A Low‐Cost Tebuconazole‐Based Screening Test for Azole‐Resistant Aspergillus fumigatus

The global emergence of azole resistance in Aspergillus fumigatus is resulting in health and food security concerns. Rapid diagnostics and environmental surveillance methods are key to understanding the distribution and prevalence of azole resistance. However, such methods are often associated with high costs and are not always applicable to laboratories based in the least‐developed countries. Here, we present and validate a low‐cost screening protocol that can be used to differentiate between azole‐susceptible “wild‐type” and azole‐resistant A. fumigatus isolates. © 2020 The Authors.


INTRODUCTION
Aspergillus fumigatus is an opportunistic fungal pathogen with an ecological niche of soil and composting vegetation, where it plays a vital role in ecosystem processes including decomposition and nutrient recycling (Rhodes, 2006;Tekaia & Latgé, 2005;Van De Veerdonk, Gresnigt, Romani, Netea, & Latgé, 2017). The fungus has a vast geographical range, facilitated by the rapid dispersion of airborne conidia that are able to tolerate a broad range of biotic stresses (Pringle et al., 2005;Sewell, Zhu, et al., 2019).
Humans inhale hundreds of conidia daily, which are frequently encountered in respiratory samples (Latgé, 1999;Mortensen et al., 2011). Typically, conidia are eliminated by the host's innate immune response, preventing attachment in the lung and subsequent infection (Balloy & Chignard, 2009). However, in immunocompromised individuals, conidia can become pathogenic resulting in a multitude of disorders including severe invasive aspergillosis (Brown et al., 2012). Current estimates indicate that more than 3 million Several similar azole compounds belonging to the same structural class as the clinical triazoles, with near identical structures, are used extensively in agriculture for crop protection. Substrates with residual azoles, which effectively support the growth and reproduction of A. fumigatus, have the potential to become hotspots for azole resistance (Dunne, Hagen, Pomeroy, Meis, & Rogers, 2017;Prigitano et al., 2014;Schoustra et al., 2019). Environmental surveillance and resistance monitoring are needed to fully appreciate the prevalence and distribution of ARAf isolates in the environment (Fisher, Hawkins, Sanglard, & Gurr, 2018). Rapid diagnostics and surveillance methods will inevitably aid outbreak recognition, response, and prevention. However, current methods are targeted specifically for clinically derived samples, are associated with high costs, and are not widely available to laboratories based in low-income developing countries (Buil et al., 2017;Kahlmeter et al., 2006;White, Posso, & Barnes, 2017).
Here, we present a screening protocol to help identify ARAf isolated from the environment at a cost of ∼£0.70 per isolate. Our method is based on the agricultural azole tebuconazole, owing to it its use in the environment, its low cost, and its ability to differentiate between azole-susceptible "wild-type" A. fumigatus and ARAf (Alvarez-Moreno et al., 2017;Chowdhary, Kathuria, et al., 2012;Sewell, Zhang, et al., 2019;Snelders, Camps, et al., 2012;Verweij, Snelders, Kema, Mellado, & Melchers, 2009)

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A. fumigatus is a Hazard Group 2 pathogen; therefore, suitable procedures and regulations for the handling of pathogenic microorganisms should always be followed. All open plate work should be carried out in a class 2 biological safety cabinet (BSC2) while wearing appropriate personal protective equipment. Extra care should be taken when handling due to the highly sporulating nature of the fungus.

Figure 1
Layout of the Tebucheck assay multi-well plate. Kelly, 1995), which is a key regulatory enzyme in the ergosterol biosynthetic pathway in fungi. Tebuconazole is classed as moderately toxic to mammals and can be dangerous in high doses (National Center for Biotechnology Information, 2020); consequently, all steps handling tebuconazole should be performed with care and while wearing single-use gloves. Table 1 provides the user with the required volumes/concentrations of tebuconazole and dimethyl sulfoxide (DMSO) for convenience. Tebuconazole stock solutions can be prepared in advance and stored in small-volume vials at −20°C for up to 1 month. To standardize the concentration of DMSO across the four wells of the assay, a supplementary volume must be added to give a final concentration of 0.032% (Table 1). Alternative methods for generating the tebuconazole-incorporated agar are acceptable; however, this may result in differential growth characteristics. The Tebucheck assay was developed using a 4-well multi-dish plate for standardization, reproducibility, and convenience. Other culture plates can be used, although discrepancies in growth characteristics may be observed. The stated volumes produce ∼32 4-well Tebucheck plates. Each well contains 1 ml Sabouraud dextrose agar (SDA) supplemented with three different concentrations of tebuconazole (Fig. 1). The first well is a drug-free control (0 mg/L tebuconazole), and wells 2, 3, and 4 contain SDA supplemented with 6, 8, or 16 mg/L tebuconazole, respectively. Tebucheck plates can be stored at 4°C for up to a month, although inspection of plates prior to use is recommended. The plates should be discarded if there is any obvious damage or contamination.

Materials
SDA (see recipe or purchase from a commercial vendor; e.g., Oxoid, cat. no.
3. Mix gently on a magnetic stirrer for 5 min using a suitable-sized magnetic stir bar.
4. Autoclave in an automated benchtop autoclave to sterilize.
5. Allow molten medium to cool to ∼60°C inside a BSC2.
6. Move a suitable water bath inside the BSC2, and set to 50°C.
8. Place tubes into the 50°C water bath using a 50-ml tube rack.
9. Using a serological pipette, transfer 40 ml molten SDA into the four conical tubes.
10. Allow SDA to equilibrate in the tubes for 5 min.

Incorporating tebuconazole
11. While the agar is equilibrating to temperature, prepare a 50 mg/ml stock solution of tebuconazole by dissolving 100 mg tebuconazole in 2 ml molecular biology-grade DMSO.
The 50 mg/ml stock solution can be stored at −20°C for up to 1 month.
13. Using a 20-μl pipette, transfer the correct volume of tebuconazole stock for each corresponding tube (Table 1).
15. Using a 20-μl pipette, transfer the correct volume of DMSO into each corresponding tube (Table 1).
17. Leave tubes to infuse before immediately moving to Basic Protocol 2.
Regularly change pipette tips to reduce the chance of volume discrepancies.
19. Once complete, allow plates to dry for 10 min in a BSC2.
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Current Protocols in Microbiology BASIC PROTOCOL 2

INOCULATION OF TEBUCHECK MULTI-WELL PLATES
Inoculation of Tebucheck plates includes two incubation steps: (1) the initial culturing of A. fumigatus isolates to be tested and (2) co.uk). After incubation of Tebucheck plates, the presence or absence of growth on the surface of the four wells can be classified using the following scale: 0, no growth (0% to <10% growth coverage); 0.5, partial/visible growth (10% to <50% growth coverage); and 1, growth (50% to 100% growth coverage).
The end point of susceptibility should be recorded where visual growth was 0% to 10% coverage. For example, an isolate that grows vigorously in well 1, grows partially in well 2, and does not grow in wells 3 and 4 would receive a final 3. Mix gently on a magnetic stir plate for 5 min using a suitable-sized magnetic stir bar.
4. Autoclave in an automated benchtop autoclave to sterilize.
5. Allow molten medium to cool to ∼60°C inside a BSC2.
6. Carefully pour ∼15 ml molten SDA into a 25-cm 2 cell culture flask or an appropriate alternative.
7. Place flasks horizontally (base down), and allow agar to solidify for 20 min.
8. Inoculate A. fumigatus sample (glycerol stock or agar punch) in the center of each cell culture flask.
9. Incubate at 37°C for 24 to 72 hr or when the colony has occupied ∼50% of the cell culture flask.
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Current Protocols in Microbiology Tebucheck screening 10. Once fully grown, add 10 ml of 0.05% (v/v) Tween 80 to each cell culture flask. 11. Gently shake each flask to disrupt the spores.
12. Leave spore suspension for 5 min to allow any aerosolized spores to settle.
13. Inoculate each well of the Tebucheck plates with 5 μl spore suspension, taking care to introduce the suspension into the center of the well.
15. Score Tebucheck end point by visually assessing presence or absence of growth on each well.
16. Analyze multiple isolates using the custom R script.

Sample data
Validation of Tebucheck was performed using a panel of 26 environmental A. fumigatus isolates (Table 2) selected from the Imperial College whole genome sequencing A. fumigatus culture collection (total collection >500 isolates). The panel represents the genetic population structure of the fungus, its vast geographical distribution, two of the most common environmentally associated azole antifungal-resistant genotypes (TR 34 /L98H [n = 8] and TR 46 /Y121F/T289A [n = 8]), and wild-type representatives (n = 10).
All wild-type isolates were susceptible to tebuconazole at 6 mg/L. Preliminary testing of the Tebucheck protocol used 4 mg/L, although this did not fully distinguish between wild-type and azole-resistant isolates. Isolates with known resistance alleles grew on the control well and exhibited various levels of growth in wells 2, 3, and 4 ( Fig. 2). Of the isolates harboring the TR 34 /L98H resistance allele, 100% had growth in well 2, and 62.5% showed partial growth in well 3 (up to 8 mg/L tebuconazole susceptibility). All TR 34 /L98H isolates were scored 0 (0 to <10% growth coverage) in well 4 (16 mg/L), suggesting an end point of tebuconazole susceptibility at 8 mg/L. Of the isolates harboring the TR 46 /Y121F/T289A allele, 100% showed growth in well 2 (6 mg/L), 100% grew in well 3, and 62.5% grew in well 4 (16 mg/L), suggesting an end point of tebuconazole susceptibility ≥16 mg/L (Fig. 3).

SDA
10 g/L mycological peptone 40 g/L glucose 15 g/L agar Adjust pH to 5.6 ± 0.2 when at 25°C Autoclave at 121°C for 15 min Store at room temperature for up to 3 months Brackin et al.

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Current Protocols in Microbiology Figure 3 An example of the Tebucheck assay displaying growth characteristics and representative Tebucheck scores of (A) an isolate with a wild-type cyp51A allele and (B) an isolate harboring the ARAf-associated cyp51A allele TR 46 /Y121F/T289A. Growth on the surface of each of the four wells can be scored using the following scale: 0, no growth (0% to <10% growth coverage); 0.5, partial/visible growth (10% to <50% growth coverage); 1, growth (50% to 100% growth coverage).

Background Information
The emergence of environmental ARAf and its associated pathological risks to immunocompromised individuals is of global concern . Currently, the majority of azole resistance monitoring studies are constrained to European countries, motivated primarily by the clinical emergence of ARAf in these regions (Snelders, van der Lee, et al., 2008;Van Der Linden et al., 2013;Verweij et al., 2009). However, there has been an increasing number of reports of ARAf in the least-developed countries (Meis, Chowdhary, Rhodes, Fisher, & Verweij, 2016) where, typically, financial assets, specialized equipment, trained staff, and testing capacity are limited. Precise estimates of prevalence are largely unknown in developing countries where knowledge is limited by a lack of surveillance and reporting. Without environmental screening it is difficult to determine whether variations in prevalence may be due to climatic conditions, fungal distributions, varying use of agrichem-ical products, or incomplete investigations. While advances in technology have resulted in a variety of diagnostic tools to save time and cost, they are not always widely available to some laboratories.
Molecular tools such as quantitative PCR, loop-mediated isothermal amplification (LAMP), and Sanger sequencing are effective diagnostic tools used in many research laboratories and are regularly used to characterize ARAf-associated resistance alleles (Sewell, Zhang, et al., 2019;White et al., 2017;Yu et al., 2019). Biological growth assays such as VIP-check TM (Buil et al., 2017) and EUCAST (Kahlmeter et al., 2006) are used to determine the organism's resistance phenotype. Many of these assays are targeted specifically at clinical ARAf isolates despite the environmental source of A. fumigatus. While these techniques are extremely effective and broadly applicable in many pathology and genomic research facilities, they can become costly when applied to large screening projects, Brackin et al.

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Current Protocols in Microbiology particularly for those included in large-scale, longitudinal studies.
For many mycology laboratories, costeffective culture-based diagnostics are paramount to research. Surveys conducted by the Asia Fungal Working Group comprising 241 mycology laboratories within seven Asian countries revealed that 89% had culture facilities, whereas only 17% performed DNA sequencing, and just 22% had molecular diagnostic facilities (Chindamporn et al., 2018). With the rise of antifungal resistance, routine susceptibility testing is essential, particularly in countries where the burden of fungal disease is high.
In order to address the issues outlined above, we present Tebucheck, a simple costeffective method for the detection of ARAf using the agricultural antifungal tebuconazole. The method is intended for the use of determining the level of azole resistance in environmental samples and to aid in the forward planning of in-depth ARAf investigations. Affordable, easy-to-use identification tools such as Tebucheck can be used to identify ARAf hotspots and their implications on public health. It allows for screening and the subsequent selection of putative ARAf to be subjected to further downstream characterization and analysis, thus reducing the number (and cost) of high-precision testing.

Critical Parameters
Tebucheck has been designed to be a basic and easy-to-use assay, meaning it has very few parameters and a low chance of user error. However, a few critical conditions should be considered. Cell density is an important consideration when handling Tebucheck. If a very turbid spore suspension is used to inoculate the Tebucheck wells, uncharacteristic growth may occur. Users should aim to have a turbidity of ∼0.5 McFarland or a cell density reading at 600 nm between 0.08 and 0.13 using a standard spectrophotometer. However, if the guidelines set out in this protocol for the generation of A. fumigatus spore suspension are followed closely (Basic Protocol 2, steps 1 to 12), then there should not be an issue at this step, and cell density readings will not need to be verified. When creating the tebuconazoleincorporated medium, it is vitally important the user allows the tebuconazole stock solution to properly mix with the molten SDA. Gently mixing SDA and allowing it to equilibrate in the water bath for 5 min is enough to ensure a consistent concentration of tebuconazole is maintained across all wells of the Tebucheck assay plates. Growth rates between A. fumigatus isolates can vary and may affect the assessment and scoring after the mandatory 48-hr incubation period. If growth in the control well (0 μg/ml tebuconazole) is underdeveloped after 48 hr, then Tebucheck plates can be left to incubate to the point at which the control well exhibits ∼50% growth coverage (maximum 5-day incubation due to drug exhaustion).

Troubleshooting
Our findings suggest that Tebucheck could be a useful addition to the current range of ARAf diagnostics available. Here, we show that it can distinguish between resistant and susceptible isolates and putatively predicted, environmentally associated resistance genotypes. Despite this finding, other resistance mechanisms can also explain ARAf phenotypes, and there may be others yet undiscovered. It is important, therefore, that Tebucheck is predominantly used to distinguish between resistant and non-resistant environmentally acquired isolates and that any putative genotype calls be properly assessed with quantitative PCR, LAMP assay, or direct sequencing of the cyp51A allele (Sewell, Zhang, et al., 2019;White et al., 2017;Yu et al., 2019).

Understanding Results
Tebucheck was validated using a panel of A. fumigatus isolates that were subjected to three concentrations of tebuconazole plus a control. Growth of all non-resistant isolates harboring the wild-type cyp51A allele was restricted to the control well only, clearly defining azole-susceptible wild-type isolates from resistant isolates. All resistant isolates were able to grow on at least one concentration of tebuconazole-incorporated growth medium. By using the scoring system outlined in Basic Protocol 2, it is possible for users to analyze a population of A. fumigatus isolates and determine its level of resistance.
The four different concentrations of tebuconazole in the Tebucheck assay can also provide insight into the level of resistance the isolates exhibit, providing the user with a more detailed characterization of their ARAf samples. During validation, it became clear that isolates with different resistance genotypes had different growth characteristics on Tebucheck, with TR 46 /Y121F/T289A genotypes more likely to grow across all four wells of the assay and TR 34 /L98H restricted to two wells (Figs. 2 and 3). It is possible, therefore, to not only gauge the isolates level of Brackin et al.

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Current Protocols in Microbiology resistance but also putatively suggest its cyp51A genotype.

Time Considerations
Time considerations for this protocol depend on the number of A. fumigatus isolates the user wishes to process using Tebucheck. Considering a single set of Tebucheck plates (32 plates), Basic Protocol 1 should take ∼30 min, while Basic Protocol 2 can take between 3 and 5 days due to the incubation steps involved in prior culturing of A. fumigatus isolates and the mandatory 48-hr Tebucheck incubation period. Generally, the total time to perform the whole protocol, including incubation steps, is between 2 and 5 days.