Abstract
Repeated quantitative measurement of bacterial DNA on whole blood has been shown to be a promising method for monitoring bloodstream infection (BSI) with selected bacterial species. To enable broad use of this method, we developed a quantitative droplet digital PCR (ddPCR) method for 16S rDNA. It was validated with species-specific ddPCRs for Staphylococcus aureus (nuc), Streptococcus pneumoniae (lytA), and Escherichia coli (uidA) on spiked whole blood samples and on repeated whole blood samples (days 0, 1–2, 3–4, 6–8, and 13–15) from 83 patients with BSI with these pathogens. In these patients, 16S rDNA and species-specific DNA were detected in 60% and 61%, respectively, at least at one time-point. The highest positivity rates were seen in S. aureus BSI, where 92% of the patients were 16S rDNA-positive and 85% nuc-positive. Quantitative 16S rDNA and species-specific DNA showed strong correlations in spiked samples (r = 0.98; p < 0.0001) and clinical samples (r = 0.84; p < 0.0001). Positivity for 16S rDNA was rapidly cleared in patients with S. pneumoniae and E. coli BSI, but more slowly and sometimes persisted, in those with S. aureus BSI. The initial 16S rDNA load was higher in BSI patients with sepsis (Sepsis-3 definition) than without sepsis (median 2.38 vs. 0 lg10 copies/mL; p = 0.031) and in non-survivors than in survivors (median 2.83 vs. 0 lg10 copies/mL; p = 0.006). 16S rDNA ddPCR appears to be a promising method for bacterial DNA monitoring during BSI. The clinical value of such monitoring should be further studied.
Highlights
Bloodstream infections (BSI) frequently lead to sepsis, one of the most common causes of death worldwide [1]
Results from the 16S rDNA and the species-specific droplet digital PCR (ddPCR) for cases shown positive by both methods were significantly correlated for S. aureus (r = 0.92, p < 0.0001) and S. pneumoniae (r = 0.83, p = 0.005), but not for E. coli (r = 0.57, p = 0.11)
In this study we developed a ddPCR for quantification of 16S rDNA in whole blood and validated it against species-specific ddPCRs by analyzing bacterial-spiked blood samples in 10-fold dilutions and blood samples from patients with S. aureus, S. pneumoniae, and E. coli BSI
Summary
Bloodstream infections (BSI) frequently lead to sepsis, one of the most common causes of death worldwide [1]. Apart from pathogen identification, some molecular methods such as quantitative PCR enable. 16S rDNA load in bloodstream infections quantification of bacterial DNA. A few other studies have shown that the dynamics of bacterial DNA load reflect the disease course and response to treatment [12,13,14]. It has been proposed that repeated quantitative measurement of bacterial DNA could be used to evaluate the appropriateness of antibiotic treatment [15]. These studies of DNA dynamics have focused on DNA from only one bacterial species at a time. A diagnostic tool for DNA dynamics should monitor DNA from many different bacterial species
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