Development and Clinical Validation of a Droplet Digital PCR Method for Detection of Acinetobacter baumannii and Klebsiella pneumonia in Patients with Suspected Bloodstream Infections

The relatively long turnaround time and low sensitivity of traditional blood culture may delay the effective antibiotic therapy in patients with bloodstream infection (BSI). To reduce the morbidity and mortality of BSI, a rapid and sensitive pathogen detection method is urgently required. Acinetobacter baumannii and Klebsiella pneumonia are two major microorganisms responsible for BSI. Here we reported a novel droplet digital PCR (ddPCR) method that can detect A. baumannii and K. pneumonia in whole blood samples within 4 h, with a specificity of 100% for each strain and limit of detection at 0.93 copies/microliter for A. baumannii and 0.27 copies/microliter for K. pneumonia. Clinical validation in 170 patients with suspected BSIs showed that, compared with blood culture that reported 4 (2.4%) A. baumannii cases and 7 (4.1%) K. pneumonia cases, ddPCR detected 23 (13.5%) A. baumannii cases, 26 (15.3%) K. pneumonia cases, and 4 (2.4%) dual infection cases, including the 11 positive patients reported by blood culture. In addition, the positive patients reported by ddPCR alone (n = 42) had significantly lower serum concentrations of procalcitonin and lactate, SOFA and APACHE II scores, and 28-day mortality than those reported by both blood culture and ddPCR (n = 11), suggesting that patients with less severe manifestations can potentially benefit from the guidance of ddPCR results. In conclusion, our study suggests that ddPCR represents a sensitive and rapid method to identify causal pathogens in blood samples and to guide the treatment decisions in the early stage of BSI.

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Development and clinical validation of a droplet digital PCR assay for detecting Acinetobacter baumannii and Klebsiella pneumoniae in patients with suspected bloodstream infections

MicrobiologyOpen ◽

10.1002/mbo3.1247 ◽

2021 ◽

Vol 10 (6) ◽

Author(s):

Yang Zheng ◽

Jun Jin ◽

Ziqiang Shao ◽

Jingquan Liu ◽

Run Zhang ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Acinetobacter Baumannii ◽

Bloodstream Infections ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Clinical Validation ◽

Pcr Assay

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A Comparison of Blood Pathogen Detection Among Droplet Digital PCR, Metagenomic Next-Generation Sequencing, and Blood Culture in Critically Ill Patients With Suspected Bloodstream Infections

Frontiers in Microbiology ◽

10.3389/fmicb.2021.641202 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bangchuan Hu ◽

Yue Tao ◽

Ziqiang Shao ◽

Yang Zheng ◽

Run Zhang ◽

...

Keyword(s):

Next Generation Sequencing ◽

Blood Culture ◽

Critically Ill ◽

Critically Ill Patients ◽

Pathogen Detection ◽

Bloodstream Infections ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Next Generation ◽

Generation Sequencing

Metagenomic next-generation sequencing (mNGS) and droplet digital PCR (ddPCR) have recently demonstrated a great potential for pathogen detection. However, few studies have been undertaken to compare these two nucleic acid detection methods for identifying pathogens in patients with bloodstream infections (BSIs). This prospective study was thus conducted to compare these two methods for diagnostic applications in a clinical setting for critically ill patients with suspected BSIs. Upon suspicion of BSIs, whole blood samples were simultaneously drawn for ddPCR covering 20 common isolated pathogens and four antimicrobial resistance (AMR) genes, mNGS, and blood culture. Then, a head-to-head comparison was performed between ddPCR and mNGS. A total of 60 episodes of suspected BSIs were investigated in 45 critically ill patients, and ddPCR was positive in 50 (83.3%), mNGS in 41 (68.3%, not including viruses), and blood culture in 10 (16.7%) episodes. Of the 10 positive blood cultures, nine were concordantly identified by both mNGS and ddPCR methods. The head-to-head comparison showed that ddPCR was more rapid (~4 h vs. ~2 days) and sensitive (88 vs. 53 detectable pathogens) than mNGS within the detection range of ddPCR, while mNGS detected a broader range of pathogens (126 vs. 88 detectable pathogens, including viruses) than ddPCR. In addition, a total of 17 AMR genes, including 14 blaKPC and 3 mecA genes, were exclusively identified by ddPCR. Based on their respective limitations and strengths, the ddPCR method is more useful for rapid detection of common isolated pathogens as well as AMR genes in critically ill patients with suspected BSI, whereas mNGS testing is more appropriate for the diagnosis of BSI where classic microbiological or molecular diagnostic approaches fail to identify causative pathogens.

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A Multiplex, Droplet Digital PCR Assay for the Detection of T-Cell Receptor Excision Circles and Kappa-Deleting Recombination Excision Circles

Clinical Chemistry ◽

10.1373/clinchem.2019.308171 ◽

2019 ◽

Vol 66 (1) ◽

pp. 229-238 ◽

Cited By ~ 1

Author(s):

Tracie Profaizer ◽

Patricia Slev

Keyword(s):

Flow Cytometry ◽

T Cell ◽

T Cell Receptor ◽

Reference Gene ◽

Cell Receptor ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Blood Samples ◽

Healthy Donors ◽

Excision Circles

Abstract BACKGROUND T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count <20 copies/μL and 17.7% had a KREC count <20 copies/μL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.

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Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR

Clinical Chemistry ◽

10.1373/clinchem.2017.272518 ◽

2018 ◽

Vol 64 (2) ◽

pp. 317-328 ◽

Cited By ~ 21

Author(s):

Charles Decraene ◽

Amanda B Silveira ◽

François-Clément Bidard ◽

Audrey Vallée ◽

Marc Michel ◽

...

Keyword(s):

Liquid Biopsy ◽

Limit Of Detection ◽

Clinical Importance ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Droplet Digital Pcr ◽

Egfr Mutations ◽

Detection Accuracy ◽

Exon 2 ◽

Exon 19

Abstract BACKGROUND Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.

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A single droplet digital PCR for ESR1 activating mutations detection in plasma

10.1101/507608 ◽

2019 ◽

Author(s):

Emmanuelle Jeannot ◽

Lauren Darrigues ◽

Marc Michel ◽

Marc-Henri Stern ◽

Jean-Yves Pierga ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Metastatic Breast Cancer ◽

Limit Of Detection ◽

Growth Factor Receptor ◽

Metastatic Breast ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Plasma Samples ◽

Activating Mutations

AbstractBackgroundActivating mutations in the estrogen receptor 1 (ESR1) gene are recurrent mechanisms of acquired resistance to aromatase inhibitors (AI), and may be the target of other selective estrogen receptor down-regulators. To assess the clinical utility of monitoring ESR1 resistant mutations, a droplet digital PCR (ddPCR)-based assay compatible with body fluids is ideal due to its cost-effectiveness and quick turnaround.MethodsWe designed a multiplex ddPCR, which combines a drop-off assay, targeting the clustered hotspot mutations found in exon 8, with another pair of probes interrogating the E380Q mutation in exon 5. We assessed its sensitivity in vitro using synthetic oligonucleotides, harboring E380Q, L536R, Y537C, Y537N, Y537S or D538G mutations. Validation of the assay was performed on plasma samples from a prospective study and compared to next generation sequencing (NGS) data.ResultsThe multiplex ESR1-ddPCR showed a high sensitivity with a limit of detection ranging from 0.07 to 0.19% in mutant allele frequency depending on the mutation tested. The screening of plasma samples from patients with AI-resistant metastatic breast cancer identified ESR1 mutations in 29% of them with perfect concordance (and higher sensitivity) to NGS data obtained in parallel. Additionally, this test identifies patients harboring polyclonal alterations. Furthermore, the monitoring of ctDNA using this technique during treatment follow-up predicts the radiological response to palbociclib-fulvestrant.ConclusionThe multiplex ESR1-ddPCR detects, in a single reaction, the most frequent ESR1 activating mutations and is compatible with plasma samples. This method is thus suitable for real-time ESR1 mutation monitoring in large cohorts of patients.Statement of translational relevanceExons 5 and 8 mutations in ESR1 are recurrent mechanisms of resistance to aromatase inhibitors (AI) in estrogen receptor (ER)-positive metastatic breast cancer and may be targeted by selective ER down-regulators. We implemented a novel droplet digital PCR, which allows for the detection of the most frequent ESR1 mutations in circulating cell-free DNA. In prospectively collected plasma samples, ESR1 mutations were found in 29% of AI-resistant patients, with excellent concordance and higher sensitivity to next generation sequencing. Moreover, circulating ESR1 mutations appear to be reliable markers for ctDNA monitoring in order to predict treatment response. Ultimately, the short turnaround time, high sensitivity and limited cost of the ESR1-ddPCR are compatible with repeated samplings to detect the onset of resistance to AI before the radiological progression. This opens a window of opportunity to develop new clinical strategies for breast cancer hormone therapy, as tested in an ongoing phase 3 trial.List of abbreviationsAIAromatase InhibitorcfDNACell-free DNActDNACirculating tumor DNAddPCRDroplet digital PCRER+ HER2-MBCER+ HER2-negative Metastatic Breast CancerEREstrogen ReceptorER+Estrogen Receptor positiveLOBLimit of blankLODLimit of detectionMAFMutant Allele FrequencyPBMCPeripheral blood mononuclear cellsPDProgressive diseaseSDStandard deviationToPTime of progressionWTWild typeHuman genesESR1: Estrogen Receptor 1HER2: Human Epidermal Growth Factor Receptor 2EGFR: Epithelial Growth Factor ReceptorKRAS: KRAS proto-oncogene, GTPaseBRAF: B-Raf Proto-Oncogene, Serine/Threonine kinase

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A Droplet Digital PCR Assay to Detect SARS-CoV-2 RNA

10.1101/2020.05.06.20090449 ◽

2020 ◽

Author(s):

Martin J. Romeo ◽

Christian P. DiPaola ◽

Cassidy Mentus ◽

Cynthia D. Timmers

Keyword(s):

Limit Of Detection ◽

Digital Pcr ◽

The United States ◽

Cross Reactivity ◽

Droplet Digital Pcr ◽

Molecular Testing ◽

Guidance Document ◽

Respiratory Pathogens ◽

Limit Of Quantification ◽

United States Food

AbstractWe describe a quantitative droplet digital PCR (ddPCR) assay for detection of SARS-CoV-2 viral ribonucleic acid (RNA) in total RNA extracted from human sputum. This method was validated using the guidance of the United States Food and Drug Administration’s Accelerated Emergency Use Authorization (EUA) Template for SARS-CoV-2 that Causes Coronavirus Disease (COVID-19) Molecular Testing of Respiratory Speciment in CLIA Certified High-Complexity Laboratories. Though our laboratory is not CLIA certified, this method met all criteria specified by the guidance document with a Limit of Detection (LOD) of 0.25 copies/μL in the final ddPCR (at least 19/20 replicates reactive), which we consider to be a Lower Limit of Quantification (LLOQ); inclusivity of all known annotated SARS-CoV-2 genomes; no cross-reactivity with other respiratory pathogens; and reactivity of all contrived positives at or above the LOD.

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Droplet Digital PCR for the Detection of Plasmodium falciparum DNA in Whole Blood and Serum: A Comparative Analysis with Other Molecular Methods

Pathogens ◽

10.3390/pathogens9060478 ◽

2020 ◽

Vol 9 (6) ◽

pp. 478

Author(s):

Elena Pomari ◽

Ronaldo Silva ◽

Lucia Moro ◽

Giulia La Marca ◽

Francesca Perandin ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Dna Analysis ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Clinical Samples ◽

Clinical Parameters ◽

Serum Samples ◽

Absolute Quantitation ◽

Blood Samples ◽

Paired Samples

Background: The estimation of Plasmodium falciparum parasitaemia can vary according to the method used. Recently, droplet digital PCR (ddPCR) has been proposed as a promising approach in the molecular quantitation of Plasmodium, but its ability to predict the actual parasitaemia on clinical samples has not been largely investigated. Moreover, the possibility of applying the ddPCR-sensitive method to serum samples has never been explored. Methods: We used, for the first time, ddPCR on both blood and serum to detect the DNA of P. falciparum in 52 paired samples from 26 patients. ddPCR was compared with loop-mediated isothermal amplification (LAMP) and rtPCR. The correlation between the ddPCR results, microscopy, and clinical parameters was examined. Results: ddPCR and microscopy were found to be strongly correlated (ρ(26) = 0.83111, p < 0.0001) in blood. Samples deviating from the correlation were partially explained by clinical parameters. In serum samples, ddPCR revealed the best performance in detecting P. falciparum DNA, with 77% positive samples among malaria subjects. Conclusion: Absolute quantitation by ddPCR can be a flexible technique for Plasmodium detection, with potential application in the diagnosis of malaria. In particular, ddPCR is a powerful approach for Plasmodium DNA analysis on serum when blood samples are unavailable.

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