Clinical evaluation of a multiplex droplet digital PCR for pathogen detection in critically ill COVID-19 patients with bloodstream infections

Abstract

Background: Nosocomial bloodstream infections (nBSI) have emerged as a clinical concern for physicians treating COVID-19 patients. In this study, we aimed to evaluate the effectiveness of a multiplex ddPCR in detecting bacterial pathogens in the blood of COVID-19 critically ill patients.

Methods: This prospective diagnostic study included RT-PCR-confirmed COVID-19 patients admitted to our hospital from December 2022 to February 2023. A multiplex ddPCR assay was used to detect common bacterial pathogens and AMR genes in blood samples of the patients, along with antimicrobial susceptibility testing (AST). The diagnostic performance of the ddPCR assay was evaluated by comparing the results with those obtained through blood culture and clinical diagnosis. Additionally, the ability of ddPCR in detecting bacterial resistance was compared with the AST results.

Results: Of the 200 blood samples collected from 184 patients, 45 (22.5%) were positive using blood culture, while 113 (56.5%) were positive for bacterial targets using the ddPCR assay. The ddPCR assay outperformed blood culture in pathogen detection rate, mixed infection detection rate, and fungal detection rate. Acinetobacter baumannii and Klebsiella pneumoniae were the most commonly detected pathogens in COVID-19 critically ill patients, followed by Enterococcus and Streptococcus. Compared to blood culture, ddPCR achieved a sensitivity of 75.5%, specificity of 51.0%, PPV of 30.9%, and NPV of 87.8%, respectively. However, there were significant differences in sensitivity among different bacterial species, where Gram-negative bacteria have the highest sensitivity of 90.3%. When evaluated on the ground of clinical diagnosis, the sensitivity, specificity, PPV and NPV of ddPCR were 78.1%, 90.5%, 94.7%, and 65.5%, respectively. In addition, the ddPCR assay detected 23 cases of bla_KPC, which shown a better consistent with clinical test results than other detected AMR genes. Compared to bla_KPC, there were few other AMR genes detected, indicating that the application of other AMR gene detection in the COVID-19 critically ill patients was limited.

Conclusion: The multiplex ddPCR assay had a significantly higher pathogen detection positivity than the blood culture, which could be an effective diagnostic tool for BSIs in COVID-19 patients and to improve patient outcomes and reduce the burden of sepsis on the healthcare system, though there is room for optimization of the panels used.- Adjusting the targets to include E. faecalis and E. faecium as well as Candida albicans and Candida glabrata could improve the ddPCR' s effectiveness. However, further research is needed to explore the potential of ddPCR in predicting bacterial resistance through AMR gene detection.

Keywords: Antimicrobial resistance; Bloodstream infections; Multiplex droplet digital PCR; Pathogen detection; Severe Acute Respiratory Syndrome Coronavirus 2.

Fig. 1

Flow-chart for patient enrollment and results analysis. *Including 3 samples judged as contaminated for isolated CoNS while present < 50% of all blood culture sets

Fig. 2

Pathogens detected by ddPCR and blood culture method. a Detection number of pathogens were compared between ddPCR and BC; Counts and percentage of co-infection in patients with ddPCR-positive (b) and blood culture-positive results (c)

Fig. 3

Comparision analysis of pathogens detected by ddPCR and BC method. a Categorization of infection events detected by ddPCR and BC method alone or simultaneously. Distribution of pathogens detected by ddPCR and BC within (b) and outside c the range of ddPCR-targeted organisms. d Within ddPCR-targeted organisms and detected to species level by BC method. b The bacteria in the blue, orange, green, and red modules are distributed in panel 1, 2, 3, and 4, respectively.