Development of a Multiplex Quantitative PCR Assay for HIV and Hepatitis B Virus, for Low- and middle Income Countries

摘要 人类免疫缺陷病毒(HIV)和乙肝病毒(乙肝)具有相同的血液传播模式 传播，主要是通过性接触和注射吸毒，以及有艾滋病毒风险的个人也是 感染乙肝病毒的风险更高。在全球范围内，估计有2.57亿人携带慢性乙肝病毒 (2015年)而在2019年，有3800万人感染艾滋病毒(PLHIV)。非洲，特别是撒哈拉以南地区 非洲(SSA)是世界上受影响最严重的地区，70%(2570万人)感染艾滋病毒，约6%的人感染艾滋病 感染乙肝病毒的成年人群。重要的是，据估计，慢性乙肝病毒感染影响了5%-20%的 PLHIV。乙肝病毒是一种无症状的肝病，其早期发现困难，导致严重的 并发症，如肝硬变、肝细胞癌和过早死亡。有关乙肝病毒状况的知识，请访问 启动艾滋病毒抗逆转录病毒疗法(ART)对于适当选择初始ART方案至关重要，因为 富马酸替诺福韦酯(TDF)应与拉米夫定(3TC)或恩曲他滨(FTC)联合使用， 它抑制艾滋病毒和乙肝病毒的复制，并用于监测治疗。马里，西非的LMIC 艾滋病毒/艾滋病流行率占总人口(2000万)和特定成人人口的1% 人群(如孕妇、学生、献血者)的HBs Ag阳性率从10%到10%不等 18.8%。而HIV RNA和HBVDNA定量分析则建议更好地指导和监测 在这种治疗下，在SSA中获得这种定量分析的机会非常有限，甚至无法获得。在大多数情况下 非洲，艾滋病毒患者只对HBs Ag进行筛查。因此，隐匿性乙肝(OBI)患者，尽管有 血清和/或肝脏中的HBVdna，但HBs Ag阴性，将无法被检测到，因此，错过了一个重要的 开始治疗的机会。OBI通常没有症状，但它的重新激活通常发生在 免疫抑制的个体，例如艾滋病毒感染者。病毒核酸的测定 在确定感染阶段、选择治疗方法方面发挥关键作用，并提供有关疗效的信息 抗病毒治疗。该K43应用寻求开发一种多重、实时、定量的聚合酶链式反应(RT- 用于同时定量HIV RNA和HBVDNA的QPCR)，该方法专门设计用于 使用经济、环保和 在SSA实验室工作人员的技术能力范围内。我提议的职业发展将是 在美国和马里导师的监督下，专注于获得设计方面的专业知识和技能 和开发新的多重PCR检测方法，评估新的诊断检测方法，以及在 新测试的实施战略，特别是与利益攸关方的参与有关的战略，以及 加强我在研究、领导和团队科学方面的职业技能。

SUMMARY Human immunodeficiency virus (HIV) and Hepatitis B virus (HBV) share the same blood borne modes of transmission, primarily through sexual contact and injection drug use and individuals at risk for HIV are also at higher risk for HBV infection. Globally, an estimated 257 million people live with chronic HBV infection (2015) while, in 2019, 38 million people were living with HIV (PLHIV). Africa, in particular sub-Saharan Africa (SSA), is the most affected region in the world with and 70% (25.7 million) of PLHIV and ~6% of the adult population infected with HBV. Importantly, it is estimated that chronic HBV infection affects 5-20% of PLHIV. HBV is an asymptomatic liver disease making its early detection difficult and leading to serious complications such as cirrhosis, hepatocellular carcinoma, and early death. Knowledge of HBV status at initiation of HIV antiretroviral therapy (ART) is essential for appropriate select an initial ART regimen, as tenofovir disoproxil fumarate (TDF) should be combined with lamivudine (3TC) or emtricitabine (FTC), which suppress both HIV and HBV replication, and for monitoring treatment. Mali, a LMIC in West Africa has a HIV/AIDS prevalence rate of >1% of the general population (>20 million) and specific adult population groups (e.g., pregnant women, students, blood donors) have HBs Ag positive rates ranging from 10 to 18.8%. While HIV RNA and HBV DNA quantification assays are recommended to better guide and monitor treatment, access to such quantification assays in SSA is very limited or not even available. In most of Africa, HIV patients screened only on HBs Ag. Thus, patients with occult hepatitis B (OBI), despite having HBV-DNA in serum and/or in liver, but HBs Ag negativity, will not be detected and, thus, miss an important opportunity to initiate treatment. OBI is usually asymptomatic but its reactivation commonly occurs in immunosuppressed individuals, such as in HIV infected persons. Measurement of viral nucleic acids plays a critical role in determining the phase of infection, selecting treatment, and is informative about the efficacy of antiviral therapy. This K43 application seeks to develop a multiplex, real time, quantitative PCR (RT- qPCR) assay for simultaneous quantification of HIV RNA and HBV DNA that is specifically designed to detect regional genetic variants, using an “open” system platform that is economically, environmentally, and within the technical capabilities of laboratory staff in SSA. My proposed career development will be supervised by both American and Malian mentors and focus on gaining expertise and skills in the design and development of new multiplexed PCR assays, methods to evaluate new diagnostic tests, and in implementation strategies for new tests, specifically as it pertains to stakeholder engagement, as well as, strengthen my career skills in research leadership and team science.