Clinical validation of a multiplex droplet digital PCR for diagnosing suspected bloodstream infections in ICU practice: a promising diagnostic tool

Abstract

Background Droplet 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. Methods A 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. Results A 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. Conclusions The 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.

Quantification of bacterial DNA in blood using droplet digital PCR: a pilot study

“…These results indicate that the ddPCR assays used in this study have a limit of detection of 1-10 CFU/mL. This very low detection limit has also been found in other studies using other ddPCR assays [2,13,14] to detect bacterial pathogens in blood.…”

“…The copyright holder for this preprint this version posted December 2, 2022. ; https://doi.org/10.1101/2022.12.02.518639 doi: bioRxiv preprint antimicrobial resistance, allowing the reduction of the time from sample collection to results from at least 24-48h to a few hours, helping to guide and improve patients' treatment [2,13,14]; iii) severity stratification of the disease: high DNA loads have been associated to faster disease progression and greater mortality in patients with BSI [8,15]; iv) distinguish between colonization and infection: quantifying bacterial DNA load might help to determine whether a certain opportunistic pathogen might be just colonizing a given site or if it is causing an infection instead (e.g. intestinal infections and lung infections); v) and finally, detection and quantification of the transcriptome of bacterial toxins: the detection and quantification of mRNA of E. coli, Shigella and Clostridioides difficile toxins might help with the diagnosis of infections by these bacterial species and also to assess disease severity, prognosis and guide treatment.…”

“…The copyright holder for this preprint this version posted December 2, 2022. ; https://doi.org/10.1101/2022.12.02.518639 doi: bioRxiv preprint identify and quantify pathogens and their antimicrobial resistances directly in the blood [2,3]. Furthermore, time to blood culture positivity has been suggested as a surrogate marker of blood bacterial load and associated with poor clinical outcome in BSIs [6,7].…”

“…Therefore, the early identification and quantification of bacterial DNA load in blood of patients with suspected BSIs or sepsis could help to assess illness severity and further guide patient's treatment [2,4,8].…”

“…Even though the previously mentioned characteristics of ddPCR make it an attractive method for the identification and quantification of pathogens directly from blood or other clinical samples [2,4] in patients with suspected BSI and sepsis, very few studies have validated its performance to assist in BSI diagnosis. In this pilot study we aimed to demonstrate the specificity and sensitivity of ddPCR to detect and quantify bacterial DNA from Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus spp directly from blood.…”