Microfluidics combined with fluorescence in situ hybridization (FISH) for Candida spp. detection

Abstract

One of the most prevalent healthcare-associated infection is the urinary tract infection (UTI), caused by opportunistic pathogens such as Candida albicans or non-albicans Candida species (NACS). Urine culture methods are routinely used for UTI diagnostics due to their specificity, sensitivity and low-cost. However, these methods are also laborious, time- and reagent-consuming. Therefore, diagnostic methods relying on nucleic acids have been suggested as alternatives. Nucleic acid-based methods can provide results within 24 h and can be adapted to point-of-care (POC) detection. Here, we propose to combine fluorescence in situ hybridization (FISH) with a microfluidic platform for the detection of Candida spp. As a case study we used C. tropicalis, which is reported as the second most common NACS urine isolate obtained from patients suspected with UTI. The microfluidic platform proposed in this study relies on hydrodynamic trapping, and uses physical barriers (e.g., microposts) for the separation of target cells from the suspension. Using a specific peptide nucleic acid (PNA) probe, the FISH procedure was applied onto previously trapped C. tropicalis cells present inside the microfluidic platform. Fluorescence signal intensity of hybridized cells was captured directly under the epifluorescence microscope. Overall, the PNA probe successfully detected C. tropicalis in pure culture and artificial urine (AU) using FISH combined with the microfluidic platform. Our findings reveal that FISH using nucleic acid mimics (PNA) in combination with microfluidics is a reliable method for the detection of microorganisms such as C. tropicalis. As such, this work provides the basis for the development of a POC detection platform in the future.

Assessment of UTI Diagnostic Techniques Using the Fuzzy–PROMETHEE Model

“…A recent study confirmed that the combination of microfluidics with FISH reduced reagent consumption and analysis time, as well as that FISH works well in microfluidic channels and is particularly effective for the diagnostics of fungal urinary tract infections [19]. In another study, it was shown that the micro-fluidic device allowed for quantitative determination and early detection of leukocyte esterase in human urine.…”

“…The hybrid complex can then be clearly visualized using a fluorescent microscope. Immunologybased tests are rapid, inexpensive tests that are particularly sensitive to Gram-negative bacteria and can detect bacteriuria, but their use is limited due to poor sensitivity against Gram-positive bacteria [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21].…”

Innovative Biosensing Approaches for Swift Identification of Candida Species, Intrusive Pathogenic Organisms

“…It offers direct target visualization amongst complex biological matrices with an epifluorescence microscope within a very short pre-enrichment time with a µL volume of samples. The FISH protocol was applied to trapped C. tropicalis cells which were permeabilized to internalize the hybridization probes [58].…”

Microfluidic systems for infectious disease diagnostics

“…The method was tested with spiked synthetic urine samples ( C. tropicalis at 10 5 cells per mL). 271 Li et al fabricated silicon nanowire surfaces functionalized with concanavalin A and integrated them into a microchannel. The high surface-to-volume ratio of the nanostructured surface enhanced the bacterial capture efficiency.…”