Ultrasensitive C.difficile toxin measurement for diagnosis and outcome prediction

 描述（由申请方提供）：最近由产酶艰难梭菌引起的疾病的全球发病率和严重程度增加是一个主要问题。C.艰难梭菌感染（CDI），从轻度腹泻相关性腹泻到致死性伪膜性结肠炎，消耗越来越多的资源用于诊断、治疗和感染控制。能够产生毒素和增加疾病严重性的流行菌株的出现进一步增加了改进CDI诊断和管理方法的紧迫性。由C.艰难梭菌是由于携带毒素基因的菌株产生毒素A和B。尽管有越来越多的证据表明粪便中毒素的检测具有最高的临床特异性和预测价值，但目前的毒素检测方法仍然不足。细胞毒性试验对毒素B具有良好的分析灵敏度[检测限（LOD）1-10 μ g/L]，但费力且不标准化。常规定性免疫测定具有高LOD（~0.8-2.5 ng/mL）和低灵敏度（52-75%）。该领域已迅速转向核酸检测，用于超灵敏地检测致突变生物体，但越来越多地认识到，其效用（如经典的金标准，致突变培养）被致突变C无症状定殖所混淆。很难考虑到这些限制，该领域准备引入一种简单的、超灵敏的粪便毒素检测试验，其将毒素检测的高分析灵敏度与临床特异性相结合。事实上，疾病的严重程度已经初步与宿主中的毒素水平相关，这表明定量宿主中毒素的能力是不可能的。 粪便对预测疾病和治疗结果以及确定需要积极治疗的患者可能很有价值。事实上，这样的测试可能会导致我们诊断和管理CDI方式的范式转变。在我们最近的NIAID R21（5 R21 AI 103612 -02）下，我们已经成功地开发了基于单分子阵列（Simoa）技术的毒素A和B的超灵敏和定量“数字ELISA”测定。我们的新型检测方法检测粪便中的天然毒素，LOD为0.45（A）和1.5（B）μ g/mL，可以在4-log动态范围内定量毒素，并检测所研究的所有主要临床菌株的毒素。有了这些分析，我们可以回答CDI领域一些最紧迫的问题。我们的目标是检验核心假设，即CDI的临床过程受结肠中毒素A和B浓度的影响，因此准确和定量的粪便毒素测量可以改善CDI的诊断，帮助预测疾病结局，并指导管理。在目标1中，我们将检验毒素水平可以预测成人CDI严重程度和治疗反应的假设;艰难毒素型对这些结果的影响;并在儿科队列中探索这些方法。在目标2中，我们将检验毒素水平可以区分定植与疾病并预测< 3岁儿童的CDI严重程度的假设。最后， 在目的3中，我们将检验宿主抗体对毒素A和B的结合和中和改变粪便和血液中的游离毒素并影响疾病表达和结果的假设。

 DESCRIPTION (provided by applicant): The recent increase in global incidence and severity of disease caused by toxigenic Clostridium difficile is of major concern. C. difficile infection (CDI), ranging from mild antibiotic-associated diarrhea to lethal pseudomembranous colitis, consumes increasing resources for diagnosis, treatment, and infection control. The emergence of epidemic strains capable of toxin hyper-production and increased disease severity have further increased the urgency of improving methods for diagnosis and management of CDI. Disease caused by C. difficile is due to production of toxins A and B by strains harboring the toxin genes. Despite mounting evidence that detection of toxin in stool has highest clinical specificity and predictive value, current methods for toxin detection remain inadequate. The cytotoxicity assay has good analytical sensitivity for toxin B [limit of detection (LOD) 1-10 g/L] but is laborious and unstandardized. Conventional qualitative immunoassays have high LODs (~0.8-2.5 ng/mL) and poor sensitivity (52-75%). The field has rapidly moved towards nucleic acid testing for ultrasensitive detection of toxigenic organisms, but is increasingly recognizing that its utility (like that of the classic gold standard, toxigenic culture) is confounded by asymptomatic colonization by toxigenic C. difficile. Given these limitations, the field is poised fr the introduction of a simple, ultrasensitive stool toxin detection test that combines high analyticl sensitivity with the clinical specificity of toxin detection. The fact that disease severity has ben preliminarily correlated to toxin levels in the host suggests that the ability to quantify toxin in stool could be valuable to predict disease and treatment outcomes, and to identify those who need aggressive therapy. Such a test could in fact lead to a paradigm shift in the way we diagnose and manage CDI. Under our recent NIAID R21 (5R21AI103612-02), we have successfully developed ultrasensitive and quantitative "digital ELISA" assays for toxins A and B based on single molecule array (Simoa) technology. Our novel assays detect native toxins in stool with LODs of 0.45 (A) and 1.5 (B) g/mL, can quantify toxin across a 4-log dynamic range, and detect toxins from all major clinical strains studied. With these assays in hand, we are positioned to answer some of the most pressing questions in the CDI field. Our goal is to test the central hypothesis that the clinical course of CDI is influenced by the concentrations of toxins A and B in the colon, and thus that accurate and quantitative stool toxin measurement can improve the diagnosis of CDI, aid prediction of disease outcomes, and guide management. In Aim 1, we will test the hypotheses that toxin levels can predict CDI severity and response to treatment in adults; evaluate the contribution of C. difficile toxinotype to these outcomes; and explore these approaches in a pediatric cohort. In Aim 2, we will test the hypothesis that toxin levels can distinguish colonization from disease and predict CDI severity in children < 3. Finally, in Aim 3 we will test the hypothesis that binding and neutralization of toxins A and B by host antibodies alter free toxin in the stool and in the blood and influence disease expression and outcomes.