Development, assessment, an implementation of a dual isothermal point-of care molecular diagnostic for both FGS and HPV

FGS是一种由感染血吸虫寄生虫引起的妇科疾病，被认为影响了生活在撒哈拉以南非洲的4000多万妇女。除了引起血尿、严重抽筋和不孕不育等症状外，嗜血杆菌感染还与性传播感染的高风险有关，是一级致癌物质。HPV是一组已知会导致生殖器疣的病毒，通常是宫颈癌的先兆。HPV是一种全球性病原体，90%以上的宫颈癌病例是由高危HPV毒株引起的。最不发达国家成功诊断和治疗的一个主要障碍是缺乏可获得的诊断方法。由于可以通过妇科样本检测到HPV和FGS，该项目旨在提供一种可在资源匮乏的情况下实施的综合诊断方法，以支持被边缘化妇女的及时治疗。回顾当前文献，以确定已开发的用于诊断FGS和HPV的等温分子分析方法。还将审查这些检测方法在低资源环境下的适用性。2.将利用开放获取的数据库中的遗传数据，选择或重新设计(如果需要)用于RPA和LAMP分析开发的合适靶标和引物-探针组。在线资源，包括BLAST和OlicoAnalyzer，将用于在实验室分析开发和测试之后以电子方式测试分析的特异性。对于HPV，一般HPV靶标和高危HPV靶标都将被调查。检测将作为单链反应进行测试和优化，然后在多路反应中进行优化。将调查内部控制目标(人DNA)，并将其纳入检测，以达到质量控制的目的。分析测试将使用合成DNA、参考样本中的gDNA以及现有的血链球菌和人乳头状瘤病毒的临床样本。所有的分子检测都将对照qPCR参考检测进行验证。我们根据它们是否符合WHO为最不发达国家诊断制定的可靠标准来评估这些检测方法。将在撒哈拉以南非洲血吸虫病/人乳头瘤病毒共同流行的环境中对检测性能和适宜性进行最后阶段评估。此外，亦会比较自拭子样本和临床拭子样本，以协助了解目标人群对抽样程序的接受程度。考虑文化和社会态度对于最准确的诊断至关重要。除了描述性统计外，还将利用潜在类别分析来分析定量聚合酶链式反应和已开发的多重分析之间的诊断能力差异。该项目将发展跨学科技能，包括分子生物学、化学、实地工作、流行病学分析和统计分析。还将发展生理学和病理学方面的知识，即被UKRI确定为脆弱的技能。重点是大学之间的合作，由于每个合作伙伴的定制专业知识，项目的很大一部分在每个机构进行。此外，与致力于支持向边缘化社区提供可获得诊断的工作的商业诊断公司和非营利组织的现有联系将指导这项研究。该项目考虑MRC LID主题的全球健康和传染病，这对MRC改善人类健康和每个人的经济繁荣的战略至关重要。血吸虫病是一种被忽视的热带疾病，在最不发达国家最为流行。HPV在全球范围内存在，但在最不发达国家获得筛查和接种疫苗的机会有限。

FGS is a gynaecological condition resulting from infection with the parasite Schistosoma haematobium and is thought to affect over 40 million women living across sub-Saharan Africa. In addition to causing symptoms such as bloody urine, severe cramping, and infertility, S. haematobium infection is associated with heightened risk of STI's and is a Grade 1 carcinogen. HPV is group of viruses known to cause genital warts and is often a precursor to cervical cancer. HPV is a global pathogen, and high-risk HPV strains cause more than 90% of all cervical cancer cases. A major hurdle in successful diagnosis and treatment in for Least Developed Countries (LDC's) is the lack of accessible diagnostics. Due to the fact that both HPV and FGS can be detected through gynaecological samples, this project aims to provide an integrated diagnostic that can be implemented in low resource settings to support timely treatment of marginalised women.Project Plan1. To review current literature to identify isothermal molecular assays that have been developed and used for the diagnosis of FGS and HPV. These assays will also be reviewed in terms of their applicability in low resource settings. 2. Suitable targets and primer-probe sets for RPA and LAMP assay development will be selected or redesigned (if needed) using genetic data from open-access databases. Online resources, including BLAST and OligoAnalyzer, will be used to in silico test the specificity of the assays followed by laboratory analytical development and testing. For HPV, a general HPV target and a high-risk HPV target would both be investigated.3. Assays will be tested and optimised as singleplex reactions before being optimised in a multiplex.4. An internal control target (human DNA) will be investigated and aimed to be incorporated into the assay for quality control purposes.5. Analytical testing will be done using synthetic DNA, gDNA from reference samples, and also available clinical samples for both S. haematobium and HPV. All molecular tests will be validated against qPCR reference tests.6. The assays we be evaluated in terms of their fit to the REASSURED criteria set out for diagnostics from the WHO for diagnostics for LDC's.7. Final stage assessment of the assay performance and suitability will be performed in a S. haematobium / HPV co-endemic setting in Sub-Saharan Africa. A comparison between self-swab and clinical swab samples will also be made to help inform the acceptability of the sampling procedure within the target population. Considering cultural and social attitudes is essential in relation to the most accurate diagnosis8. As well as descriptive statistics, differences in diagnostic capabilities between qPCR and the developed multiplex assay would be carried out using latent class analysis. The project will develop interdisciplinary skills including molecular biology, chemistry, field work, epidemiological analysis, and statistical analysis. Knowledge of physiology and pathology, skills identified as vulnerable by the UKRI, will also be developed.There is emphasis on intercollegiate collaboration, with significant portions of the project being undertaken at each institution due the bespoke expertise of each partner. Additionally, existing connections with commercial diagnostic companies and non-for-profit organisations that are dedicated to supporting work towards the provision of accessible diagnostics to marginalized communities, will guide this research.The project considers the MRC LID themes of global health and infectious disease, which are essential to the MRC strategy of improving human health and economic prosperity for everyone. Schistosomiasis is a neglected tropical disease and is most prevalent in LDC's. HPV is present globally, but access to screening and vaccination is limited in LDCs.