A Rapid Differential Diagnostic Method for Detection of Post-Neurosurgical Infections

Abstract: This work pertains to the application of a biochemical assay with the purpose of monitoring for and early discovery of an active bacterial infection through direct sampling and rapid testing of the Cerebrospinal Fluid (CSF). A diagnostic device is described and evaluated, which aids in quickly discerning, verifying, or ruling out active infection at the injury site. We have applied it on post-neurosurgical wounds, the state of which, due to their complexity, is challenging to determine with certainty, using conventional means. Infection is the successful invasion and degeneration of the body tissue by pathogenic microorganisms. Even successfully performed invasive treatments are susceptible to infections that may not be discovered until severe complications arise. The primary sampling access to the brain for diagnostic purposes is via the CSF, which is also highly valuable for the present end. Notwithstanding this possibility, it is difficult to distinguish between cases with aseptic inflammation and meningitis, despite available methods such as cell counting, culture, and protein analysis. Therefore, current treatment options rely mainly on broad and/or prophylactic measures with few options for monitoring the results. Under normal circumstances, the body reacts immediately to infection in an immune-competent host. The initial event of the body defense is the recruitment of neutrophils to the injury site, where inflammation induces increased permeability of vessels, development of edema, and increased internal pressure inside the afflicted tissue. The brains constrained, well-protected intracranial environment has the disadvantage that swelling of the brain early causes increased pressure, which potentially exerts irreversible damaging effects on the brain tissues. Septic and aseptic inflammations and related hemorrhages that cause increased intracranial pressure are important reasons for invasive interventions to decrease the pressure. Infection is a major cause of inflammation, swelling, and increased intracranial pressure. Timely diagnosis of infection and eliminating the causative pathogens is strongly related to survival and recovery. Therefore, a prompt diagnosis of infection is essential for the immediate and appropriate initiation of antibiotic therapy and monitoring treatment effects. Further diagnostic procedures are used to identify the specific bacterial pathogens and optimize the treatment. Early discovery of infections and proper antibiotic treatment reduces complications and the need for invasive and/or repeated treatments. Non-specific immune response mediated by activated neutrophils reacts specifically and immediately to the presence of invading pathogens. The currently described diagnostic method uses the biochemical characteristics of this natural defensive mechanism. We have developed platforms for diagnostic assays to immediately discover an infection and monitor the efficacy of the appropriate antibiotic treatment against the infection. Neutrophil Extracellular Traps (NETs) are chromatin particles extruded from activated neutrophils to the site of infection to trap and kill the pathogenic microorganisms. NETs contain extracellular polyanion DNA with a high affinity to aniline dyes, as well as for proteins. This activity correlates with the expression of acute-phase proteins. The interaction of mesenchymal and epithelial cells after organ injuries enhances cytokine production, such as hepatocyte growth factor (HGF). HGF is a glycoprotein with unique properties that contribute to wound healing after injuries. The release and role of HGF during infectious diseases have been investigated and are presented here with particular emphasis on pneumonia and meningitis. In the current thesis, both clinical and preclinical assays are employed. Initially, through a preclinical experiment, we have found that upon activation by bacteria, healthy neutrophils extrude DNA particles in vitro. In a pre-mixed pink-red dextran sulfate and toluidine blue solution, the addition of DNA particles causes a visible color change to blue and a change in wavelength for absorbance of light from 542 to 620 nm. This reaction is inhibited when the bacteria in the in vitro experiment are pre-incubated with effective antibiotics. Furthermore, in clinical studies, we have compared the cerebrospinal fluid (CSF) from patients with post neurosurgical bacterial meningitis with controls that had aseptic inflammation post neurosurgery. We have assessed the clinical performance of this diagnostic platform to distinguish the cases with post neurosurgical infection such as post neurosurgical meningitis (PNM) or ventilator-associated pneumonia by analysis of CSF or tracheal secretion long before the other tests can identify the infection. Simultaneous enhanced production of HGF locally at the site of injury was demonstrated.  For a comprehensive assessment, diagnosis and treatment decisions must consider the case history and development of the patient’s condition over time. Experienced neurosurgeons with independent studying habits that have conducted comparative studies on patient records, regarding which conditions and indications precede and follow the incidence of which specific diseases, have diligent routines for monitoring results of their medical decisions, and are competent at utilizing numerous diagnostic options correctly and thus at arriving at the most appropriate conclusions.  

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