Faculty of Medicine, University of Sciences, Techniques and Technologies of Bamako (USTTB), Bamako BP 1805, Mali.
University Clinical Research Center, International Centers for Excellence in Research (UCRC), University of Sciences, Techniques and Technologies of Bamako, Bamako, Mali.
J Virol Methods. 2024 Dec;330:115026. doi: 10.1016/j.jviromet.2024.115026. Epub 2024 Sep 2.
Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16-74.3] for HBV and 82 % [78.09-85.90] for HIV and an overall specificity of 100 % [94.95-100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26-88.73] and >1000 IU/mL for HBV was 100 % [95.51-100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.
由于传播途径相同，包括性接触和垂直传播，HIV 和 HBV 合并感染很常见，特别是在撒哈拉以南非洲地区。测量 HIV 和 HBV 的病毒载量（VL）对于确定它们的感染阶段和监测抗病毒治疗的反应至关重要。在资源有限的国家，实施病毒载量检测是一个重大挑战，特别是由于成本和可用性问题。我们设计了针对 HIV 和 HBV 基因组保守区域的引物，这些引物专门适应于在西非流行的 HIV-1 亚型 CRF02AG 和 HBV 基因型 E 病毒株。我们首先验证了两种用于单独定量的单重 qPCR 检测方法，然后开发了一种用于同时定量两种病毒的多重 qPCR 检测方法。使用针对两种病毒的一步逆转录-PCR 反应，在单个试管中进行 HIV RNA 和 HBV DNA 的扩增。该多重 qPCR 检测方法的性能特征，如定量范围、灵敏度和特异性，与 HIV 和 HBV 病毒载量定量的参考 qPCR 检测方法进行了比较。该多重 qPCR 检测方法使用来自合并或单纯感染患者的临床样本进行了验证，分别与 HIV 和 HBV 参考检测方法进行了可比的病毒载量定量。该多重 qPCR 检测方法对 HBV 的总灵敏度为 71.25%[68.16-74.3]，对 HIV 的总灵敏度为 82%[78.09-85.90]，对两种病毒的总特异性为 100%[94.95-100]。尽管 HIV 和 HBV 检测方法的总灵敏度低于商业比较检测方法，但 HIV 的>1000 拷贝/mL 的临床决策范围的灵敏度为 80%[71.26-88.73]，HBV 的>1000 IU/mL 的灵敏度为 100%[95.51-100]，这表明检测结果可用于指导治疗决策。这种内部开发的多重 qPCR 检测方法是一种有用的诊断工具，因为它可以在马里目前用于 HIV 或 SARS-CoV-2 感染监测的负担得起的"开放"实时 PCR 平台上进行。
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