Novel Method to Detect Pathogens for the Diagnosis of Chronic Wound Infection

检测病原体以诊断慢性伤口感染的新方法

基本信息

批准号：
7270191
负责人：
James Comolli
金额：
$ 20.83万
依托单位：
ECI BIOTECH
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-01 至 2008-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7270191
关键词：
Amino Acid Sequence Amputation Bacteria Bacterial Infections Biological Assay Biosensor Cellulitis Cessation of life Chronic Cleaved cell Clinical Clip Color Complication Condition Depth Detection Development Devices Diagnosis Diagnostic Diagnostic Procedure Diagnostic Specificity Disease Early Diagnosis Elastases Elderly Endopeptidases Engineering Enzymes Focal Infection Frequencies Goals Healed Healthcare Systems Hour Human In Vitro Incidence Individual Infection Inflammation Inflammatory Intervention Laboratories Lead Length Leukocyte Elastase Limb structure Liquid substance Localized Masks Measures Methods Morbidity - disease rate Numbers Osteomyelitis Pain Pancreatic Elastase Pathogen detection Pathway interactions Patient Care Patients Peptide Hydrolases Peptides Phase Population Process Proteins Proteolysis Pseudomonas aeruginosa Rate Research Sampling Septicemia Site Source Standards of Weights and Measures Swab Symptoms Systemic infection Technology Virulence Factors Visit Visual Work Wound Infection aging population base cross reactivity design diabetic experience healing improved novel pathogen pathogenic bacteria point of care sensor wound

项目摘要

DESCRIPTION (provided by applicant): Localized infection is a frequent complication of chronic wounds that interferes with the healing process and can lead to systemic infection, limb amputation, or death. Compounding the problem is an inability of the clinician to accurately detect localized infection by clinical observation, which is still the primary diagnostic method. This is because the usual signs of infection are masked by inflammation or the patients' underlying conditions or disease. The main objective of the proposed studies is to develop and validate a new assay that will enhance the ability of clinicians to detect chronic wound infection earlier and with more accuracy. The long-term goal is to incorporate this assay into a simple, inexpensive, point-of-care device to aid in diagnosis during a patient visit. This device will help individuals with chronic wounds receive appropriate treatment sooner, allow healing to resume, relieve their pain and discomfort, and avoid the spread of infection. The diagnostic device will be based on a novel detection method for pathogenic bacteria that was developed at ECI Biotech. This method measures the activity of protein-degrading enzymes (proteases) that infection-causing pathogens release into wound fluid. Since proteases are specific toward particular sites within proteins, ECI was able to design a single target peptide that contains cleavage sites for proteases from the most common wound pathogens. The resulting assay will detect the total number of pathogens but not discern between species. In previous studies, roughly 80% of clinical isolates from chronic wounds produced proteases in vitro that cleaved this target peptide. Also, over 90% of clinical samples taken directly from infected wounds were shown to have significant protease activity toward the target peptide. This work also revealed some cross-reactivity of the target peptide with the human neutrophil elastase (HNE), a host inflammatory protease present at high levels in chronic wounds. The first goal of this proposal is to alter the amino acid sequence of the target peptide to reduce interference by HNE while maintaining sensitivity toward bacterial proteases. The next goal is to demonstrate the efficacy of the new target peptide in detecting pathogens in chronic wounds. Protease activity in samples from patients with chronic wounds occupied by 105 or more pathogenic bacteria (infected) will be compared to those that are not (non-infected). In addition to verifying that this assay can differentiate infected from non- infected chronic wounds, this study will define a cutoff activity level that distinguishes the two. The last goal of our proposal is to correlate target peptide cleavage in vitro with known quantities of purified bacterial proteases. This information will be necessary to generate standards for conversion of proteolysis activity into a simple yes-or-no readout (visible by a color change) that will be used in the diagnostic device. Overall, the studies in this proposal will improve the assay for pathogen detection and demonstrate that it is suitable to aid in the early diagnosis of wound infection. /Relevance The goal of this research is to develop a simple, inexpensive diagnostic device to help clinicians determine if a chronic wound is infected at the point-of-care. By improving the ability for early detection of chronic wound infection, patients will receive appropriate treatment sooner and at a greater frequency. This will limit the spread of local wound infection, reduce the incidence of deep or systemic wound-related infections, and restore healing.

描述（由申请人提供）：局部感染是慢性伤口的常见并发症，它会干扰愈合过程，并可能导致全身感染、截肢或死亡。使问题更加复杂的是临床医生无法通过临床观察准确检测局部感染，而临床观察仍然是主要的诊断方法。这是因为通常的感染迹象被炎症或患者的潜在状况或疾病所掩盖。拟议研究的主要目的是开发和验证一种新的检测方法，以提高临床医生更早、更准确地检测慢性伤口感染的能力。长期目标是将这种检测方法整合到一种简单、廉价的即时护理设备中，以帮助患者就诊期间进行诊断。该设备将帮助慢性伤口患者更快地接受适当的治疗，恢复愈合，减轻疼痛和不适，并避免感染扩散。该诊断设备将基于 ECI Biotech 开发的一种新型病原菌检测方法。该方法测量引起感染的病原体释放到伤口液中的蛋白质降解酶（蛋白酶）的活性。由于蛋白酶对蛋白质内的特定位点具有特异性，因此 ECI 能够设计出单一目标肽，其中包含来自最常见伤口病原体的蛋白酶的裂解位点。由此产生的检测将检测病原体的总数，但不能区分物种。在之前的研究中，大约 80% 的慢性伤口临床分离物在体外产生可裂解该目标肽的蛋白酶。此外，超过 90% 直接取自感染伤口的临床样本显示出对目标肽具有显着的蛋白酶活性。这项工作还揭示了目标肽与人中性粒细胞弹性蛋白酶（HNE）的一些交叉反应性，HNE是一种在慢性伤口中高水平存在的宿主炎症蛋白酶。该提案的第一个目标是改变目标肽的氨基酸序列，以减少 HNE 的干扰，同时保持对细菌蛋白酶的敏感性。下一个目标是证明新目标肽在检测慢性伤口病原体方面的功效。慢性伤口被 105 种或更多病原菌（感染）占据的患者样本中的蛋白酶活性将与那些未被感染（未感染）的患者样本进行比较。除了验证该测定可以区分感染与未感染的慢性伤口外，本研究还将定义区分两者的截止活性水平。我们建议的最后一个目标是将体外目标肽切割与已知数量的纯化细菌蛋白酶相关联。这些信息对于生成将蛋白水解活性转换为将在诊断设备中使用的简单的是或否读数（通过颜色变化可见）的标准是必要的。总体而言，本提案中的研究将改进病原体检测方法，并证明其适合帮助伤口感染的早期诊断。 /相关性这项研究的目标是开发一种简单、廉价的诊断设备，帮助临床医生在护理点确定慢性伤口是否被感染。通过提高早期发现慢性伤口感染的能力，患者将更快、更频繁地接受适当的治疗。这将限制局部伤口感染的蔓延，减少深部或全身伤口相关感染的发生率，并恢复愈合。