Bloodstream infection (BSI) and its progression to sepsis and septic shock represent major causes of global mortality (Rudd et al., 2020). Substantial evidence indicates that delayed or inappropriate initial antibiotic administration significantly reduces both short-term and long-term survival in sepsis patients (Peltan et al., 2019; Kollef et al., 2021). Elderly individuals face increased risk of missed or incorrect BSI diagnosis due to atypical clinical manifestations and subtle alterations in infection markers (Hyernard et al., 2019; Laupland et al., 2021; Leibovici-Weissman et al., 2021).

Current guidelines (Evans et al., 2021) advocate prompt initiation of broad-spectrum antibiotics for patients with high suspicion of sepsis or septic shock to enhance clinical outcomes. Nevertheless, extended broad-spectrum antibiotics use may elevate adverse event risks (Ong et al., 2017; Tamma et al., 2017; Baggs et al., 2018; Branch-Elliman et al., 2019), particularly among geriatric populations (Leibovici-Weissman et al., 2021). Consequently, rapid and accurate etiological diagnosis of BSI is critical for antibiotic regimen optimization and outcome improvement in elderly BSI patients.

Blood culture (BC) remains the diagnostic gold standard for bacterial and fungal BSI. However, this method suffers from prolonged turnaround times (Ehren et al., 2020; Kim et al., 2021). In early-stage of BSI, BC sensitivity decreases significantly due to low microbial concentrations in circulation and prior antibiotic administration (Pilecky et al., 2019). Emerging molecular detection technologies, including meta-genomic second-generation sequencing (mNGS), real-time quantitative polymerase chain reaction (RT-qPCR) and droplet digital polymerase chain reaction (ddPCR), aim to overcome these limitations (Tjandra et al., 2022). As the third-generation PCR technology succeeding RT-qPCR (Chen et al., 2021), ddPCR offers several advantages: reference curve independence, minimal blood PCR inhibitors interference, high sensitivity (≤10 CFU/mL), rapid detection speed (TAT ≤ 1 hour), and cost-effectiveness (Abram et al., 2020; Merino et al., 2022). Given these characteristics, ddPCR demonstrates utility not only for early BSI diagnosis but also for monitoring pathogen load dynamics to evaluate disease progression and prognosis. Unlike mNGS, ddPCR remains limited by fundamental PCR principles, restricting detection of uncommon and unidentified pathogens. Nevertheless, within its detectable spectrum, ddPCR offers superior speed and sensitivity compared to conventional methods (Hu et al., 2021).

Microorganisms including Acinetobacter baumannii (A. baumannii), Streptococci, coagulase-negative Staphylococci (CoNS) and Candida species frequently yield positive test results due to non-infectious factors such as contamination or transient bacteremia/fungemia, despite their pathogenic potential in immunocompromised and hospitalized populations (Fournier and Richet, 2006; Mea et al., 2021; Knauf et al., 2022; Lopes and Lionakis, 2022). Elderly patients exhibit increased susceptibility to false-positive results (transient bacteraemia and specimen contamination) attributable to extended healthcare facility exposure, compromised vascular integrity, and diminished skin barrier function. While BC benefits from standardized interpretation protocols developed through extensive clinical application (Centers For Disease Control And Prevention), ddPCR as an emerging technology currently lacks comparable established criteria.

Clinical trials have validated the diagnostic efficacy, accuracy, and sensitivity of ddPCR for etiological identification (Hu et al., 2021; Wu et al., 2022; Lin et al., 2023), along with its prognostic predictive value (Ziegler et al., 2019; Shao et al., 2022). Existing investigations, however, have predominantly concentrated on adult patients, with scarce data available for elderly patients. Moreover, prior studies failed to examine the diagnostic impact of false-positive findings.

A single-center retrospective study was conducted to compare the diagnostic efficacy and antibiotic guidance capability between BC and ddPCR, assess the prognostic value of ddPCR, and determine the threshold for distinguishing non-pathogenic microbiota detected by ddPCR. The analytical spectrum of ddPCR in this investigation encompassed Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella species, Escherichia coli, A. baumannii, Staphylococcus aureus, CoNS, Enterococcus species, Streptococcus species and Candida species.

This investigation evaluated the efficacy of ddPCR and BC for rapid pathogen identification, antimicrobial therapy guidance, and prognostic assessment in elderly BSI patients. ddPCR demonstrates superior resistance to blood-derived PCR inhibitors while maintaining high sensitivity and rapid turnaround time, exhibiting substantial diagnostic potential for pathogen detection (Chen et al., 2021). Although previous studies have validated its clinical utility (Ziegler et al., 2019; Hu et al., 2021; Shao et al., 2022; Wu et al., 2022; Lin et al., 2023; Liu et al., 2023; Li et al., 2024), limited data exist regarding application in elderly BSI populations, which exhibit distinct clinical manifestations, diagnostic challenges, and disease outcomes (Leibovici-Weissman et al., 2021). Considering the differential clinical presentation, diagnostic challenges, and prognosis of BSI in elderly patients compared to the general adult population, the enhanced sensitivity and specificity of ddPCR may provide particular diagnostic advantages for this vulnerable patient demographic.

Existing literature reported ddPCR positive rates of ranging from 41.1% to 83.3% in suspected BSI patients (Hu et al., 2021; Wu et al., 2022; Li et al., 2024). The current investigation demonstrated pathogen detection in 59% of elderly BSI patients using ddPCR versus pathogens 21% BC, with and combined methodology achieving 65% positivity. Analysis revealed 94.82% of all bloodborne pathogens identified fell within the ddPCR detection range, indicating that although the detection range of ddPCR remains limited, it covers the majority of clinically relevant microorganisms and satisfies routine diagnostic requirements.

BC, currently regarded as the diagnostic gold standard for BSI, demonstrates limited sensitivity (approximately 20%) (Hu et al., 2021; Sun et al., 2022) and requires prolonged turnaround time (1-3 days) (Ehren et al., 2020; Kim et al., 2021), creating substantial obstacles for rapid clinical diagnosis. This present study employed clinical assessment as the reference standard to evaluate the diagnostic efficiency of ddPCR and BC. Comparative analysis revealed markedly superior sensitivity for ddPCR (59.61% versus 20.57%). To mitigate potential subjective in the clinical interpretation, discordant results between ddPCR and BC were systematically analyzed using supplementary etiological evidence. This validation process showed enhanced ddPCR sensitivity (90.63%) and significantly higher PPV value compared to BC ( Table 2 ). Despite its advantages, ddPCR exhibits detection limitations for specific pathogens including Haemophilus influenzae and Bacteroides fragilis. Expanding the detection range of ddPCR based on clinical epidemiology data could enhance its diagnostic utility. The median TAT for ddPCR and BC was 7.38 h and 19.60 h respectively, showing discrepancy with results of Yin et al (Yin et al., 2024). This discrepancy may be due to our study defining the BC TAT endpoint as the reporting of positive results rather than AST results.

This investigation further examined the distribution of ddPCR copy numbers across BC results. Quantitative analysis revealed significantly elevated ddPCR copy numbers in BC+ samples compared to BC-, though this relationship did not demonstrate adequate fit for logistic regression modeling. Limited sample size precluded detailed evaluation of species-specific ddPCR copy number correlations with BC outcomes. Notably, while ddPCR exhibited superior sensitivity to BC, discordant results were observed in 12 BC+/ddPCR- cases. Subsequent clinical evaluation confirmed 7 of these as true positives, all involving pathogens within the theoretical detection range of the ddPCR assay. As a retrospective study, this investigation included paired ddPCR and BC tests conducted within 24 hours but not necessarily simultaneously. Given the cyclical nature of pathogenemia (Forrester), sample collection timing significantly impacts detection sensitivity, with peak pathogenemia period yielding higher positivity rates compared to trough phases. Furthermore, analogous discrepancies have been documented in prospective studies employing simultaneous blood samples collection (Wu et al., 2022). These observations suggest potential technical influences, including variations in ddPCR testing or inherent instrument detection characteristics.

As the diagnostic gold standard for BSI, BC serves dual critical functions: establishing definitive etiological diagnosis and guiding antibiotic selection. Clinical evidence demonstrates the potential complementary role of ddPCR in antimicrobial stewardship. Lin et al. found that antibiotic regimen modifications in 51.2% of patients based on ddPCR results (Lin et al., 2023), while Li et al. reported observed inadequate empirical antimicrobial coverage relative to ddPCR results in 63.3% of patients (Li et al., 2024). These findings indicate ddPCR may provide valuable therapeutic guidance, particularly when BC results remain negative or inconclusive. In this study, the antibiotic adjustment rates for ddPCR and BC were 24.59% versus 20.93%, and effectiveness rates were 60.13% versus 53.33%, with no significant differences. Despite this, ddPCR’s higher sensitivity may offer better antibiotic guidance for elderly BSI patients and support earlier antibiotic adjustments. While antibiotic adjustments typically follow BC and AST, this study found ddPCR-based changes needed no later revision, and BC/AST-based changes were not influenced by prior ddPCR results. These findings suggest that while ddPCR, due to its higher sensitivity, demonstrates certain advantages in guiding antibiotic use, BC combined with AST remains an important basis for antibiotic decision-making.

Previous investigations by Shao, Ziegler, et al. established significant associations between elevated microbial DNA loads in BSI patients and three clinical parameters: increased inflammation, more severe disease, and worse prognosis (Ziegler et al., 2019; Shao et al., 2022). The current findings corroborate these observations, demonstrating statistically significant differences in both maximum copy numbers and more species/genera than those with favorable outcomes. During early in BSI, reduced pathogen loads correlate with both diminished BC positive rates and absence of organ dysfunction. Timely antibiotic use based on ddPCR can prevent severe complications and improve prognosis. During disease progression, increasing microbial loads correlate with enhanced BC positive rates, yet may coincide with missed therapeutic windows, leading to suboptimal treatment responses and unfavorable clinical outcomes. Notably, while significant differences in pathogen copy numbers exist between patient groups, absolute quantitative values demonstrate limited predictive capacity for individual prognosis, a finding consistent with previous reports by Zhao, Shao, et al (Shao et al., 2022; Zhao et al., 2024). The dynamic changes of microbial DNA copy number may provide valuable prognostic supplementation. However, the limited sample size of current investigation for serial monitoring necessitates further validation studies to establish the clinical utility of copy number kinetics in prognosis prediction.

The present study addressed the critical diagnostic challenge of differentiating true positives from false positives (including contamination and transient bacteremia/fungemia) in clinical microbiological testing, with particular focus on Streptococcus or CoNS, A. baumannii, and Candida. These organisms can asymptomatically colonize patients but also cause infections in immunocompromised individuals (Beigi et al., 2004; Bougnoux et al., 2006; Grice et al., 2009; Knauf et al., 2022; Lopes and Lionakis, 2022). Comparative analysis revealed that optimal diagnostic performance for Streptococcus & CoNS, A. baumannii complex, and Candida species required elevated threshold values (132.545, 182.70/262.24, and 174.775 copies/mL, respectively) compared to the overall diagnostic threshold of 45 copies/mL. At these optimal thresholds, the sensitivity and specificity were: 0.882 versus 0.791, 0.944 versus 0.889 (0.0.889 versus 0.944), 0.667 versus 1. The current threshold analysis presents two notable limitations: the restricted sample size and potential supervisor bias in clinical determination of true and false positive results. These constraints necessitate further investigation through larger-scale studies employing more rigorous, predefined clinical criteria to validate the present findings.

This study demonstrates that ddPCR, as an emerging pathogen detection methodology, provides superior sensitivity and rapid turnaround time, facilitating early diagnosis and therapeutic intervention for BSI in elderly populations, ultimately leading to improved clinical outcomes. While the inherent limitations of PCR technology constrain its detectable spectrum, this approach nevertheless encompasses the predominant clinically relevant microorganisms responsible for BSI. This investigation represents the first comprehensive comparison establishing advantages of ddPCR over BC in elderly BSI patients, while proposing that early implementation of ddPCR testing may confer prognostic benefits. Furthermore, the study systematically assesses ddPCR performance for microorganisms with high false positives potential and establishes novel diagnostic thresholds to enhance clinical interpreting of results.

The current investigation faces constraints in evaluating the diagnostic and prognostic utility of ddPCR value at the species/genus levels due to limited sample size. The retrospective design precluded simultaneous collection of ddPCR and BC samples, with potential confounding variables beyond methodological differences potentially contributing to observed result variations. These limitations necessitate prospective validation studies to confirm these findings. For clinical relevance, the study employed physician-determined clinical assessment as the reference standard for BSI diagnosis and discrimination between true and false positives results. Despite employing independent case assessments by multiple experienced clinicians to ensure reproducibility, inherent subjective in clinical diagnostic process remains unavoidable. This methodological limitation may lead to partial overestimation of the diagnostic advantages attributed to ddPCR. Consequently, the reported diagnostic superiority of ddPCR requires additional verification through rigorous studies. Future investigations should incorporate predefined composite reference standards in accordance with contemporary best practices for diagnostic accuracy research.

BSI in elderly patients frequently manifests with nonspecific clinical presentations, while collected specimens demonstrate increased susceptibility to contamination. The enhanced analytical sensitivity of ddPCR relative to conventional BC confers significant advantages for the early BSI detection in this demographic. Nevertheless, positive results from both ddPCR and BC require careful interpretation alongside clinical presentation and supplementary diagnostic evaluations. Implementation of composite clinical adjudication incorporating predefined criteria represents a critical methodological advancement aligned with contemporary diagnostic standards. Further refinement of pathogen-specific detection thresholds, particularly for microorganisms associated with contamination or transient bacteremia, would improve the reliability of positive results.