Abstract
Guidelines on the diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scans, culture, fungal biomarker tests, microscopy and fungal PCR. The first-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of azole-resistant isolates has negatively impacted the management of IA. Failure to detect azole-resistance dramatically increases the mortality rates of azole-treated patients. Despite drug susceptibility tests not being routinely performed currently, we suggest including resistance testing whilst diagnosing Aspergillus disease. Multiple tools, including DNA sequencing, are available to screen for drug-resistant Aspergillus in clinical samples. This is particularly beneficial as a large proportion of IA samples are culture negative, consequently impeding susceptibility testing through conventional methods. Pyrosequencing is a promising in-house DNA sequencing method that can rapidly screen for genetic hotspots associated with antifungal resistance. Pyrosequencing outperforms other susceptibility testing methods due to its fast turnaround time, accurate detection of polymorphisms within critical genes, including simultaneous detection of wild type and mutated sequences, and—most importantly—it is not limited to specific genes nor fungal species. Here we review current diagnostic methods and highlight the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes.
Highlights
Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, NIHR Manchester Biomedical Research Centre (BRC) at the Manchester Academic Health Science Centre, The University of Manchester, Manchester M23 9LT, UK
To summarise, completing one pyrosequencing run includes DNA extraction of the clinical sample, followed by PCR amplification of the four target gene fragments and ends with the pyrosequencing reactions of biotinylated PCR products (Figure 2). This pyrosequencing assay generates reads of up to 150 bp per reaction and provides a large number of sequence reads in a single run, resulting in significant sampling depth. This allows the detection of the most numerous specific A. fumigatus cyp51A sequences, and of the lower-abundance reads, which is especially important in processing clinical samples where there is a potential mixture of genomes from human tissue, fungal hyphae of different A. fumigatus genotypes, other fungi and other microorganisms
Available PCR assays can be useful for time-pressing samples in smaller clinical laboratories, but the range of polymorphisms they are designed to detect is very limited
Summary
Mutations in genes involved in the A. fumigatus ergosterol biosynthetic pathway, including hmg, erg, cyp51A and cyp51B among others, are described as having a role in azole resistance [26,46,47,48,49,50,51]. Azole-resistant strains harbour genes with specific point mutations in combination or absence of tandem repeats in the promoter region. The most common pan-azole resistance mechanism in A. fumigatus cyp51A was found to be a combination of a 34-bp long tandem repeat (TR). The TR46/Y121F/T289A is a less widespread but emerging azole resistance mutation, which was first reported in the Netherlands in 2009 [60]. Other resistance mechanisms are described to be a combination of point mutations or the less common TR53 [89,95,96,97,98,99,100,101,102]. The single point mutation T289A does not result in azole resistance, whereas the single mutation TR46 has a slight reduced pan-azole susceptibility. Pan-azole resistance is reported in combination with TR34/L98H
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