Infections – Diagnostics

With next-generation sequencing (NGS) technologies, several hundred million fragments in a sample can be simultaneously sequenced and matched with known gene sequences – and thus the entire genome of organisms can be analyzed within just a few hours.

On the basis of these technologies researchers at Fraunhofer IGB have developed an alternative diagnostic platform for sepsis. This enables them to identify bacteria, fungi or viruses directly by means of a sequence analysis of their DNA or RNA – without having to cultivate the pathogens beforehand in the laboratory. In clinical studies carried out in cooperation with Heidelberg University Clinic, the diagnostic method was validated with blood samples from sepsis patients. Within only 24 hours, the NGS diagnostic method provided information about which pathogens the patients were infected with.

Further advantages: If samples contain not only DNA of bacteria, but also that of viruses or fungi, these are sequenced, analyzed and identified as well. For example, in this study a viral pathogen could be identified as the cause of a patient’s illness. Yet the patient’s blood culture showed a negative result because with blood culture only bacteria and fungi can be detected. The resistance of bacteria to commonly used antibiotics such as methicillin, vancomycin or tetracycline is deduced from corresponding resistance genes. Therefore high-throughput sequencing makes it possible, in the same analysis, to identify both the biological species of the pathogen and its resistance genes. This also helps the physician treating the patient to set in motion a targeted therapy.

Infection diagnostics 3.0

To further reduce the time to diagnosis, the scientists are investigating how the method can be transferred to most recent sequencing platforms. With the help of latest single molecule DNA sequencing technologies of the third generation, which the IGB was involved in testing, DNA can be sequenced in even shorter time than before: We have been able to show that microbial DNA can be analyzed in real time during sequencing and that pathogen identification can thus be reduced to six to eight hours [1].

Thus, the reliability of sequencing-based diagnostics can be combined with the speed advantage of real-time analysis in order to provide patients with optimal antibiotic therapy as quickly as possible in the future.

_{[1] Grumaz, C; Hoffmann, A.; Vainshtein, Y.; Kopp, M.; Grumaz, S.; Stevens, P.; Decker, S. O.; Weigand, M. A.; Hofer, S.; Brenner, T.; Sohn, K. (2020) Rapid next generation sequencing-based diagnostics of bacteremia in septic patients. Journal of Molecular Diagnostics 22 (3): 405 DOI: 10.1016/j.jmoldx.2019.12.006}

Lateral Flow Assays (LFA) are based on the chromatographic separation of a diverse range of molecules. Since they can be carried out even in absence of infrastructure they are suitable as point-of-care tests (POCT). While numerous LFA formats already exist for a long time, nucleic-acid based detection methods are under-represented and require further development for diagnostic implementation.

Therefore, a Nucleic Acid Lateral Flow Assay (NALFA) for the detection of pathogens was established at Fraunhofer IGB in cooperation with the High Performance Center for Mass Personalization with the detection of Herpes simplex virus 1 as a proof of concept. For this purpose, the genomic DNA of the virus was specifically amplified using a polymerase chain reaction (PCR) and modified for detection in the LFA. By modifying the amplified genomic region of a pathogen, a color signal is generated during hybridization with the immobilized and complementary capture DNA, which serves for specific detection.

Based on these results, further developments are planned, which on the one hand include an infrastructure-independent amplification of the pathogen genome and on the other hand the detection of several pathogens on one test strip. This should enable rapid on-site diagnostics of infectious pathogens even in regions with poor infrastructure, so that a specific therapy can be initiated promptly and further spread of the pathogens can be counteracted.

DNA microarrays are highly effective detection systems for nucleic acids. They can be used to detect and discriminate against infectious agents. This is particularly important for the diagnostics of diseases where a highly parallel detection and discrimination of pathogens is required.

One focus of the institute is the rapid identification of pathogens and the detection of antibiotic resistance using DNA arrays. For more than 10 years we have been developing DNA microarrays for customers from companies, clinics and research institutions, individually adapted to specific indications. The infrastructure in the institute enables the complete production, from the establishment of multiplex PCR and amplification of pathogen targets, probe design and immobilization of probes by contact printing to the hybridization of target DNAs for fluorescence-based identification of pathogens.

The basic principle of a microarray is the ordered, point-like immobilization of up to thousands of short defined DNA probes on a solid support. Using hybridization, the immobilized DNA probes which are complementary to the pathogen target sequence allow to capture the genomic target of respective pathogens. To this end, target sequences of pathogens present in a patient sample are amplified by PCR and detected by binding to specific immobilized probes. Simultaneously present probes, each specific for a particular pathogen, allow the parallel analysis of pathogens or resistances. In this way, complex causes of disease can be detected and discriminated against in just a few hours in a single sample and and a single diagnostic approach.

For example, in the BMBF-funded project Fungal Yeast Identification, we have developed a fully integrated lab-on-a-chip system for the rapid determination of approximately 50 yeast and fungi infections in cooperation with the companies Euroimmun, Multi Channel Systems MCS and Bosch as well as the Heart and Diabetes Centre North Rhine-Westphalia. In addition, a DNA microarray platform was developed in close cooperation with GATC Biotech AG, with which sepsis pathogens (bacteria and fungi) as well as their relevant resistances to antibiotics can be detected and identified in parallel. On behalf of Immundiagnostik AG Bensheim, we have developed DNA-based microarrays for the highly parallel diagnosis of fungal infections and sexually transmitted infectious diseases caused by fungi, bacteria, viruses or protozoa.

Different classes of immune receptors are of key importance in the innate system. As the name pattern-recognition-receptors (PRRs) suggests, these receptors recognize conserved molecular patterns of infectious pathogens and isolated chemical structures. Stimulating these receptors activates various signaling cascades and transcription factors that induce an immune response. Thus, the PRRs play an important role in fighting pathogens. However, by inducing pro-inflammatory cytokines, they can also contribute to pathologic processes in acute and chronic inflammatory diseases in humans.

The largest and best-known family of PRRs are the toll-like-receptors (TLRs). The IGB was able to develop test systems for pathogens and microbial components (e.g. in pharmaceutical products or medical engineering equipment) by targeted use of these sensors. In addition to cell-based assays, the ImmuStick also demonstrated a competitive immunoassay as a test strip for the detection of pyrogens with immobilized immune receptors.These assays are also well suited to detect microbial contaminations. This invention was developed and patented (patent WO002008003489A8). The use of the ImmuStick test strip for the classification of sepsis pathogens directly on the patient is a further possibility.

Similarly, whole cell biosensor assays were established. These can identify new immune modulatory molecules, which are potential drug candidates for the treatment of the diseases mentioned above. This approach is described in the chapter "Drug development".