FYI-Chip – Detection of human fungal pathogens using a lab-on-a-chip device

Infections by yeasts and mold fungi lead to severe illnesses, especially in immunocompromised patients. With a mortalityrate of between 30 to 80 percent, the rapid detection of a pathogen, including its resistance spectrum, plays a particularly decisive role in the success of treatment. The classical detection of pathogens using culture-based methods (Microdilution,Etest®) can take up to 14 days for yeasts and mold fungi. Furthermore, clinical studies have shown that phenotypic resistance testing is subject to an error rate of up to15 percent. Cultivation often fails completely, even when the patient displays clear clinical symptoms. These cases require the initiation of a therapy of suspicion, which cannot be specifically matched to the pathogen.

Because of this, molecular biology methods such as sequencing, fluorescence-in-situ-hybridization (FISH), PCR or quantitative real-time PCR (qRT-PCR) are increasingly being used for the identification of pathogens. However, these methods have a limited multiplex capability. This means that only a small number of a large amount commonly occurring pathogensor resistances can be tested for (≤ 10 parameters). This makes numerous cost-intensive tests necessary, reducing the time advantage of the method.

It is possible to compensate for this diagnostic gap by the use of DNA microarrays, which enable the simultaneous examination of up to several thousand parameters. Such tests were previously rarely used in routine diagnostics due, among other things, to the high experimental and instrument costs of the processing of microarrays. These problems can be minimized by the application of microsystems that combine the entire testing process into a so-called lab-on-a-chip (LOC).

The Fraunhofer IGB and the Institute for Interfacial Engineering IGVT of the University of Stuttgart, in combination with partners from medicine, science and industry and within the BMBF-funded research project “FYI-Chip – Fungi Yeast Identification”, are therefore developing a fully integrated lab-on-a-chip system for the rapid identification of fungal infections in respiratory secretions and primarily sterile body fluids in immunocompromised patients. The scientists at the Fraunhofer IGB and IGVT are therefore closely working with the company Euroimmun, based in Lübeck, Germany, with doctors at the Heart and Diabetes Centre NRW, as well as with developers at the Reutlinger Multi Channel Systems MCS GmbH and Robert Bosch GmbH, Gerlingen. The aim is to combine the individual functional components such as sample preparation, microfluidics and the detection of pathogenic DNA into a single fully integrated LOC.

Within the project, the IGVT has developed PCR systems and DNA probes for over 50 relevant fungal pathogens (including Candida or Aspergillus species.) In order to identify this amount of pathogens PCR systems have been developed for genes that show highly conserved areas, but are also variable enough to enable discrimination between the species. These are currently being tested for their suitability in LOC evaluation models. The Fraunhofer IGB is developing LOC-compatible cell disruption techniques for fungal species. The use of disposable cartridges for the chip makes the system flexible and economical.

As a miniature laboratory, the LOC combines sample preparation directly on the chip with highly sensitive and rapid molecular biological diagnostics of yeasts and mold fungi and their resistances. This enables them to support clinicians in their diagnoses and facilitate the rapid and adequate initiation or adjustment of therapy. The LOC system is designed as an open system that might be used for further sample materials, such as biopsy tissues or for the detection of bacterial pathogens and their antibiotic resistance markers in the future.

• Euroimmun Medizinische Labordiagnostika AG, Lübeck (coordinator)
• Heart and Diabetes Centre NRW, Bad Oeynhausen
• Institute for Interfacial Engineering IGVT, University of Stuttgart
• Multi Channel Systems MCS GmbH, Reutlingen
  
• Robert Bosch GmbH, Gerlingen

We would like to thank the German Federal Ministry of Education and Research (BMBF) for their funding of the project “FYI – Fungi Yeast Identification”, promotional reference 01EZ1113F.