Assessment of the impact of environmental azoles on the development of resistance to medical azoles by Aspergillus spp

Methods

Summary

BAL sample

Galactomannan detection in BAL

This test will be performed according to the manufacturer's instructions. Briefly, 70µL of BAL sample will be pipetted into the cassette test and the result read after 15 minutes.

culture

A good quantity of BAL pellet will be inoculated in duplicate on the Sabouraud’s dextrose agar + antibiotics and incubated aerobically at 25 to 30°C and 37°c for up to 96 hours. Positive results will be confirmed and lactophenol cotton blue (LPCB)/methylene blue wet mount.

Environmental and food samples

Air

The prepared culture medium (SDA) will be exposed to ambient air for some hours at different localisation and incubated at 37°C, 50°C and 25°C for up to four days. Positive results will be confirmed and lactophenol cotton blue (LPCB)/methylene blue wet mount.

Ground

The ground will be collected from different locations and filtered to remove large materials. A small amount of each sample will later be inoculated in duplicate on SDA and incubated at 37°C, 50°C and 25°C for up to four days. Positive results will be confirmed and lactophenol cotton blue (LPCB)/methylene blue wet mount.

Culture of common foods

A small piece of each sample (previously washed with 70% alcohol) will be cut and be inoculated in duplicate on SDA and incubated at 37°C, 50°C and 25°C for up to four days. Positive results will be confirmed and lactophenol cotton blue (LPCB)/methylene blue wet mount.

Galactomannan detection in common foods samples

 For solid and semi-solid foods, 500 mg of the food products will be homogenized with 500 µl sterile distilled water using a bead beater. For liquid food products, 500 µl samples will be homogenized with an equal volume of sterile distilled water. The mixture of both types of food products will be then spun down at 10.000 rpm for 10 minutes. The supernatant will be used for GM antigen detection.

Antifungal susceptibility of isolates

The susceptibility to two classes of azoles (itraconazole, and voriconazole) and amphotericin B of every growth will be performed based using diffusion strips.