55.1% of samples from patients with chronic pulmonary aspergillosis or ABPA (Denning et al, 2011).…”

In vivo Efficacy of Posaconazole (POS) against Voriconazole Resistant (VCZ-R) Aspergillus flavus in an Inhalational Neutropenic Murine Model of Invasive Pulmonary Aspergillosis

“…Molecular studies and continued surveillance for azole-resistance from clinical isolates of A. flavus is imperative [21]. An understanding of the mechanisms of azole-resistance and establishing a correlation between specific cyp51 mutations and azoleresistance may have therapeutic implications [22][23][24][25][26]. The recent introduction of phenotypic and genotypic assays for rapid and timely identification of specific cyp51 mutations from clinical specimens will serve as a valuable tool in this field.…”

Aspergillus collected in specific indoor settings: their molecular identification and susceptibility pattern

“…These fungi can produce mycotoxins and a high amount of small conidia easily dispersed in the air. Aspergillus species are associated with several health problems, like mycotoxicosis, allergies, asthma, allergic bronchopulmonary aspergillosis, aspergilloma, necrotizing aspergillosis and invasive pulmonary aspergillosis (Dudakova et al 2017). The frequency of these fungal respiratory infections has been more frequently reported during the last decades because of an increasing life expectancy of immunocompromised patients, cystic fibrosis patients (Nasri et al 2019).…”

“…These cryptic species may have an intrinsic resistance to specific antifungals, like azoles (Van Der Linden et al 2011;Vermeulen et al 2015). The most frequently used medical azoles are voriconazole, itraconazole and posaconazole (Dudakova et al 2017). However, new combinational therapies with clinically licensed drugs against fungal infections are being tested as potential alternatives (Fakhim et al 2018).…”