Droplet digital polymerase chain reaction for rapid broad-spectrum detection of bloodstream infections. 2020

Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
Intestinal Failure Unit, Department of Gastroenterology and Hepatology, Radboud University Medical Centre, Nijmegen, The Netherlands.

The droplet digital polymerase chain reaction (ddPCR) is a novel molecular technique that allows rapid quantification of rare target DNA sequences. Aim of this study was to explore the feasibility of the ddPCR technique to detect pathogen DNA in whole blood and to assess the diagnostic accuracy of ddPCR to detect bloodstream infections (BSIs), benchmarked against blood cultures. Broad-range primers and probes were designed to detect bacterial 16S rRNA (and Gram stain for differentiation) and fungal 28S rRNA. To determine the detection limit of ddPCR, 10-fold serial dilutions of E. coli and C. albicans were spiked in both PBS and whole blood. The diagnostic accuracy of ddPCR was tested in historically collected frozen blood samples from adult patients suspected of a BSI and compared with blood cultures. Analyses were independently performed by two research analysts. Outcomes included sensitivity and specificity of ddPCR. Within 4 h, blood samples were drawn, and DNA was isolated and analysed. The ddPCR detection limit was approximately 1-2 bacteria or fungi per ddPCR reaction. In total, 45 blood samples were collected from patients, of which 15 (33%) presented with positive blood cultures. The overall sensitivity of ddPCR was 80% (95% CI 52-96) and specificity 87% (95% CI 69-96). In conclusion, the ddPCR technique has considerable potential and is able to detect very low amounts of pathogen DNA in whole blood within 4 h. Currently, ddPCR has a reasonable sensitivity and specificity, but requires further optimization to make it more useful for clinical practice.

UI MeSH Term Description Entries
D002176 Candida albicans A unicellular budding fungus which is the principal pathogenic species causing CANDIDIASIS (moniliasis). Candida albicans var. stellatoidea,Candida stellatoidea,Dematium albicans,Monilia albicans,Myceloblastanon albicans,Mycotorula albicans,Parasaccharomyces albicans,Procandida albicans,Procandida stellatoidea,Saccharomyces albicans,Syringospora albicans
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000328 Adult A person having attained full growth or maturity. Adults are of 19 through 44 years of age. For a person between 19 and 24 years of age, YOUNG ADULT is available. Adults
D012336 RNA, Ribosomal, 16S Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis. 16S Ribosomal RNA,16S rRNA,RNA, 16S Ribosomal,Ribosomal RNA, 16S,rRNA, 16S
D012339 RNA, Ribosomal, 28S Constituent of the 60S subunit of eukaryotic ribosomes. 28S rRNA is involved in the initiation of polypeptide synthesis in eukaryotes. 28S Ribosomal RNA,28S rRNA,RNA, 28S Ribosomal,Ribosomal RNA, 28S
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide
D018805 Sepsis Systemic inflammatory response syndrome with a proven or suspected infectious etiology. When sepsis is associated with organ dysfunction distant from the site of infection, it is called severe sepsis. When sepsis is accompanied by HYPOTENSION despite adequate fluid infusion, it is called SEPTIC SHOCK. Bloodstream Infection,Pyaemia,Pyemia,Pyohemia,Blood Poisoning,Poisoning, Blood,Septicemia,Severe Sepsis,Blood Poisonings,Bloodstream Infections,Infection, Bloodstream,Poisonings, Blood,Pyaemias,Pyemias,Pyohemias,Sepsis, Severe,Septicemias

Related Publications

Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
November 2015, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc,
Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
December 2018, Journal of medical virology,
Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
July 2021, Analytical chemistry,
Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
January 2020, Frontiers in microbiology,
Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
October 2021, Microbiological research,
Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
July 2020, Malaria journal,
Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
August 2018, Analytical chemistry,
Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
February 2019, Analytical chemistry,
Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
April 2006, Journal of the Formosan Medical Association = Taiwan yi zhi,
Yannick Wouters, and Daisy Dalloyaux, and Anke Christenhusz, and Hennie M J Roelofs, and Heiman F Wertheim, and Chantal P Bleeker-Rovers, and René H Te Morsche, and Geert J A Wanten
February 2024, Hematology, transfusion and cell therapy,
Copied contents to your clipboard!