Development of Improved Serological Diagnostic and Parasite Genotyping Tools for Congenital Chagas Disease

项目总结： 改进的先天性血吸虫血清学诊断和寄生虫基因分型方法的研制 恰加斯病 恰加斯病是一种被忽视的疾病，由原生动物寄生虫克氏锥虫引起。这是一个 拉丁美洲和美国南部的严重公共卫生问题。慢性心绞痛的诊断 感染的患者，包括先天性和母体病例，是基于抗体检测的。PA-18-031 克氏锥虫的优先事项包括开发新的高效度诊断试验和 及时检测先天性和母体旋毛虫感染的可靠性。事实上，世界卫生组织 建议至少进行两次阳性检测以确定诊断，因为敏感性和 特异性强，结果经常不一致。部分不和谐可以归因于非常大的 克鲁兹毛滴虫的遗传和抗原多样性，已划分为7个离散的分型单位 (DTU)TCI-TcVI和Tcbat。事实上，目前的血清学测试是基于一组非常有限的寄生虫 不能反映克氏毛滴虫多样性和多克隆感染的全部抗原/菌株。 因此，迫切需要基于一组保守抗原的更可靠的测试，以允许 检测所有克氏锥虫的基因型别。另一方面，虽然寄生虫的基因分型在 在寄生虫培养和媒介样本中，目前基于聚合酶链式反应的技术对血液的敏感性较低 查格症患者的样本。因此，另一个关键的需要是识别新的分子标记 T.ruzi DTUS，允许在临床样本中进行更敏感的基因分型。在目标1中，我们将改进 克氏毛滴虫母源和先天性感染的血清学诊断新保守型克氏毛滴虫 抗原。在目标2中，我们将通过以下方式提高目前用于基因分型的分子工具的灵敏度 孕妇和新生儿临床标本中克氏毛滴虫的序列分析我们将表演一场 从多个DTU中筛选克氏锥虫基因组序列寻找新抗原和新抗原 分子标记结合多肽阵列和多重高通量免疫筛查 聚合酶链式反应和测序。将使用在酶联免疫吸附试验平台中确定用于诊断的抗原 在快速检测平台上准确诊断克氏毛滴虫感染。我们还将表演一场 母婴寄生虫基因分型及多株感染的回顾性分析 恰加斯病。在这些研究完成后，我们预计将确定一套保守的 用于可靠诊断的新的快速检测的抗原和一个或多个分子标志物 孕妇和新生儿所有克氏锥虫DTU测序的基因分型。这些进展将 也通过进一步探索，推动了对克氏锥虫的分子研究，远远超出了我们自己的研究重点 寄生虫进化和遗传多样性使我们能够理解 寄生虫特征、先天传播和临床症状。

PROJECT SUMMARY: Development of improved serological diagnostic and parasite genotyping tools for congenital Chagas disease Chagas disease is a neglected disease caused by the protozoan parasite Trypanosoma cruzi. It is a serious public health issue in Latin America and the southern United States. Diagnosis of chronically infected patients, including congenital and maternal cases, is based on antibody detection. PA-18-031 priorities for Trypanosoma cruzi include the development of new diagnostic tests with high validity and reliability for timely detection of congenital and maternal T. cruzi infection. Indeed, the WHO recommends a minimum of two positive tests to establish diagnosis, due to limited sensitivity and specificity and frequent discordant results. Part of the discordances may be attributed to the very large genetic and antigenic diversity of T. cruzi, which has been divided into seven discrete typing units (DTUs) TcI-TcVI and Tcbat. Indeed, current serological tests are based on a very limited set of parasite antigens/strains which do not reflect the entire range of T. cruzi diversity and multiclonal infections. There is thus a critical need for more reliable tests based on panels of conserved antigens allowing detection of all T. cruzi genotypes. On the other hand, while parasite genotyping is straightforward in parasite culture and in vector samples, current PCR-based techniques have a low sensitivity with blood samples from chagasic patients. Another critical need is therefore to identify new molecular markers of T. cruzi DTUs, allowing a more sensitive genotyping in clinical samples. In aim 1, we will improve serological diagnostic of maternal and congenital T. cruzi infection identifying new conserved T. cruzi antigens. In Aim 2, we will improve the sensitivity of current molecular tools for genotyping by sequencing of T. cruzi in clinical samples from pregnant women and newborns. We will perform a bioinformatics screening of T. cruzi genome sequences from multiple DTUs for new antigens and molecular markers coupled with high-throughput immuno-screening with peptide arrays and multiplex PCR and sequencing, respectively. Antigens identified for diagnostic in an ELISA platform will be used in a rapid test platform for the accurate diagnostic of T. cruzi infection. We will also perform a retrospective analysis of parasite genotype and multi-strain infection on maternal and congenital Chagas disease. At the completion of these studies, we expect to have identified a set of conserved antigens for a novel rapid test for a reliable diagnostic and one or more molecular markers for genotyping by sequencing of all T. cruzi DTUs in pregnant women and newborns. These advances will also spur on molecular research on T. cruzi much beyond our own research focus, by further exploring parasite evolution and genetic diversity allowing us the to understand the potential relations between parasite characteristics, congenital transmission and clinical symptoms.