A Broad-Spectrum Infection Diagnostic that Detects Pathogen-Associated Molecular Patterns (PAMPs) in Whole Blood

Abstract

Background: Blood cultures, and molecular diagnostic tests that directly detect pathogen DNA in blood, fail to detect bloodstream infections in most infected patients. Thus, there is a need for a rapid test that can diagnose the presence of infection to triage patients, guide therapy, and decrease the incidence of sepsis.

Methods: An Enzyme-Linked Lectin-Sorbent Assay (ELLecSA) that uses magnetic microbeads coated with an engineered version of the human opsonin, Mannose Binding Lectin, containing the Fc immunoglobulin domain linked to its carbohydrate recognition domain (FcMBL) was developed to quantify pathogen-associated molecular patterns (PAMPs) in whole blood. This assay was tested in rats and pigs to explore whether it can detect infections and monitor disease progression, and in prospectively enrolled, emergency room patients with suspected sepsis. These results were also compared with data obtained from non-infected patients with or without traumatic injuries.

Results: The FcMBL ELLecSA was able to detect PAMPS present on, or released by, 85% of clinical isolates representing 47 of 55 different pathogen species, including the most common causes of sepsis. The PAMP assay rapidly (<1h) detected the presence of active infection in animals, even when blood cultures were negative and bacteriocidal antibiotics were administered. In patients with suspected sepsis, the FcMBL ELLecSA detected infection in 55 of 67 patients with high sensitivity (>81%), specificity (>89%), and diagnostic accuracy of 0·87. It also distinguished infection from trauma-related inflammation in the same patient cohorts with a higher specificity than the clinical sepsis biomarker, C-reactive Protein.

Conclusion: The FcMBL ELLecSA-based PAMP assay offers a rapid, simple, sensitive and specific method for diagnosing infections, even when blood cultures are negative and antibiotic therapy has been initiated. It may help to triage patients with suspected systemic infections, and serve as a companion diagnostic to guide administration of emerging dialysis-like sepsis therapies.

Keywords: Biomarker; Blood culture; C-reactive protein; Infection diagnostic; Mannose binding lectin; Sepsis.

Fig. 1

Patient enrollment. Out of the total of 291 patients enrolled, 126 were excluded due to blood samples being stored for longer than 24 h or results of assays not meeting internal validation of 95% confidence interval of slope and background determined from the curve fit analysis of the internal mannan standard (see Supplemental Fig. 1). Of the remaining 165 patients, 78 sepsis patients (67 who entered the study at time 0, and 34 at 24 h [11 patients enrolled at 24 h]), 35 non-infected control patients without evidence of infection or trauma (n = 23 hospital controls and n = 12 healthy volunteers), and 52 patients with trauma but no clinical suspicion of infection were analyzed.PLEASE REMOVE the paragraph this is duplicated from the article Patient enrollment. Out of the total of 291 patients enrolled, 126 were excluded due to blood samples being stored for longer than 24 h or results of assays not meeting internal validation of 95% confidence interval of slope and background determined from the curve fit analysis of the internal mannan standard (see Supplemental Fig. 1). Of the remaining 165 patients, 78 sepsis patients (67 who entered the study at time 0, and 34 at 24 h [11 patients enrolled at 24 h]), 35 non-infected control patients without evidence of infection or trauma (n = 23 hospital controls and n = 12 healthy volunteers), and 52 patients with trauma but no clinical suspicion of infection were analyzed.

Fig. 2

Development of the FcMBL-based infection diagnostic. (a) Schematic representation of the FcMBL ELLecSA methodology in which PAMPs or whole pathogens contained in blood are captured using FcMBL-coated magnetic microbeads (FcMBL-beads) in combination with applied magnetic fields, and detection of bound material by application of recombinant human MBL linked to horse radish peroxidase (rhMBL-HRP), followed by addition of the chromogenic substrate, tetramethylbenzidine (TMB), which generates a positive signal in infected blood samples. (b) Mannan standard curve showing FcMBL binding to mannan measured as optical density at 450 nm, which was used to determine PAMP units (1 PAMP unit is equivalent to FcMBL binding to 1 ng/ ml mannan). Graph showing detection of the carbohydrate PAMP LPS (c) when diluted in whole blood using the FcMBL ELLecSA. Graphs showing enhanced detection of Klebsiella oxytoca (d), Klebsiella pneumonia (e), Enterobacter cloacae (f), and Escherichia coli (g) bacteria using the FcMBL ELLecSA in combination with (dark grey) or without (light grey) mechanical disruption (30 Hz for 10 min in a beadmill) or treatment with the antibiotic cefepime (100 μg/ml) (f) or amikacin (250 μg/ml) for 4 h (g) (* p < 0.05; Unpaired t-test); data are presented as PAMP units. Bacteria were cultured in RPMI/10% Glucose from a single colony and harvested at log phase (to 0.5 McF; 10⁸ CFU/ml).

Fig. 3

Detection of infection in animals by measuring PAMPs in blood using the FcMBL ELLecSA. a) Graph showing PAMP levels measured with the FcMBL ELLecSA (open circles) versus white blood cell (WBC) counts (open triangles) and positive blood culture results (grey shaded area) and in samples obtained from pigs injected with Escherichia coli (0.5 × 10⁷ CFU/kg) and then infused with E. coli (5 × 10⁷ CFU/kg) over 6 h or vehicle (no E. coli) at the start of the 36 h experiment. b) Pathogen loads measured in cultures of lung, liver, and spleen after the organs were removed at the end of the study and bead-milled to release harbored bacteria (4 different regions of each organ were processed, and pathogen loads are normalized per gram of organ tissue). c) Rats were infected intraperitoneally with Escherichia coli (2 × 10⁹ CFU) and then treated with the antibiotic cefepime (60 mg/kg) starting 4 h after pathogen injection. The FcMBL ELLecSA was used to measure released PAMPs at the conclusion of the experiment (8 h) and compared to control rats that were infected but received no antibiotic (no ABX) (*** p < 0.0001; unpaired t-test).

Fig. 4

The FcMBL ELLecSA enables detection of patients with sepsis compared to healthy controls based on higher PAMP levels. (a) PAMP levels were significantly increased in blood samples from patients with suspected sepsis at both study entry (0 h) and 1 day later (24 h) compared with non-infected control donor samples (**, p < 0.001; Wilcoxon Rank Sum test). (b) Change in PAMP scores for paired sequential blood samples at 0 and 24 h for sepsis patients (ns, no significant difference; Wilcoxon Signed Rank Test). (c) Receiver operating characteristic (ROC) comparison of the FcMBL ELLecSA data for sepsis patients versus non-infected controls. Areas under the ROC curve for sepsis patient bloods at 0 and 24 h were = 0.870 + 0.041 (mean + SEM; p < 0.0001) and 0.889 + 0.050 (p < 0.0001), respectively. (d) Performance characteristics of the FcMBL ELLecSA for detection of infection in patients suspected of sepsis versus healthy non-infected controls using a PAMP threshold of > 0.45 units, as determined by ROC analysis.

Fig. 5

The FcMBL ELLecSA enables detection of patients with sepsis compared to non-infected trauma patients. (a) PAMP levels were extremely low in trauma patients, and significantly increased in blood samples from patients with suspected sepsis at both study entry (0 h) and 1 day later (24 h) compared with non-infected trauma patient samples (***, p < 0.0001; Wilcoxon Rank Sum test). (b) CRP levels measured in the same sepsis samples using a CRP ELISA also were increased in blood samples from patients with suspected sepsis at both study entry (0 h) and 1 day later (24 h) compared with non-infected trauma patient samples (***, p < 0.0001; *, p < 0.003; Wilcoxon Rank Sum test). (c) Comparison of the performance characteristics of the FcMBL ELLecSA versus the CRP ELISA revealed that while both assays have similar high sensitivity, the ELLecSA has a much higher specificity. CRP performance characteristics were determined by ROC analysis of CRP ELISA data using a 5 mg/ml cutoff value (Fig. 6).

Fig. 6

Receiver operating characteristic (ROC) curves used to compare the ability of the FcMBL ELLecSA and CRP assays to differentiate patients with systemic infection or sepsis from those with sterile trauma. Plasma extracted from the same patient samples analyzed by the FcMBL ELLecSA were then diluted and analyzed using the CRP ELISA. (a) FcMBL ELLecSA. Areas under the ROC curves at 0 and 24 h for the septic patient samples compared to those with trauma were 0.831 + 0.038 (p < 0.0001) and 0.867 + 0.045 (p < 0.0001); area under the ROC curve for the non-infected control versus trauma was 0.589 + 0.061 (p < 0.156). (b): CRP ELISA. Areas under the ROC curves at 0 and 24 h for the septic patient samples compared to those with trauma were 0.793 + 0.045 (p < 0.0001) and 0.740 + 0.063 (p < 0.003); area under the ROC curve for the non-infected control versus trauma was 0.741 + 0.064 (p < 0.0016). (c) FcMBL ELLecSA PAMP levels were not significantly increased in blood samples from patients with non-infected trauma compared with non-infected control (Wilcoxon Rank Sum test). (d) CRP levels were significantly increased in blood samples from patients with non-infected trauma compared with non-infected control (p < 0.0013; Wilcoxon Rank Sum test).