NeoChip for specific and rapid identification of congenital CMV and neonatal HSV infections on minimal sample volume

项目摘要 先天性巨细胞病毒（cCMV）和新生儿单纯疱疹病毒（nHSV）对儿童的健康构成重大威胁。 新生儿虽然CMV和HSV是具有潜伏期和再激活期的终身感染，但大多数产妇 由于非特异性或缺乏临床症状，感染仍然无法诊断。在美国，一个孩子 即使10个婴儿中有9个在出生后没有症状， 仍未确诊。另一方面，侵袭性nHSV是一种罕见的新生儿感染， 一系列临床症状，包括可能危及生命的症状。病毒培养和DNA检测， 聚合酶链反应（PCR）已成为诊断cCMV和nHSV感染的“金标准”， 尽管新生儿中PCR测定的灵敏度差并且培养技术耗时（长达5-7天）。 使用高分辨率熔解（U-HRM）对病原体基因组序列进行通用基因分型提供了一种简单， 低成本、快速和现代化的替代病毒培养和PCR技术。通过测量 当PCR扩增的病原体DNA或RNA片段被加热并解离时， 在闭管反应中用单核苷酸分辨率产生确定的解链曲线或“指纹”。 这些独特的微生物“指纹”然后使用机器学习自动识别和量化 该技术在称为NeoChip的平台上以99-100%的准确度在最小血液体积（1 mL）上进行。目前， 我们已经建立了40种细菌物种和抗菌素抗性基因的独特特征熔解曲线， 通常感染新生儿。此外，NeoChip已扩展到区分单独扩增的熔解 根据多种微生物感染的要求，对多种病原体进行鉴定和定量的曲线特征。 在这个方案中，我们将通过整合CMV的临床菌株来建立NeoChip的综合数据库 和具有可作用的抗病毒抗性基因的HSV。因为NeoChip能识别核酸的变异 因此，CMV和HSV毒株的个体区分和定量是可能的。我们也会翻译 NeoChip用于cCMV和nHSV感染的特异性和快速诊断的大型前瞻性临床研究， 孕妇及其后代，以及直接比较平台与标准定量核酸 酸性试验（QNAT）检测、IgG/IgM抗体检测和临床结局指标的统计学一致性 （预测值）。最后，我们将验证和翻译NeoChip用于cCMV检测和临床相关性， 干血斑（DBS）样本，用于纳入标准的通用新生儿筛查程序。NeoChip的 目的是提供一个准确和有效的测试，及时诊断病原体病因（病毒，细菌， 真菌）在单一测试中对广泛的组织基质有效，并能够告知微生物耐药性，从而 有利于靶向抗菌剂和治疗剂的早期给药。该提案直接针对 通过应用多学科方法来解决快速准确地解决生物医学挑战， 诊断cCMV和nHSV以促进疾病诊断和早期治疗或干预方案。

Project Summary Congenital cytomegalovirus (cCMV) and neonatal herpes simplex virus (nHSV) impose major health threats on neonates. Although CMV and HSV are lifelong infections with periods of latency and reactivation, most maternal infections remain undiagnosed due to nonspecific or absent clinical symptoms. In the United States, a child is permanently disabled by cCMV infection every hour, even though 9 of 10 infants are asymptomatic after birth and remain undiagnosed. Invasive nHSV, on the other hand, is a rare neonatal infection that presents with a broad range of clinical symptoms, including those that may be life-threatening. Viral culture and DNA detection by polymerase chain reaction (PCR) have become the “Gold Standard” for the diagnosis of cCMV and nHSV infection, despite poor sensitivity of PCR assays in neonates and time consuming culture techniques (up to 5-7 days). Universal genotyping of pathogen genomic sequences using High Resolution Melt (U-HRM) provides a simple, low cost, rapid, and modern alternative to viral cultures and PCR techniques. By measuring the fluorescence of an intercalating dye as PCR-amplified pathogen DNA or RNA fragments are heated and disassociate, sequence defined melt curves, or “fingerprints”, are generated with single-nucleotide resolution in a closed-tube reaction. These unique microbial “fingerprints” are then automatically identified and quantified using machine learning technology, with an accuracy of 99-100% on minimal blood volume (1 mL), in a platform called NeoChip. Presently, we have established unique signature melt curves for 40 bacterial species and antimicrobial resistance genes that commonly infect neonates. Additionally, NeoChip has been expanded to distinguish individually amplified melt curve signatures for multiple pathogen identification and quantification, as required for polymicrobial infection. In this proposal, we will build out NeoChip’s comprehensive database by incorporating clinical strains of CMV and HSV with actionable antiviral resistance genes. Because NeoChip identifies variances in nucleic acid sequences, individual differentiation and quantification of CMV and HSV strains are possible. We will also translate the NeoChip for specific and rapid diagnosis of cCMV and nHSV infection in a large prospective clinical study of pregnant women and their offspring(s), as well as directly compare the platform to standard quantitative nucleic acid test (QNAT) assays, IgG/IgM antibody testing, and clinical outcome measures for statistical concordance (predictive value). Finally, we will validate and translate NeoChip for cCMV detection and clinical correlation using dried blood spot (DBS) samples for incorporation into standard universal newborn screening programs. NeoChip’s goal is to provide an accurate and valid test for the timely diagnosis of pathogen etiology (viral, bacterial, and fungal) in a single test with efficacy on broad tissue matrices and capacity to inform microbial resistance, thereby facilitating early administration of targeted antimicrobials and therapeutics. This proposal directly addresses the funding call by applying a multidisciplinary approach to address the biomedical challenges of rapidly and accurately diagnosing cCMV and nHSV to facilitate disease prognostication and early therapeutic or intervention programs.