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.
Highlights
Bloodstream infections remain one of the major challenges in intensive care units, leading to sepsis or even septic shock in many cases
Elevated levels of cell-free DNA (cfDNA) in septic patients reveal microbial DNA fragments In order to test the diagnostic potential of cfDNA to identify infecting microorganisms in septic patients, in total 62 plasma samples from septic patients (S, n = 7), healthy volunteers (V, n = 12), and patients that underwent abdominal surgery (P, n = 6) at different time points were analyzed in this study
Due to the small cohort size, it was not possible to draw statistically relevant conclusions; concentrations of cfDNA tend to be increased in patients with septic shock (S, mean 197.23 ng/ml), especially at the onset of sepsis (S T0, mean 377.30 ng/ml) (Fig. 1), which is consistent with other reports [21, 22]
Summary
Bloodstream infections remain one of the major challenges in intensive care units, leading to sepsis or even septic shock in many cases. Despite massive efforts in sepsis research, new therapeutic approaches are rare and mortality in patients with septic shock still remains unacceptably high [1, 2]. The identification of Grumaz et al Genome Medicine (2016) 8:73 context, culture-independent molecular diagnostic procedures (e.g., PCR-based techniques) have already been introduced for the identification of the causative pathogen in infected patients [5,6,7,8,9]. The concept of our work was to develop an alternative diagnostic platform for the identification of infectious microorganisms based on unbiased sequence analyses of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS). We demonstrate the applicability of NGS for the detection of antibiotic resistance markers in plasma
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