Rapid and sensitive identification of pleural and peritoneal infections by droplet digital PCR

Abstract

Pleural and peritoneal infections cause substantial morbidity and mortality. Traditional diagnostic methods rely on the cultivation of clinical samples, which usually takes days to obtain report and holds a low detection sensitivity. In this study, we evaluated a 5-fluorescent-channel droplet digital PCR (ddPCR) system and 5 assay panels for culture-independent rapid pathogen detections directly from pleural and peritoneal fluid samples. Traditional culture of the same sample was used as reference. A total of 40 pleural fluid samples and 19 peritoneal fluid samples were tested in this study. Twenty-five positives including 4 polymicrobial infections by culture and 26 positives including 11 polymicrobial infections by ddPCR were detected for pleural fluid samples; 14 positives including 2 polymicrobial infections by culture and 15 positives including 3 polymicrobial infections by ddPCR were detected for peritoneal fluid samples. Klebsiella pneumoniae was the most common bacterium detected both in pleural and in peritoneal fluid samples. The sensitivity of the ddPCR assay for pleural and peritoneal fluid samples was 96% (95% confidence interval (CI) = 79.65 to 99.90%) and 92.86% (95% CI = 66.13 to 99.82%), respectively. The turnaround time of the ddPCR assay was approximately 3 h comparing with 38.30 ± 22.44 h for culture-based identifications. Our results demonstrated that the ddPCR assay is a rapid and sensitive method for identifying pathogens responsible for pleural and peritoneal infections and would be a promising approach for early diagnosis and optimizing treatment of infections.

Digital PCR applications for the diagnosis and management of infection in critical care medicine

“…Compared to the gold standard cultures of pleural and peritoneal fluids, dPCR showed a sensitivity of 96% and 93%, specificity of 87% and 60%, a positive predictive value of 92% and 87% and a negative predictive value of 93% and 75%, respectively. These results support dPCR as a rapid and sensitive alternative for the detection of pleural and peritoneal fungal infections [ 19 ].…”

“…dPCR has also been applied to the detection of pathogens in clinical samples other than blood. For instance, Zhou et al [ 19 ] tested dPCR to detect bacteria and fungi in pleural and peritoneal fluids of critically ill patients, comparing the obtained results with culture methods. Compared to the gold standard cultures of pleural and peritoneal fluids, dPCR showed a sensitivity of 96% and 93%, specificity of 87% and 60%, a positive predictive value of 92% and 87% and a negative predictive value of 93% and 75%, respectively.…”

Recent Insights into the Management of Pleural Infection

“… 60 Digital droplet PCR, which employs similar methodology to multiplex PCR, has been successfully used to identify bacteria in pleural fluid, with sensitivity higher than standard cultures. 61 Moreover, commercially available multiplex-PCR methods used in CAP 62 have proved more sensitive to conventional cultures in the bacteriological diagnosis of pleural infection. Multiplex-type techniques carry the advantages of utilizing widely available equipment and being relatively inexpensive.…”

“…Multiplex-type techniques carry the advantages of utilizing widely available equipment and being relatively inexpensive. They have short turnaround times (a few hours) and are particularly sensitive for polymicrobial infection 61 and in patients who have started antibiotics. 62 A pleural infection–specific multiplex-PCR panel that incorporates genetic sequence of anaerobes commonly implicated in pleural infection may transform the diagnosis and treatment decisions in pleural infection as this technology becomes more readily available at bench and bedside.…”

Evaluation of droplet digital PCR rapid detection method and precise diagnosis and treatment for suspected sepsis (PROGRESS): a study protocol for a multi-center pragmatic randomized controlled trial

“…Wousters et al [ 11 ] applied ddPCR to 45 blood samples collected from patients with suspected BSI, and the overall sensitivity and specificity was 80% (95% CI 0.52–0.96) and 87% (95% CI 0.69–0.96) compared with blood culture, respectively. Zhou et al [ 27 ] evaluated the ddPCR detection method using pleural and peritoneal fluid samples, and the sensitivity was 96% (95% CI = 79.65–99.90%) and 92.86% (95% CI = 66.13–99.82%), respectively. Furthermore, Hu et al [ 14 ] performed a head-to-head comparison between the ddPCR assay and metagenomic NGS in 45 critically ill patients.…”