Beijing Key Laboratory for HIV/AIDS Research, Clinical and Research Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China.
Chin Med J (Engl). 2024 Mar 20;137(6):729-736. doi: 10.1097/CM9.0000000000003081. Epub 2024 Mar 4.
Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.
The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed.
The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts.
This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.
总人类免疫缺陷病毒（HIV）DNA 和整合的 HIV DNA 广泛用作 HIV 持续存在的标志物。液滴数字聚合酶链反应（ddPCR）可用于绝对定量，而无需标准曲线。在这里，我们开发了双 ddPCR 检测法来检测和定量总 HIV DNA 和整合的 HIV DNA。
通过含有 HIV 长末端重复（LTR）和人类 CD3 基因（用于总 HIV DNA）和 ACH-2 细胞（用于整合的 HIV DNA）的质粒构建体评估检测限、动态范围、灵敏度和重现性。在总 HIV DNA 和整合的 HIV DNA 中检测了 42 例稳定抑制性抗逆转录病毒治疗（ART）的病例。对与 DNA 拷贝数相关的数据以及 CD4 + T 细胞计数、CD8 + T 细胞计数和 CD4/CD8 T 细胞比值分别进行相关系数分析。还评估了检测的线性动态范围和最低检测限（LLOQ）。
该检测法可以在 6.3 拷贝/反应的浓度下 100%检测到 HIV-1 拷贝的存在，ddPCR 检测法的估计 LLOQ 为 4.4 HIV DNA 拷贝/反应（95%置信区间 [CI]：3.6-6.5 拷贝/反应），总 HIV DNA 检测法的线性度为 5 个对数 10 单位范围。对于整合的 HIV DNA 检测法，LLOQ 为 8.0 HIV DNA 拷贝/反应（95%CI：5.8-16.6 拷贝/反应），线性度为 3 个对数 10 单位范围。CD4 + T 细胞中的总 HIV DNA 与整合的 HIV DNA 呈正相关（r = 0.76，P <0.0001）。同时，CD4 + T 细胞中的总 HIV DNA 和整合的 HIV DNA 均与 CD4/CD8 比值呈负相关，但与 CD8 + T 细胞计数呈正相关。
该 ddPCR 检测法可以高效、稳健、灵敏地定量总 HIV DNA 和整合的 HIV DNA。它可以很容易地适应非 B 谱系的 HIV DNA 检测，这可能有助于临床试验中的检测。
