Rapid Detection of Legionella pneumophila in Drinking Water, Based on Filter Immunoassay and Chronoamperometric Measurement

Abstract

Legionella is a pathogenic bacterium, ubiquitous in freshwater environments and able to colonise man-made water systems from which it can be transmitted to humans during outbreaks. The prevention of such outbreaks requires a fast, low cost, automated and often portable detection system. In this work, we present a combination of sample concentration, immunoassay detection, and measurement by chronoamperometry. A nitrocellulose microfiltration membrane is used as support for both the water sample concentration and the Legionella immunodetection. The horseradish peroxidase enzymatic label of the antibodies permits using the redox substrate 3,3′,5,5′-Tetramethylbenzidine to generate current changes proportional to the bacterial concentration present in drinking water. Carbon screen-printed electrodes are employed in the chronoamperometric measurements. Our system reduces the detection time: from the 10 days required by the conventional culture-based methods, to 2–3 h, which could be crucial to avoid outbreaks. Additionally, the system shows a linear response (R2 value of 0.99), being able to detect a range of Legionella concentrations between 101 and 104 cfu·mL−1 with a detection limit (LoD) of 4 cfu·mL−1.

High-Throughput Flow-Through Direct Immunoassays for Targeted Bacteria Detection

“…The main differences of the FTDI assays compared to other immunoassays (e.g., conventional ELISAs) are the following: (i) FTDI assays can analyze large volumes of sample (theoretically any volume) compared to 100–200 μL typically analyzed on conventional immunoassays, (ii) FTDI assays are direct immunoassays so they do not require capture antibodies, (iii) total analysis time of rapid FTDI assays is almost half to that of conventional ELISA while the assay is at least 100 times more sensitive, (iv) FTDI assays involves fewer washing steps and is therefore simpler to perform compared to an ELISA, and (v) some steps of FTDI assay can be easily semiautomated (by using a filter plate vacuum manifold and a multichannel peristaltic pump). On the contrary to other assays that used conventional filtration membranes for bacteria detection and an immunoassay for detection purposes, , all the steps of the FTDI assays (sample, addition, bacteria proliferation, reagents incubation, washing steps, signal measurement) are performed in a single well of a 96-well filter plate and neither the samples or the membranes are removed outside of the detection well at any point. Since all the steps are performed inside a single well of a 96-well filter plate, (i) the analytical protocol is easy to be performed, (ii) the risk of user exposure to the bacteria is minimal, (iii) the risk of sample contamination is low, (iv) microplate readers can measure the analytical signal (i.e., fluorescence) from inside the well, and (v) the waste production is low.…”

Water Sampling Module for Collecting and Concentrating Legionella pneumophila from Low-to-Medium Contaminated Environment

“…We argue that with a CF = 5 produced by WSM employed for processing CTW with L. pneumophila , and assisted by the chemotaxis effect [ 63 ], an LOD approaching 10 CFU/mL could be obtained reproducibly with a workstation designed for automated biosensing. An LOD of 4 was reported recently for the detection of L. pneumophila in freshwater environments, which are less turbid sources, by using a combination of sample concentration, immunoassay detection and measurement by chronoamperometry [ 64 ].…”

Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide

“…Boss et al (2018) transferred starved bacteria into a fresh nutritional medium that stimulates them, resulting in a boosted rRNA synthesis, unlike non-viable cells, followed by RT-PCR. Ezenarro et al (2020) presented a combination of sample concentration, immunoassay detection, and measurement by chronoamperometry. A nitrocellulose microfiltration membrane is used as support for both the water sample concentration and the Legionella immunodetection.…”