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)对人类健康构成重大威胁 新生儿。虽然CMV和HSV是终生感染，有潜伏期和再活跃期，但大多数母体 由于非特异性或缺乏临床症状，感染仍未得到诊断。在美国，孩子是 每小时因CCMV感染而永久残疾，即使10个婴儿中有9个在出生后和 仍未确诊。另一方面，侵袭性nHSV是一种罕见的新生儿感染，表现为广泛的 一系列临床症状，包括那些可能危及生命的症状。病毒培养和DNA检测 聚合酶链式反应已成为诊断CCMV和nHSV感染的“金标准”， 尽管新生儿的PCR检测灵敏度不高，培养技术也很耗时(长达5-7天)。 使用高分辨率熔融(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、现代的病毒培养和聚合酶链式反应技术的替代品。通过测量分子的荧光 插入染料作为聚合酶链式反应扩增的病原体DNA或RNA片段被加热并解离，序列 定义的熔融曲线或“指纹”是在闭管反应中以单核苷酸分辨率产生的。 然后使用机器学习自动识别和量化这些独特的微生物“指纹” 技术，在一个名为NeoChip的平台上，对最小血量(1毫升)的准确率为99%-100%。目前， 我们已经为40种细菌和抗菌素耐药性基因建立了独特的签名熔融曲线 通常感染新生儿。此外，新芯片已经扩展到区分单独放大的熔体 用于多病原体鉴定和定量的曲线特征，如多微生物感染所需。 在这项建议中，我们将通过纳入CMV的临床毒株来建立NeoChip的综合数据库 和具有可操作的抗病毒抗性基因的HSV。因为新芯片识别出了核酸的变异 CMV和HSV毒株的序列、个体分化和量化是可能的。我们还将翻译 CCMV和nHSV感染快速诊断新芯片的临床应用 孕妇及其后代(S)，以及直接将平台与标准定量核进行比较 酸试验(QNAT)、免疫球蛋白抗体/免疫球蛋白M抗体检测和临床结果测量的统计一致性 (预测值)。最后，我们将验证和翻译新芯片用于CCMV检测和临床相关性 用于纳入标准通用新生儿筛查计划的干血斑(DBS)样本。新芯片公司 目标是为病原体(病毒、细菌和病毒)的及时诊断提供准确有效的测试 真菌)在单一测试中对广泛的组织基质有效，并具有告知微生物耐药性的能力，从而 促进靶向抗菌剂和治疗药物的早期使用。这项建议直接针对 通过应用多学科方法快速、准确地应对生物医学挑战来筹集资金 诊断CCMV和nHSV有助于疾病预测和早期治疗或干预计划。

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.