16S rDNA droplet digital PCR for monitoring bacterial DNAemia in bloodstream infections

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.

Droplet digital PCR for the detection and monitoring of Legionella pneumophila

“…However, although this represents a limit for culture methods and for molecular methods based on RT-qPcR, here we demonstrated that ddPcR is not affected by the presence of fragmented dNA or cell debris thanks to the nanopartitions of gene targets and the dilutions of contaminants into thousands of droplets. In this context, other studies support our findings and the use of ddPcR for the detection of bacterial dNAemia during infection or for the monitoring of bacterial load in contaminated samples with PcR inhibitors (27,28).…”

Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels

“…In the present study, we applied ddPCR to DNAs from single bacterial strains, bacterial mocks and metagenomic samples, demonstrating that it allows accurate absolute quantification of 16S rRNA gene copy numbers. Moreover, using DNA templates with different integrity levels, we demonstrated that 16S copy number quantification is strongly affected by DNA quality, a relevant issue that has not been addressed in previous studies [16, 20, 21, 29, 30]. Remarkably, we determined a precise correlation between quantification underestimation and DNA degradation levels, measured as DIN values, and demonstrated that a correction of the DNA mass, based on the underestimation value, allows one to estimate the 16S copy number accurately even in the most degraded metagenomic DNAs.…”

“…In the present study, we present data showing that ddPCR provides accurate quantification of 16S rDNA copy number in metagenomic DNAs. ddPCR is characterized by precise quantification, higher reproducibility compared to qPCR and higher sensitivity in low-copy-number detection [14, 20, 47]. Furthermore, ddPCR mitigates the effects of the presence of PCR inhibitors that may affect PCR sensitivity in 16S metabarcoding sequencing analysis [27, 48].…”

“…The method we propose to accurately quantify the total microbial content also makes suitable adjustments for DNA quality. Indeed, although ddPCR has already been applied for absolute quantification of bacteria [16, 20, 21] and viruses [29, 30], a suitable correction of input mass for DNA quality, which may vary substantially in different samples and conditions, has never been addressed.…”

“…Recently, these limitations have been overcome through the development of droplet digital PCR (ddPCR), which allows absolute DNA quantification without a standard curve and functions over a wide dynamic range with high sensitivity in low copy number detection [14–19]. Indeed, several examples of the application of ddPCR have recently been reported in the literature for the absolute quantification of microbial species [16, 20, 21].…”