Antifungal Susceptibility Testing of Aspergillus niger on Silicon Microwells by Intensity-Based Reflectometric Interference Spectroscopy

Abstract

There is a demonstrated and paramount need for rapid, reliable infectious disease diagnostics, particularly those for invasive fungal infections. Current clinical determinations for an appropriate antifungal therapy can take up to 3 days using current antifungal susceptibility testing methods, a time-to-readout that can prove detrimental for immunocompromised patients and promote the spread of antifungal resistant pathogens. Herein, we demonstrate the application of intensity-based reflectometric interference spectroscopic measurements (termed iPRISM) on microstructured silicon sensors for use as a rapid, phenotypic antifungal susceptibility test. This diagnostic platform optically tracks morphological changes of fungi corresponding to conidia growth and hyphal colonization at a solid–liquid interface in real time. Using Aspergillus niger as a model fungal pathogen, we can determine the minimal inhibitory concentration of clinically relevant antifungals within 12 h. This assay allows for expedited detection of fungal growth and provides a label-free alternative to broth microdilution and agar diffusion methods, with the potential to be used for point-of-care diagnostics.

Shining light in blind alleys: deciphering bacterial attachment in silicon microstructures

“…39,40 While many efforts focus on understanding mammalian cell interfaces with materials, [41][42][43][44] in this work, we investigate bacterial cell responses to manipulated surfaces of silicon topologies by intrinsic phase-shift reflectometric interference spectroscopy measurements, termed PRISM. 45,46 In this method, bacterial cells colonize on diffractive, patterned Si microstructures, while zero-order reflectance spectra are continuously collected during illumination with a broadband white light source. [45][46][47][48][49][50][51] The resulting reflectance spectra exhibit optical interference fringes due to the reflection of the incident light at the two interfaces of the Si microstructures (top and bottom).…”

Paving the Way to Overcome Antifungal Drug Resistance: Current Practices and Novel Developments for Rapid and Reliable Antifungal Susceptibility Testing

“…A recent approach for rapid AST employs optical sensors based on photonic silicon arrays for label-free monitoring of bacte-rial and fungal behavior during exposure to antimicrobials in real time. [60,[83][84][85] These sensors are based on diffraction gratings, consisting of periodic micropatterned silicon architectures of micrometer-dimensions, which are used as the optical transducer element and preferential surface for microbial colonization as presented in Figure 6A. These sensors were first demonstrated to optically track the growth of Escherichia coli (E. coli) and determine MIC values within 2-3 h (compared to 8 h with state-of-the-art automated methods) by monitoring bacterial growth patterns in the presence of varying concentrations of clinically relevant antibiotics.…”

“…Therefore, a significant research effort is directed toward expediting phenotypic AFST, and new techniques, relying on mass spectrometry, flow cytometry, calorimetry, fluorescence microscopy, and optical on-chip assays, are emerging. [60][61][62][63][64] Table 2 summarizes these new AFST approaches and briefly describes their concept and typical assay time. The table also provides a short comparison of these methods' main advantages and disadvantages, as discussed in the next paragraphs.…”

“…There are several microfluidic-assisted phenotypic AFST assays reported [60,108,132] ; yet, some of these were only used for qualitative antifungals screening with no MIC determination.…”

“…There are several microfluidic‐assisted phenotypic AFST assays reported [ 60,108,132 ] ; yet, some of these were only used for qualitative antifungals screening with no MIC determination. For example, a highly parallelized microfluidic system was developed for the screening of small molecule compounds that enhance the antifungal effect of amphotericin B against C. albicans .…”