Point-of-care HPV mRNA test for cervical cancer screening in low-resource settings

摘要： 非常需要改善全球获得宫颈癌筛查的机会。在全球范围内，大约 每年有570，000名妇女被诊断患有宫颈癌，311，000名妇女死于宫颈癌。近90% 宫颈癌死亡的20%发生在低收入和中等收入国家，主要原因是 实施有效的筛查计划。同样，2010年医疗服务不足地区的宫颈癌发病率 美国仍然很高。例如，在得克萨斯州的格兰德河流域，宫颈癌发病率为55 高于美国平均水平，约10%的合格妇女接受筛查。普遍一致 高危人乳头瘤病毒（hrHPV）检测是改善全球筛查工作的最佳方法。一个 提供比DNA更好的灵敏度和更好的特异性的HPV生物标志物是hrHPV mRNA。然而，在这方面， 目前可用的hrHPV mRNA检测仍然过于复杂和昂贵（例如，设备和 每项测试大约74美元），以便在资源匮乏和医疗服务不足的环境中有效实施。 等温扩增和侧流检测的进展为发展即时检测提供了机会 (POC)hrHPV mRNA检测是准确的，负担得起的，可以在低资源环境中进行。目标 该建议的一个优点是将联合收割机等温扩增和侧流检测结合在一个集成的点- 护理设备，以显着降低hrHPV mRNA检测的成本。我们将开发一种低成本的POC hrHPV E7 mRNA测试需要最少的实验室设备，并执行以及商业RNA测试。 等温扩增降低了通常与核酸扩增相关的仪器成本和复杂性。 放大，只需要一个单一的温度加热器。侧流检测集成了样品操作 处理并吸取所有检测试剂通过预定义的测试区域，产生简单的比色读数。 我们提出的概念验证测试将检测HPV 16和18型，这两种类型负责70%的宫颈癌。 癌症，我们估计在小批量生产中每个测试的成本<3美元。 与R21提案的探索/发展目标一致，我们的目标是（1）设计和优化HPV 16和18 E7 mRNA扩增测定和侧流检测;（2）将联合收割机mRNA扩增和 检测到单个POC设备中;（3）使用合成和 临床样本。我们将利用我们的跨学科团队的专业知识，其中包括设计 用于LMIC、宫颈癌护理、HPV诊断和流行病学的技术，以开发、验证和 翻译这个新的筛选测试。我们打算从这个概念验证测试中建立，将HPV mRNA 未来将对31、33、35、45、52和58型进行检测，以实现对引起>90%的HPV类型的检测。 子宫颈癌总的来说，这项研究将导致开发和实施一个可扩展的，成本- 有效的筛查测试是全球消除宫颈癌的关键和必要的一步。

Abstract: There is a significant need to improve global access to cervical cancer screening. Globally, approximately 570,000 women are diagnosed with and 311,000 women die from cervical cancer each year. Nearly 90 percent of cervical cancer deaths occur in low- and middle-income countries (LMICs), due mainly to challenges in implementing effective screening programs. Likewise, cervical cancer rates in medically underserved areas in the US remain high. For example, in the Rio Grande Valley of Texas, cervical cancer incidence rates are 55% higher than the US average, and approximately 10% of eligible women are screened. There is broad consensus that high-risk human papillomavirus (hrHPV) testing is the best approach to improve global screening efforts. An HPV biomarker that provides excellent sensitivity and better specificity than DNA is hrHPV mRNA. However, currently available hrHPV mRNA testing remains too complex and costly (e.g. >$45,000 for equipment and roughly $74 per test) for effective implementation into low-resource and medically underserved settings. Advances in isothermal amplification and lateral flow detection offer an opportunity to develop a point-of-care (POC) hrHPV mRNA test that is accurate, affordable, and can be performed in low-resource settings. The goal of this proposal is to combine isothermal amplification and lateral flow detection within an integrated point-of- care device to dramatically lower the cost of hrHPV mRNA testing. We will develop a low-cost, POC hrHPV E7 mRNA test that requires minimal laboratory equipment and performs as well as commercial RNA tests. Isothermal amplification reduces the instrumentation cost and complexity typically associated with nucleic acid amplification requiring only a single-temperature heater. Lateral flow detection integrates sample manipulation processes and wicks all detection reagents past pre-defined test zones, producing a simple, colorimetric readout. Our proposed proof-of-concept test will detect HPV types 16 and 18, the two types responsible for 70% of cervical cancer, and we estimate will cost <$3 per test in low-volume production. Consistent with the exploratory/developmental goals of an R21 proposal, we aim to (1) design and optimize HPV 16 and 18 E7 mRNA amplification assays and lateral flow detection; (2) combine mRNA amplification and detection into a single POC device; (3) evaluate performance of the developed mRNA test using synthetic and clinical samples. We will leverage the expertise of our interdisciplinary team, which includes designing technologies for LMICs, cervical cancer care, HPV diagnostics, and epidemiology, to develop, validate, and translate this novel screening test. We intend to build from this proof-of-concept test to incorporate HPV mRNA detection for types 31,33, 35, 45, 52, and 58 in the future to achieve detection of the HPV types that cause >90% of cervical cancer. Collectively, this research will lead to the development and implementation of a scalable, cost- effective screening test, a critical and necessary step toward the global elimination of cervical cancer.