采用源自商业 HIV-1 RNA 定量检测的标准化方法，对未治疗和已治疗患者外周血中的 HIV-1 总库进行定量分析。

Quantification of the HIV-1 total reservoir in the peripheral blood of naïve and treated patients by a standardised method derived from a commercial HIV-1 RNA quantification assay.

机构信息

Department of Molecular and Clinical Sciences, Polytechnic University of Marche, Ancona, Italy.

Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Italy.

出版信息

Clin Chem Lab Med. 2020 Apr 28;59(3):609-617. doi: 10.1515/cclm-2020-0142. Print 2021 Feb 23.

DOI:10.1515/cclm-2020-0142

PMID:33326413

Abstract

OBJECTIVES

HIV-1 DNA can persist in host cells, establishing a latent reservoir. This study was aimed to develop an extraction and amplification protocol for HIV-1 DNA quantification by modifying a quantitative commercial assay.

METHODS

HIV-1 DNA was extracted on an Abbott m2000 instrument, using an open-mode protocol. Two calibrators, spiked with a plasmid containing HIV-1 genome (10 and 10 cps/mL), were extracted and amplified to generate a master calibration curve. Precision, accuracy, linear dynamic range, limit of quantification (LOQ) and limit of detection (LOD) were determined. A cohort of patients, naïve or chronically infected, was analysed.

RESULTS

Calibration curve was obtained from 42 replicates of standards (std); precision was calculated (coefficients of variability [CVs] below 10%); accuracy was higher than 90%. Linearity covered the entire range tested (10-10 copies per reaction), and LOD (95%) was 12 copies per reaction. HIV-1 DNA was significantly higher (p < 0.0001) in drug-naïve (62) than in chronically treated patients (50), and proviral loads correlated with lymphocytes (p = 0.0002) and CD4 (p < 0.0001) counts only in naïve patients. Both groups displayed a significant inverse correlation between CD4 nadir and proviral loads. A significant correlation (p < 0.0001) between viraemia and HIV-1 reservoir was disclosed. No significant difference was obtained from the comparison between proviral loads on whole blood and peripheral blood mononuclear cells (PBMCs) from the same patient.

CONCLUSIONS

The novelty of our approach relies on the selection of appropriate reference standard extracted and amplified as clinical specimens avoiding any underestimation of the reservoir. Results confirm HIV-1 DNA as a marker of disease progression, supporting the relationship between the width of latent reservoir and the immunological status of the patient.

摘要

目的

HIV-1 DNA 可在宿主细胞中持续存在，建立潜伏库。本研究旨在通过修改一种定量商业检测方法，开发一种用于 HIV-1 DNA 定量提取和扩增的方案。

方法

在 Abbott m2000 仪器上采用开放式方案提取 HIV-1 DNA。提取并扩增含有 HIV-1 基因组的质粒（10 和 10 cp/mL）的两个校准品，以生成主校准曲线。测定精密度、准确性、线性动态范围、定量下限（LLOQ）和检测下限（LOD）。分析了一组初治或慢性感染的患者。

结果

从 42 个标准品（std）的重复检测中获得了校准曲线；计算了精密度（变异系数 [CV] 低于 10%）；准确性高于 90%。线性范围涵盖了整个测试范围（10-10 个拷贝/反应），LOD（95%）为 12 个拷贝/反应。与慢性治疗组（50）相比，初治组（62）的 HIV-1 DNA 显著更高（p<0.0001），只有初治患者的病毒载量与淋巴细胞（p=0.0002）和 CD4（p<0.0001）计数相关。两组患者的 CD4 最低点与前病毒载量之间均存在显著负相关。病毒血症与 HIV-1 储存库之间存在显著相关性（p<0.0001）。同一患者的全血和外周血单个核细胞（PBMCs）中的前病毒载量之间的比较没有得到显著差异。