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

FGS是一种由血血吸虫感染引起的妇科疾病，据认为，生活在撒哈拉以南非洲的4000多万妇女受到该病的影响。除了引起血尿、严重痉挛和不孕症等症状外，血氧梭菌感染还与性传播感染的风险增加有关，是一级致癌物。HPV是一组已知会引起生殖器疣的病毒，通常是宫颈癌的前兆。HPV是一种全球性的病原体，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HPV和FGS都可以通过妇科样本检测到，因此该项目旨在提供一种综合诊断方法，可在资源匮乏的环境中实施，以支持对边缘化妇女的及时治疗。项目Plan1。回顾目前的文献，以确定已经开发和用于诊断FGS和HPV的等温分子分析。还将审查这些测定法在低资源环境中的适用性。2. 将使用开放获取数据库中的遗传数据选择或重新设计RPA和LAMP测定开发的合适靶标和引物探针集。在线资源，包括BLAST和OligoAnalyzer，将用于在实验室分析开发和测试之后对分析的特异性进行硅测试。对于HPV，一般HPV靶点和高危HPV靶点都将进行研究。在多路反应中进行优化之前，检测将作为单路反应进行测试和优化。将研究一个内部控制目标（人类DNA），目的是将其纳入分析以进行质量控制。分析测试将使用合成DNA、参考样本的gDNA以及可用的临床样本来进行，包括血氧梭菌和人乳头状瘤病毒。所有分子检测将与qPCR参比检测进行验证。我们将根据其是否符合世卫组织为最不发达国家的诊断而确定的诊断标准对检测方法进行评估。将在撒哈拉以南非洲的嗜血杆菌/人乳头瘤病毒共流行环境中对检测性能和适用性进行最后阶段评估。自我拭子样本和临床拭子样本之间的比较也将进行，以帮助告知在目标人群中抽样程序的可接受性。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，qPCR和开发的多重分析之间的诊断能力差异将使用潜在类分析进行分析。该项目将培养跨学科技能，包括分子生物学、化学、实地工作、流行病学分析和统计分析。生理学和病理学的知识，被UKRI确定为脆弱的技能，也将得到发展。强调校际合作，由于每个合作伙伴的定制专业知识，项目的重要部分在每个机构进行。此外，与商业诊断公司和非营利组织的现有联系将指导这项研究，这些组织致力于为边缘化社区提供可获得的诊断。该项目考虑了MRC LID的全球健康和传染病主题，这对MRC改善人类健康和经济繁荣的战略至关重要。血吸虫病是一种被忽视的热带病，在最不发达国家最为普遍。HPV在全球都存在，但在最不发达国家获得筛查和疫苗接种的机会有限。

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.