NGS-based diagnostics of sepsis pathogens

Sepsis is caused by infections of the bloodstream and is responsible for approximately 60,000 deaths every year in Germany alone. In order to quickly and reliably diagnose the pathogens that cause the disease, the innovation field is working with leading hospitals in Germany to develop and evaluate a diagnostic method based on high-throughput sequencing (NGS) of circulating nucleic acids in plasma.

Disadvantages of microbial methods of pathogen identification

Classical methods for pathogen identification usually are based on microbiological detection of the pathogen using so-called blood cultures with subsequent identification of the corresponding species using microscopic, biochemical or mass spectroscopic methods. Disadvantages of the microbiological approach are low detection rates and longer times required for an unambiguous diagnosis. In addition, the result is often negative even though the disease is actually caused by an infection, because some pathogens cannot be cultivated at all or only under laboratory conditions.

NGS diagnosis of pathogens

For this reason, we develop innovative molecular methods for pathogen diagnostics on behalf of and in cooperation with customers and partners from industry and hospitals. These new methods are based on molecular analyses of the genetic information of pathogens. The three-stage process comprises optimal sample preparation, high-throughput sequencing (Next-Generation Sequencing, NGS) and bioinformatics analysis using proprietary diagnostic algorithms.

Advantage: Fast and reliable diagnosis

The new technology avoids time-consuming cultivation procedures and makes possible the detection of all pathogens: viruses, parasites and bacteria that do not grow on the culture media used. This not only makes the diagnosis faster, but also significantly more reliable. The NGS data can also be used for pathogen diagnostics, or for the identification of new biomarkers.

Application areas of NGS diagnostics

We have extensive experience in the indications of sepsis, endocarditis, amniotic fluid infections, as well as in biomarker screening, the genome characterization of pathogens and microbial studies (e.g. on the skin).

Thanks to its comprehensive, open platform character, this type of infection diagnostics already holds the potential for universal use in hospitals. With third-generation sequencers, immediate point-of-care diagnosis is also within reach, as we have been able to show in successful pilot studies.

© Fraunhofer IGB
Three-stage process for molecular infection diagnostics using NGS.

More reliable next-generation diagnostics for sepsis pathogens

Sepsis diagnosis
© Fraunhofer IGB
Automated processing of clinical samples for next-generation sequencing diagnostics.

In sepsis management, fast treatment using the correct antibiotic is crucial for patient survival. This, however, is not always possible in a targeted manner, since using the current standard (blood culture) pathogen detection is successful in only approximately 30 percent of cases. Therefore, a novel, molecular diagnostic and bioinformatic method has been developed at Fraunhofer IGB to detect DNA fragments of pathogens in patient blood using parallel sequencing (next-generation sequencing, NGS) and bioinformatic algorithms, allowing for highly-specific and sensitive identification of pathogens [2, 3].

 

Sepsis study in collaboration with the University Hospital Heidelberg

As part of a recent clinical study in collaboration with the University Hospital Heidelberg, we were successful in achieving significantly more positive results on pathogen identification with this technology compared to using blood culture (71 percent compared to 11 percent) in 50 patients with septic shock [1]. A jury of independent intensive care medics considered 96 percent of the positive NGS results to be plausible.

According to the jury, treatment of 53 percent of the patients would have been adapted subsequently on the basis of these results, since they were often over- or undertreated due to the empirical antibiosis. In this group of patients not receiving adequate treatment, the mortality rate was increased by 13 percent [1].

These concrete effects on patient treatment success convincingly demonstrate the enormous potential of more reliable and sensitive pathogen diagnostics. The retrospective observations will now be validated in a multicenter study with approximately 15 hospitals in Germany, expected to start in January 2019.

Infection diagnostics 3.0

DNA high-throughput sequencer
DNA high-throughput sequencer of the 3rd generation.

Reliable and timely diagnosis of pathogens in critically ill patients still poses major problems for intensive care medicine. By establishing a diagnostic procedure based on high-throughput sequencing (NGS) of microbial DNA from circulation of patient blood, Fraunhofer IGB has already been able to establish a procedure for the reliable diagnosis of pathogens in the past, which has a detection rate five to six times better than that of culture-based procedures [1, 3].

 

Third generation single molecule DNA sequencing technologies for real-time point-of-care diagnostics

With the help of latest single molecule DNA sequencing technologies of the third generation, this method has now been further developed so that real-time analysis of microbial DNA is already possible during sequencing. Consequently, the identification of pathogens can be reduced to six to eight hours [4]. 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.

Literature

[1] Grumaz, S.; Grumaz, C.; Vainshtein, Y.; Stevens, P.; Glanz, K.; Decker, S.O.; Hofer, S.; Weigand, M.A.; Brenner, T.; Sohn, K. (2019) Enhanced performance of next-generation sequencing diagnostics compared to standard of care microbiological diagnostics in patients suffering from septic shock, Critical Care Medicine, doi: 10.1097/CCM.0000000000003658

[2] Decker, S.O.; Sigl, A.; Grumaz, C.; Stevens, P.; Vainshtein, Y.; Zimmermann, S.; Weigand, M.A.; Hofer, S.; Sohn, K; Brenner, T. (2017) Immune-response patterns and next generation sequencing diagnostics for the detection of mycoses in patients with septic shock – results of a combined clinical and experimental investigation, International Journal of Molecular Sciences 18, 1796, doi: 10.3390/ijms18081796

[3] Grumaz, S.; Stevens, P.; Grumaz, C.; Decker, S.O.; Weigand, M.A.; Hofer, S.; Brenner, T.; von Haeseler, A.; Sohn, K. (2016) Next-generation sequencing diagnostics of bacteremia in septic patients, Genome Medicine 8:73, doi: 10.1186/s13073-016-0326-8

[4] 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