Ultrasensitive C.difficile toxin measurement for diagnosis and outcome prediction

 描述(由申请人提供)：最近由产毒艰难梭菌引起的全球发病率和疾病严重程度的增加是主要关注的问题。艰难梭菌感染(CDI)，从轻微的抗生素相关性腹泻到致死性伪膜性结肠炎，消耗了越来越多的诊断、治疗和感染控制资源。能够超产毒素的流行菌株的出现和疾病严重性的增加进一步增加了改进CDI诊断和管理方法的紧迫性。艰难梭菌引起的疾病是由于含有毒素基因的菌株产生毒素A和B。尽管越来越多的证据表明，检测粪便中的毒素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存在不足。细胞毒性试验对毒素B具有较好的分析灵敏度[检出限为1-10g/L]，但操作繁琐且不规范。传统的定性免疫分析方法具有较高的灵敏度(52-75%)和较高的检测灵敏度(~0.8-2.5 ng/mL)。该领域已迅速转向核酸检测，以超灵敏地检测产毒生物，但越来越多地认识到，其实用性(如经典的黄金标准，产毒培养)被产毒艰难梭菌无症状的定植所混淆。鉴于这些局限性，该领域将引入一种简单、超灵敏的粪便毒素检测测试，该测试结合了高分析灵敏度和毒素检测的临床特异性。疾病的严重程度与寄主中的毒素水平初步相关的事实表明，对寄主体内的毒素进行量化的能力 粪便可能对预测疾病和治疗结果以及确定需要积极治疗的人有价值。事实上，这样的测试可能会导致我们诊断和管理CDI的方式发生范式转变。在我们最新的NIAID R21(5R21AI103612-02)的指导下，我们成功地基于单分子阵列(SIMOA)技术开发了超灵敏和定量的毒素A和B的数字ELISA法。我们的新检测方法检测粪便中的天然毒素，检测灵敏度分别为0.45(A)和1.5(B)g/mL，可以在4对数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，我们就可以回答CDI领域一些最紧迫的问题了。我们的目标是验证这一中心假设，即CDI的临床病程受到结肠中毒素A和B浓度的影响，因此准确和定量的粪便毒素检测可以提高CDI的诊断，帮助预测疾病结果，并指导治疗。在目标1中，我们将测试毒素水平可以预测成人CDI严重程度和治疗反应的假设；评估艰难梭菌毒素类型对这些结果的贡献；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疾病和定居的假设，并预测儿童CDI的严重程度。 在目标3中，我们将测试这样一个假设，即宿主抗体结合和中和毒素A和B会改变粪便和血液中的游离毒素，并影响疾病的表现和预后。

 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.