Next-generation sequencing diagnostics of bacteremia in septic patients

Abstract

BackgroundBloodstream infections remain one of the major challenges in intensive care units, leading to sepsis or even septic shock in many cases. Due to the lack of timely diagnostic approaches with sufficient sensitivity, mortality rates of sepsis are still unacceptably high. However a prompt diagnosis of the causative microorganism is critical to significantly improve outcome of bloodstream infections. Although various targeted molecular tests for blood samples are available, time-consuming blood culture-based approaches still represent the standard of care for the identification of bacteria.MethodsHere we describe the establishment of a complete diagnostic workflow for the identification of infectious microorganisms from seven septic patients based on unbiased sequence analyses of free circulating DNA from plasma by next-generation sequencing.ResultsWe found significant levels of DNA fragments derived from pathogenic bacteria in samples from septic patients. Quantitative evaluation of normalized read counts and introduction of a sepsis indicating quantifier (SIQ) score allowed for an unambiguous identification of Gram-positive as well as Gram-negative bacteria that exactly matched with blood cultures from corresponding patient samples. In addition, we also identified species from samples where blood cultures were negative. Reads of non-human origin also comprised fragments derived from antimicrobial resistance genes, showing that, in principle, prediction of specific types of resistance might be possible.ConclusionsThe complete workflow from sample preparation to species identification report could be accomplished in roughly 30 h, thus making this approach a promising diagnostic platform for critically ill patients suffering from bloodstream infections.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0326-8) contains supplementary material, which is available to authorized users.

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform

“…It is unknown whether these bacterial organisms were actually present within the patient or alternatively their nucleic acids were introduced via laboratory reagents (51) or were sample carryover from a prior sequencing run. However, some of the detected bacteria such as Propionibacterium , a common skin inhabitant, or Ralstonia have been shown before to be present in human blood (52,53). The contig-based taxonomy analyses also clearly showed Ebola virus to be present, and confirmed that many contigs belonged to the same bacterial groups identified by read-based analyses.…”

Translational metagenomics and the human resistome: confronting the menace of the new millennium

“…A recent study used sequence-based metagenomics to detect free-circulating DNA from plasma samples of septic patients [86]. The authors analysed 62 samples from seven sepsis patients, six patients who underwent abdominal surgery and 12 healthy volunteers at different time points.…”

Bacterial sepsis

“…Discrimination between infection and colonization is made possible by calculating the Sepsis Indicating Quantifier (SIQ) score [98]. Analogous to PCR-based techniques, a further advantage of NGS is the prediction of particular resistance phenotypes [98]. NGS will become increasingly interesting in the future, since with this method-analogous to blood cultures-there exists the possibility of a widescale search for the pathogen which is independent of the pathogen growth rate.…”

“…In this method, cellfree DNA (cfDNA) is extracted from plasma samples, and all pathogens are detected and sequenced. Discrimination between infection and colonization is made possible by calculating the Sepsis Indicating Quantifier (SIQ) score [98]. Analogous to PCR-based techniques, a further advantage of NGS is the prediction of particular resistance phenotypes [98].…”