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

Abstract

BackgroundBlood 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.MethodsAn 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.ResultsThe 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 (< 1 h) 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.ConclusionThe 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.

Modular biomaterials vaccine technology protects against multiple pathogens and septic shock

“…2A). To avoid this toxicity, we then captured the E. coli PAMPs from the lysate using FcMBLbeads (13,14). When FcMBL-bead-captured PAMPs containing an equivalent amount of PAMPs as the E. coli cell lysate (20.5 PAMP/mL vs 22.9 PAMPs/mL in lysate) was injected into rabbits ( Fig.…”

“…2E). We used the previously described sandwich ELISA-like assay (13,14) to quantify and equalize the total amount of captured PAMPs added to each vaccine in all following studies to ensure equivalent PAMP dosing in different experiments, and with different bacterial species. The ciVAX forms a subcutaneous depot with little signs of necrosis, and the MPS lasts for longer than 7 days and dissolves spontaneously by 37 days (Supplementary Materials Fig.…”

“…Our goal was to develop a broad-spectrum infection vaccine that is potent, durable and safe, using complex killed bacterial components that do not require cold chain storage, and that also can be used for ring vaccination in biothreat or pandemic emergencies, without having to first identify or isolate the pathogen. To address this challenge, we combined two technologies that are currently in clinical development for other applications ¾ a broad spectrum engineered opsonin known as Fc-Mannose Binding Lectin (Fc-MBL) (12)(13)(14) that rapidly binds to carbohydrate-containing PAMP antigens (including glycoproteins and glycolipids) expressed on the surface of over 120 different pathogen species and toxins, including some that are known to trigger innate immunity (e.g., lipopolysaccharide endotoxin; LPS) (15), and a biomaterial scaffold-based vaccine being used for cancer immunotherapy (16)(17)(18) ¾ to create a new composite infection vaccine technology (ciVAX) (Fig. 1).…”

“…FcMBL beads. The captured PAMPs bacterial antigens captured on FcMBL beads were quantified using an Enzyme-Linked Lectin Sorbent Assay (ELLecSA) as previously published(13). Briefly, FcMBL was biotinylated at the N terminus of the Fc protein using an Nterminal amino-oxy reaction and then coupled to streptavidin superparamagnetic beads (1µM MyOne Dynabead (Thermo Fisher Scientific, USA).…”

Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

“…For instance, Ingber’s group reported a broad-spectrum sepsis diagnostic using micro-bead-modified mannose binding lectin linked to the Fc portion of human IgG1 that detects pathogen-associated molecular patterns (PAMPs) in blood. 156 Automated matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS (e.g., bioMérieux VITEK MS and Bruker Daltonics MALDI Biotyper [Billerica, MA]) has recently been introduced to clinical microbiology labs for rapid microorganism ID based on distinct protein and peptide mass spectrum, compared with a reference database. MS can also be used for AST profiling by measuring antibiotic resistance markers such as β-lactamases that degrade antibiotics, and antibiotic degradation due to resistance enzymes.…”