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

Abstract

treating IFI, rising resistance to these drugs is a cause of major concern. [1,3] For example, the globally emerging multidrug-resistant species Candida auris is now recognized by the Centers for Disease Control and Prevention (CDC) as an urgent threat. [4] Thus, its proper treatment is therefore crucial for both individual therapeutic outcomes and preventing its spread. [5] The increasing and undirected use of antifungals in medicine and agriculture is associated with the rising numbers of acquired resistance in fungi. [3] The readers are referred to the following excellent reviews on fungal pathogens [6,7] and antifungal resistance [3] for further reading. The latter, in combination with the challenges associated with developing novel antifungals, emphasizes the need for rapid disease detection and adequate antifungal therapy protocols. [3,8] Implementing such measures is part of a proper antimicrobial stewardship aimed at reducing the excessive usage of antimicrobial therapies in clinical settings by encouraging physicians to prescribe appropriate antimicrobials only when truly required. [9] Antimicrobial susceptibility testing (AST), specifically antifungal susceptibility testing (AFST), is employed to reveal the susceptibility or resistance of fungal pathogens to clinically relevant antifungals. [10,11] In these tests, pathogenic fungi are exposed to various concentrations of a panel of antifungals to determine the minimum inhibitory concentration (MIC) values, which are typically defined as the lowest drug concentration inhibiting the pathogen's growth. [12] MIC values allow physicians to predict the success of antifungal treatments. [13] As such, rapid AFST is an essential tool to improve antifungal therapy by choosing the correct and most effective antifungal drug in a timely manner. Early therapy initiation is also crucial for enhancing the therapeutic outcome of patients with invasive fungal infections, such as life-threatening bloodstream infections. [14] Clinical AFST is conducted according to standardized methods and protocols, published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or the Clinical and Laboratory Standards Institute (CLSI). [12,15] Yet, as these gold-standard methods are laborious and typically require at least 24 h (and in many cases, several days) for completion, [12,16] significant research efforts are now being directed toward the development of more expedited phenotypic and molecular AFST techniques. [17,18] The past year has established the link between the COVID-19 pandemic and the global spread of severe fungal infections; thus, underscoring the critical need for rapid and realizable fungal disease diagnostics. While in recent years, health authorities, such as the Centers for Disease Control and Prevention, have reported the alarming emergence and spread of drug-resistant pathogenic fungi and warned against the devastating consequences, progress in the diagnosis and treatment of fungal infections is limited. Early diagnosis and patient-tailored t...

A 3D-printed microfluidic gradient generator with integrated photonic silicon sensors for rapid antimicrobial susceptibility testing

“…The successful miniaturization, integration, and automation of the various steps required in AST methods (e.g., generation of two-fold antimicrobial dilution series, cell inoculum, sensing, and MIC determination) are considered essential prerequisites for providing rapid and easy-to-use AST assays for point-of-care applications. 14,52 We thus envisioned a microfluidic system that automatically generates the desired antimicrobial concentrations and also integrates the sensing elements for convenient and time-saving real-time optical AST directly within the microfluidic channels of the GGsystem. Before integrating photonic silicon chips as sensing elements for on-chip AST into our device, we first test the compatibility of the 3D-printed GG with the gold-standard BMD assay (schematically illustrated in Fig.…”

“…This value is typically defined as the lowest drug concentration that inhibits the pathogens' growth and helps physicians to differentiate between resistant and susceptible isolates and choose the adequate antimicrobial for treatment. [13][14][15] Yet gold-standard reference AST techniques, such as broth microdilution (BMD) and agar-based methods in which growth can be determined by absorbance measurements 16 or visually, 17,18 are labor-intensive procedures with long (≥16 h) wait times and are not wellsuited for point-of-care (PoC) purposes. [16][17][18][19] To overcome these limitations, extensive research is directed towards the development of microfluidic devices for miniaturized and rapid AST assays 20,21 using various sensing approaches (e.g., chromogenic agar, 22 oxygen consumption, 23,24 membrane integrity, 25 single-cell imaging, 26 or metabolic activity/metabolite analysis [27][28][29] ).…”

“…This value is typically defined as the lowest drug concentration that inhibits the pathogens' growth and helps physicians to differentiate between resistant and susceptible isolates and choose the adequate antimicrobial for treatment. 13–15 Yet gold-standard reference AST techniques, such as broth microdilution (BMD) and agar-based methods in which growth can be determined by absorbance measurements 16 or visually, 17,18 are labor-intensive procedures with long (≥16 h) wait times and are not well-suited for point-of-care (PoC) purposes. 16–19…”

Microbiological Profiles of Ocular Fungal Infection at an Ophthalmic Referral Hospital in Southern China: A Ten-Year Retrospective Study

“…Moreover, the development of antifungal drug resistance highlights the need for in vitro susceptibility testing. 21 There have been various methods for in vitro antifungal susceptibility testing used, including macro- and micro-dilution, E-tests, and disk diffusion, based on the standards of the Clinical and Laboratory Standards Institute (CLSI) or The European Committee on Antimicrobial Susceptibility Testing (EUCAST). 18 , 22 , 23 Different fungal isolates have different susceptibilities for antifungal agents.…”

Photonic Si microwell architectures for rapid antifungal susceptibility determination of Candida auris

“…9 Specifically, antifungal susceptibility testing (AFST), in which pathogenic fungi are exposed to varying antifungals at increasing concentrations, and the minimum inhibitory concentration (MIC) is determined, can help physicians to guide treatment decisions. 10 However, current AFST methods, like agar-based tests or the gold standard broth microdilution (BMD), are lengthy and require 424 h. 10 Also, commercially available state-of-the-art automated methods such as the Vitek2 (bioMe ´rieux) typically provide results only within 12-18 h, 11,12 a limited set of antifungals is available, and MIC values have not always been accurately determined. 13 Thus, there is an urgent clinical need for rapid and reliable novel AFST methods for Candida species.…”