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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.

SUBMITTER: Ziegler I

PROVIDER: S-EPMC6853374 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Ziegler Ingrid I Lindström Sofia S Källgren Magdalena M Strålin Kristoffer K Mölling Paula P

PloS one 20191113 11

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 ...[more]

PMID: 31721817

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Project description:BackgroundDroplet digital PCR (ddPCR) has emerged as a promising tool of pathogen detection in bloodstream infections (BSIs) in critical care medicine. However, different ddPCR platforms have variable sensitivity and specificity for diverse microorganisms at various infection sites. There is still a lack of prospective clinical studies aimed at validating and interpreting the discrepant ddPCR results for diagnosing BSI in intensive care unit (ICU) practice.MethodsA prospective diagnostic study of multiplex ddPCR panels was conducted in a general ICU from May 21, 2021, to December 22, 2021. Paired blood cultures (BCs) and ddPCRs (2.5 h) were obtained synchronously to detect the 12 most common BSI pathogens and three antimicrobial resistance (AMR) genes. Firstly, ddPCR performance was compared to definite BSI. Secondly, clinical validation of ddPCR was compared to composite clinical diagnosis. Sensitivity, specificity, and positive and negative predictive values were calculated. Thirdly, the positive rate of AMR genes and related analysis was presented.ResultsA total of 438 episodes of suspected BSIs occurring in 150 critical patients were enrolled. BC and ddPCR were positive for targeted bacteria in 40 (9.1%) and 180 (41.1%) cases, respectively. There were 280 concordant and 158 discordant. In comparison with BCs, the sensitivity of ddPCR ranged from 58.8 to 86.7% with an aggregate of 72.5% in different species, with corresponding specificity ranging from 73.5 to 92.2% with an aggregate of 63.1%. Furthermore, the rate of ddPCR+/BC- results was 33.6% (147/438) with 87.1% (128 of 147) cases was associated with probable (n = 108) or possible (n = 20) BSIs. When clinically diagnosed BSI was used as true positive, the final sensitivity and specificity of ddPCR increased to 84.9% and 92.5%, respectively. In addition, 40 blaKPC, 3blaNDM, and 38 mecA genes were detected, among which 90.5% were definitely positive for blaKPC. Further, 65.8% specimens were predicted to be mecA-positive in Staphylococcus sp. according to all microbiological analysis.ConclusionsThe multiplexed ddPCR is a flexible and universal platform, which can be used as an add-on complementary to conventional BC. When combined with clinical infection evidence, ddPCR shows potential advantages for rapidly diagnosing suspected BSIs and AMR genes in ICU practice.

Dataset Information

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

Publications

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

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