The faster, the better
Currently, sepsis is one of the main causes of health loss – up to 50 million cases of sepsis and 11 million deaths due to sepsis per year are registered annually. Reliable and early identification of the pathogen helps to quickly select the most appropriate antibiotic, increasing the likelihood of better treatment results and patient survival. Currently, standard diagnostics is still based on microbiological cultivation of pathogens, which in most cases (from 70 to 90%) does not allow to obtain a result in a timely and accurate manner.
The next-generation sequencing technique in real time, created by researchers from the University of Heidelberg and the Institute of Interfacial Engineering and Biotechnology. Fraunhofer, Germany, analyzes a small amount of microbial extracellular DNA in the plasma of patients with sepsis, making it possible to accurately diagnose the causative agents of sepsis within a few hours after taking blood.
It includes several innovative processes used to sequence nucleotides in DNA.
In their previous work, the researchers described a system using next-generation sequencing to detect pathogens in the blood, which was more sensitive and faster than standard blood culture (the result was ready within 28 hours). However, the ultimate goal of the researchers was to develop an even faster test for earlier initiation of treatment.
To overcome this limitation, the researchers used high-performance nanopore sequencing of microbial DNA of the third generation for diagnostics. Typically, nanopore sequencing is used to analyze a sufficient number of long DNA fragments. However, microbial extracellular DNA circulates in the blood in small fragments and in small quantities. Nanopore sequencing enables real-time analysis, significantly reducing the time required to obtain results. The researchers also created specially adapted bioinformatic procedures for reliable identification of pathogenic microorganisms.
The new method is based on the use of a MinION manual sequencer.
A nanopore sequencer used to analyze the DNA of microorganisms in patients with sepsis. Source: Fraunhofer IGBI.
In this study, the authors analyzed plasma taken from four sepsis patients and three healthy people. The DNA of each sample was sequenced using two technologies: standard Illumina sequencing and new nanopore sequencing. In the second case, all samples of patients with sepsis tested positive for the corresponding bacterial, viral or fungal pathogens.
After additional improvements, the new technology increased the sequencing throughput by 3.5 times – up to 5-6 hours. With the Illumina technique, the final results were available only after the sequencing was completed, after 24-30 hours.
The new system can facilitate the choice of antibacterial intervention in intensive care units and intensive care units, giving results within one medical shift. This, in turn, can significantly improve patient outcomes.
The researchers also compared the probability of pathogen detection using the MinION system with the highly accurate and clinically validated Illumina technique in a retrospective analysis of 239 samples taken from patients with sepsis. Although the accuracy of nanopore sequencing was lower than using Illumina (approximately 85% vs. 99%), they found a strong correlation between the results obtained by both methods.
Blood culture shows results in a few days at best, is prone to errors and contamination, but it is still considered the standard for the diagnosis of sepsis. The nanopore sequencing platform can reduce the diagnostic time to several hours. The authors hope that it will soon replace the sepsis diagnostic standard.
Article by C.W.Stratton et al. Diagnosing Bacteremia in Real Time Using Next-Generation Sequencing–Based Technology is published in the Journal of Molecular Diagnostics.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on Elsevier: New next-generation sequencing technique dramatically shortens diagnosis of sepsis.