Quantifying Legionella in Shorter Time

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KNF Neuberger GmbH

rqmicro AG

It is questionable that the vast majority of information on the spread and behavior of pathogens in water systems has been compiled using these conventional, cultivation-dependent methods. To the best of our knowledge, this means that the accuracy and reliability of the data is neither satisfactory nor sufficient for the assessment of drinking water hygiene.

The new method – faster and more precise

The rapid analysis of the total cell count of bacterial cells in water samples by flow cytometry is already established in water laboratories worldwide [16, 17]. rqmicro, a spin-off of the Federal Institute of Technology (ETH) Zurich, set itself the objective of optimizing this method for various pathogens and matrices. Thanks to this ingenious sample preparation process developed by the interdisciplinary team at rqmicro, it is possible to isolate, purify and quantify Legionella from water samples in record time.

The method is based on the immunomagnetic separation (IMS) of target cells using magnetic particles and subsequent flow cytometric detection — in less than two hours from sampling to result. In contrast to the established methods, the new method detects all potentially infectious Legionella — including cells that do not grow on agar plates, the VBNC cells.

Magnetic particles bind to the surface of Legionella via antibodies specifically developed for the immunomagnetic separation. The application of a magnetic field is used to separate the target cells from the sample matrix. This step is necessary as one liter of drinking water commonly contains about 100 million bacterial cells. The detection of only one hundred Legionella that might be present in a sample, which corresponds to a concentration of 0.0001%, is equivalent to the proverbial search for the needle in a haystack.

In order to quantify the target cells by flow cytometry after the separation step, they are labeled with antibodies bound to a fluorescent dye. In addition to the accurate evaluation and faster quantification, the flow cytometric detection reveals the physiological state of cells. Membrane-damaged and thus mostly dead cells can be distinguished from viable cells. This is achieved by using a viability dye that only penetrates and stains damaged cells.

The ability to assess not only the presence but also the physiological conditions of cells is particularly useful for disinfection measures. It creates the basis for reassessing and improving today's disinfection and remediation measures, which unfortunately do not always lead to sustainable problem solving [4, 15]. New test methods also enable the optimization of sanitary and building technology, for example by improving piping systems and critical sampling points.

Automation using microfluidic technology

The rqmicro Cell-Stream device performs immunomagnetic cell separation automatically and efficiently. The integration of microfluidics in disposable cartridges notably reduces application errors and working time. The workflow is simple. After filtration of a water sample of up to one liter, the cells are resuspended by vortexing the filter in three mL buffer solution. Antibodies bound to magnetic particles or fluorophores respectively, are added to this cell suspension. After incubation, the samples are transferred onto the cartridge and processed automatically by the Cell-Stream device.

The fluid in the cartridge moves through the microfluidic channels by means of a patented microair pressure process — the fluid always remains in the cartridge and does not come into contact with the instrument.

There will be other kits launched in 2018

The Cell-Stream has been commercially available since mid-2016 with a kit for Legionella pneumophila SG1. In the first half of 2018, kits for L. p. SG1-15 and L. spp. will be launched. Further kits for the detection of Pseudomonas aeruginosa, E. coli, Salmonella, Giardia lamblia and Cryptosporidium parvum are under development.

After sample preparation with the Cell-Stream the samples can also be used for today's cultivation methods — with significant advantages. The separation and purification eliminates more than 95% of the competing flora that accompanies Legionella in surface waters, wastewater and cooling towers.

In addition, the acid and heat treatments required for the standard process but potentially detrimental to Legionella, become obsolete thanks to the sample preparation. This fact favours that Legionella present in a sample actually are able to grow on agar plates. Internal and external validation studies are underway and already show promising results. Alternatively, after sample preparation, target cells can also be used for PCR, MALDI-TOF or fluorescence microscopy.

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