Discussion
By studying 165,593 blood specimens from 13 USA hospitals, Tabak et al. have shown that the median time to identify BSI pathogens using traditional blood culture was 44.0 h, with a sensitivity of approximately 70% in critically ill patients and even lower for fastidious microorganisms (Tabak, et al., 2018). In this study, to overcome the shortcomings of the blood culture in BSI diagnosis, we developed a culture-independent ddPCR method to rapidly and accurately identify A. baumannii and K. pneumonia in blood samples of patients with suspected BSI. Our results showed that ddPCR could identify 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/µL for A. baumannii and 0.27 copies/µL for K. pneumonia . Clinical validation in 170 patients with suspected BSIs showed that ddPCR not only detected the positive patients who were identified by blood culture but also detected the patients who were missed by blood culture. Of note, compared with the patients reported by both blood culture and ddPCR assay, the patients reported by ddPCR alone had less severe clinical manifestations and better clinical outcomes, suggesting that these patients benefit from the guidance of ddPCR results in the early stage of BSI. Thus, ddPCR may serve as a rapid and reliable method to identify causal pathogens in BSI and to guide the treatment decisions in the early stage of BSI.
In BSI, the human immune system and antibiotic treatment kill invading pathogens, leading to the release of the nucleic acids from the pathogens into the blood, which become a part of circulating cell-free DNA (cfDNA) (Lo, et al., 1999, Diehl, et al., 2008, Lu, et al., 2018, Papadopoulos, 2020). Thus, the presence of specific pathogenic DNA in cfDNA can reflect the presence of pathogens in the bloodstream. Accumulating evidence have demonstrated the feasibility of next-generation sequencing (NGS) of plasma cfDNA to identify the pathogens in BSI (Blauwkamp, et al., 2019, Farnaes, et al., 2019, Grumaz, et al., 2019, Rossoff, et al., 2019). However, the typical turnaround time of 2 days and the high cost of NGS remain barriers for cfDNA NGS in clinical practice (Long, et al., 2016, Simner, et al., 2018, Chiu and Miller, 2019, Crawford, et al., 2019). In this study, we took advantage of the ultra-high sensitivity of ddPCR and the feasibility of cfDNA in pathogen identification to develop a ddPCR-based method using cfDNA as the template. The turnaround time of ddPCR to diagnosis was 4 h, which was significantly shorter than that of NGS (2–3 d) or blood culture (90.6 ± 12.9 h in this study).
Wouters et al. have developed a ddPCR method to detect bacteria or fungi using metagenomic DNA as the template and broad-range primer-probe sets; however, the overall specificity in clinical validation was only 80% (Wouters, et al., 2019). In this study, we used cfDNA as the template and designed specific primer-probe sets for A. baumannii andK. pneumonia , respectively. We achieved 100% specificity in well-characterized ATCC isolates of each strain, which was also higher than other reported PCR-based methods, such as SeptiFast (50%, (Warhurst, et al., 2015); 85.5%, (Korber, et al., 2017)), Magicplex (29%) (Zboromyrska, et al., 2019), TAC assay (79.4%) (Zhang, et al., 2018), T2Bacteria (90%) (Maki, 2019, Nguyen, et al., 2019), and cfDNA NGS (93.7%) (Blauwkamp, et al., 2019).
The sensitivity of blood culture is typically lower than expected. The blood culture positivity rate of septic patients was 51% over 22 years in the United States (Martin, et al., 2003). Cheng et al. have reported a 71.7% blood culture positivity rate in severe sepsis patients from ten university hospitals in China (Cheng, et al., 2007). Similarly, 70% of infected patients in a one-day international investigation in ICU have been reported positive by blood culture test (Vincent, et al., 2009). The low sensitivity of blood culture may attribute to the low bacteria abundance in the blood, the antibiotic treatment before sampling, and the culture techniques. Molecular detection methods are less affected by these factors, thus usually have higher positivity rates than blood culture. The positivity rates of different molecular methods are 1.56 – 6.45-fold higher than that of blood culture (Long, et al., 2016, Korber, et al., 2017, Farnaes, et al., 2019, Grumaz, et al., 2019, Nguyen, et al., 2019). In the present study, the positivity rates of A. baumannii and K. pneumonia were 6.8-fold ((23+4)/4) and 4.3-fold ((26+4)/7) higher than those of blood culture, respectively. Thus, molecular detection methods may disclose the missed diagnosis in blood culture, allowing timely diagnosis and appropriate antibiotic treatment for the patients with BSI.
In this study, the 53 positive patients detected by ddPCR had typical manifestations resulting from BSI, including body temperature greater than 38.5℃, abnormally elevated serum levels of C-reactive protein and procalcitonin, hemodynamic instability, and severe organ dysfunction. Of note, the positive patients reported by ddPCR alone exhibited less severe manifestations than those reported by both ddPCR and blood culture, suggesting that ddPCR is more sensitive than blood culture for early diagnosis of BSI.
In conclusion, we developed a novel ddPCR method to detect two major pathogens in patients with suspected BSI. Clinical validation revealed that our method outperformed the blood culture in specificity, sensitivity, and turnaround time, serving as a promising method for early and accurate diagnosis of BSI. However, in this pilot study, we only included two major Gram-negative bacteria responsible for BSI. More clinically important pathogens will be further investigated in future studies.