Innovative and sensitive detection of microbial cells.

This project focuses on the development of procedures that allow rapid detection and analysis of low numbers of cells and / or microbes from large complex suspensions.

Innosieve, as partner in this project, focuses on the development of a procedure to allow the detection and quantificatrion of microbes in blood.

Currently analysis of a blood sample relies on culturing using enrichment bottles and takes 24-48 hours. In case of presence of microbes a microscopy slide is prepared of the sample, next to plating the sample for microbial growth. These tests require an additional 1-2 days. In recent years a lot of efford has been put in direct detection and identification of the microbes directly after the first enrichment. Methods that have been applied are for example FISH, Q-PCR, MALDITOF etc. The advantage of these methods is that within several hours the micro-organism can be identified.

However, the first enrichment is still required, so no earlier than 24 hours after sampling an inidication can be given about the presence / absence of microbes in the blood sample. Up to now, no method has yet achieved a reduction in this first enrichment time, or even has made in possible to omit the enrcihment step.

The microsieve concept might fullfill this need. The diagnostic principle is based on an innovative microsieve as diagnostic platform, which is analysed using a novel scanning device, the µScan. In total filtration and detection require only 30 minutes until results. All micro-organisms in a blood sample are concentrated on the microsieve surface, stained specifically including viability staining and detected / quantified using the LED-based µScan device.

To accomplish this, it is of utmost importance that the bacteria are separated from the blood cells before application onto the microsieve. This sample preparation is essential as it is recognized that the blood cells will hamper the diagnostics too significantly. Several approaches will be evaluated based on density, size separation or fractionated filtration.

The initial principle consists thus of three steps; 1: blood pretreatment / preparation 2: filtration of suspension with micro-organisms using a microsieve 3: specific staining of bacteria followed by detection In this project these different steps will be evaluated / developed, during which a lot of attention will be put in blood sample preparation prior application onto the microsieve.