Rapid and unambiguous detection of infection in a miniature laboratory
Bacteria or fungi that have caused an infection need to be detected as rapidly as possible in order to save the lives of people with serious and life-threatening complications. Up until now, blood cultures have been used to detect the presence of pathogens. However, such tests take far too long and often do not provide an unambiguous result. In cooperation with industrial partners, researchers at the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB in Stuttgart have spent many years developing innovative diagnostic tools for use in infection biology. Among these are microsystems that combine complete test procedures on a “lab-on-a-chip” (LOC) and enable simultaneous analysis of several thousand parameters relatively quickly and with little effort. The scientists hope that these systems will soon be ready for application in routine diagnostics.
The human body copes with infections – either bacterial or fungal – more or less well depending on whether the infected person is healthy and on the type of pathogen that has caused the infection. A bacterial or fungal infection can quickly become life-threatening in immunocompromised, elderly or intensive care patients. In order to stop this happening, rapidly detecting the pathogen, and ideally also its resistance spectrum, is crucial for successful treatment. However, standard detection of pathogens still uses culture-based methods that can take up to several weeks to deliver results. This is often far too long. Cultivation often fails completely even when the patient displays clear clinical symptoms. Such cases require suspicion-based therapy which cannot be matched specifically to the pathogen. It is then impossible to tell whether the drug is effective or not.
For this reason, molecular biology methods are used to identify the pathogens and potential resistances whenever possible. Although techniques such as polymerase chain reaction (PCR) and sequencing can provide a treating physician with relevant information within a relatively short time, they are quite complex and only have limited multiplex capability in everyday clinical routines, which means that only a small number of the large number of commonly occurring pathogens or resistances can be tested for. Diagnosis therefore boils down to a game of chance: if the disease-causing pathogen is identified at the first attempt, therapy can be adjusted specifically to the pathogen. If not, either many more cost-intensive tests will need to be carried out, reducing the time advantage of the method, or drugs will need to be prescribed on suspicion.
LOC: miniature laboratory the size of a microscope slide detects infections
Researchers in the Department of Molecular Biotechnology at the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB in Stuttgart have therefore been focussing on techniques that can be used to identify pathogens rapidly and unambiguously, and are at the same time simple and cheap to use in daily laboratory routine. The researchers’ goal is to develop a so-called lab-on-a-chip (LOC), “an entire laboratory in a minute device,” as PD Dr. Susanne Bailer, who heads up the research group, calls it. Ideally, the patient sample, blood for example, is pipetted directly into the device. The components to be tested, i.e. the pathogen cells, are broken up and separated from the host components fully automatically. The pathogen DNA is then isolated and PCR used to amplify a specific region of the DNA. PCR uses fluorescence-labelled primers to amplify this particular region. Pathogen-specific probes consisting of only a few nucleic acids are used to detect the pathogen-specific region of the DNA, and hence the presence or absence of a particular pathogen on a microarray.
The probes are immobilised in small spots in rows and columns on a glass surface. Such an arrangement is called a microarray. The hybridisation (i.e. complementary binding) of the probe and the target can then be detected using a specific scanner (reader) that visualises fluorescence. A detectable signal means that the correct base pairing has occurred and the pathogen that has caused the disease has been identified. “This can be done with several genomes and several thousand parameters in parallel,” says Bailer, adding: “Ideally, everything is interlinked in such a way that the device will only spit out the definite diagnosis.”
Reactions on microfluidic surfaces
An investigation carried out within a short timescale, while remaining affordable and applicable in daily laboratory routine, can only be achieved through the miniaturisation of devices and the use of small amounts of samples and chemicals. The systems developed by the Fraunhofer IGB and its industrial, scientific and medical partners are therefore based on the principles of microfluidics: all reactions are performed on microfluidic surfaces in miniature laboratories the size of microscope slides. “For example, the sample is applied to tiny chambers and fine channels where the cells are then broken up,” says the infection biologist, explaining how one of their tests works. “The enzymes required by the PCR are also included, as the entire system is a closed one. To amplify the target region, the reaction mixtures are pumped between chambers with different temperatures. Everything is controlled microelectronically. It is all highly technical.”
In the development of the miniature laboratories, the IGB is responsible for PCR systems and DNA probes. For example, in the BMBF-funded project “FYI – Fungi Yeast Identification”, the IGB researchers have already developed systems that can differentiate between different yeast and fungus species or Actinobacter subtypes. The system allows the simultaneous identification of several species. The rapid and unambiguous detection of pathogens is made possible by amplification reactions that can be run in parallel (multiplexing). “We focus on the biological aspects of the LOCs,” says Bailer. “We are responsible for the design of the probes and their specificity and sensitivity as well as the design of the PCR systems. As part of the FYI project, we have developed an array that can be used to differentiate over 50 different species and their resistances within a short time."
Development of customised systems for industry
The IGB researchers are very flexible as far as the design of their system is concerned. “We can develop a system to suit a particular industry’s needs, whatever they may be,” says the scientist. “For example, we have also developed a system for diagnosing sepsis. Sepsis is a life-threatening disease that needs to be treated rapidly and if possible independently from a central laboratory. This is perfect for lab-on-a-chip systems.” Bailer also mentions LOC development projects that targeted cancer, but were not pursued as intensively as LOCs for detecting infectious diseases. Cancer does not need to be diagnosed as rapidly as infectious diseases. Moreover, in the case of cancer, it would be good to have an expert there to explain the results. “In general, LOC approaches have been around for quite a while. However, I get the impression that this field of research is currently being pushed to the fore to a greater extent than before, so other concrete applications will probably be available within the foreseeable future,” concludes Bailer.