Department of Pediatrics, Division of Pneumonology-Immunology, Charité University Medical Center, Berlin, Germany.
BMC Infect Dis. 2013 Jan 2;13:1. doi: 10.1186/1471-2334-13-1.
The impact of extended use of ART in developing countries has been enormous. A thorough understanding of all factors contributing to the success of antiretroviral therapy is required. The current study aims to investigate the value of cross-sectional drug resistance monitoring using DNA and RNA oligonucleotide ligation assays (OLA) in treatment cohorts in low-resource settings. The study was conducted in the first cohort of children gaining access to structured ART in Peru.
Between 2002-5, 46 eligible children started the standard regimen of AZT, 3TC and NFV Patients had a median age of 5.6 years (range: 0.7-14y), a median viral load of 1.7·105 RNA/ml (range: 2.1·10(3) - 1.2·10(6)), and a median CD4-count of 232 cells/μL (range: 1-1591). Of these, 20 patients were classified as CDC clinical category C and 31/46 as CDC immune category 3. At the time of cross-sectional analysis in 2005, adherence questionnaires were administered. DNA OLAs and RNA OLAs were performed from frozen PBMC and plasma, RNA genotyping from dried blood spots.
During the first year of ART, 44% of children experienced virologic failure, with an additional 9% failing by the end of the second year. Virologic failure was significantly associated with the number of resistance mutations detected by DNA-OLA (p < 0.001) during cross-sectional analysis, but also with low immunologic CDC-scores at baseline (p < 0.001). Children who had been exposed to unsupervised short-term antiretrovirals before starting structured ART showed significantly higher numbers of resistance mutations by DNA-OLA (p = 0.01). Detection of M184V (3TC resistance) by RNA-OLA and DNA-OLA demonstrated a sensitivity of 0.93 and 0.86 and specificity of 0.67 and 0.7, respectively, for the identification of virologic failure. The RT mutations N88D and L90M (NFV resistance) detected by DNA-OLA correlated with virologic failure, whereas mutations at RT position 215 (AZT resistance) were not associated with virologic failure.
Advanced immunosuppression at baseline and previous exposures to unsupervised brief cycles of ART significantly impaired treatment outcomes at a time when structured ART was finally introduced in his cohort. Brief maternal exposures to with AZT +/- NVP for the prevention of mother-to-child transmission did not affect treatment outcomes in this group of children. DNA-OLA from frozen PBMC provided a highly specific tool to detect archived drug resistance. RNA consensus genotyping from dried blood spots and RNA-OLA from plasma consistently detected drug resistance mutations, but merely in association with virologic failure.
在发展中国家，ART 的长期使用产生了巨大的影响。因此，我们需要深入了解所有有助于抗逆转录病毒治疗成功的因素。本研究旨在探讨在资源匮乏的环境下，使用 DNA 和 RNA 寡核苷酸连接分析（OLA）进行横断面耐药性监测在治疗队列中的价值。该研究是在秘鲁第一批接受结构化 ART 的儿童中进行的。
2002 年至 2005 年期间，46 名符合条件的儿童开始接受 AZT、3TC 和 NFV 的标准方案治疗。患者的中位年龄为 5.6 岁（范围：0.7-14 岁），中位病毒载量为 1.7·105 RNA/ml（范围：2.1·10(3) - 1.2·10(6)），中位 CD4 计数为 232 个/μL（范围：1-1591）。其中，20 名患者被归类为 CDC 临床类别 C，31/46 名患者被归类为 CDC 免疫类别 3。在 2005 年进行横断面分析时，我们进行了问卷调查，检测了冷冻 PBMC 和血浆中的 DNA OLA 和 RNA OLA，以及从干血斑中进行 RNA 基因分型。
在 ART 的第一年，44%的儿童出现病毒学失败，第二年又有 9%的儿童失败。病毒学失败与 DNA-OLA 检测到的耐药突变数量显著相关（p < 0.001），但也与基线时较低的免疫 CDC 评分显著相关（p < 0.001）。在开始结构化 ART 之前接受过未经监督的短期抗逆转录病毒治疗的儿童，通过 DNA-OLA 检测到的耐药突变数量显著增加（p = 0.01）。RNA-OLA 和 DNA-OLA 检测到的 M184V（3TC 耐药）的敏感性分别为 0.93 和 0.86，特异性分别为 0.67 和 0.7。RT 位置 88D 和 L90M（NFV 耐药）的突变通过 DNA-OLA 检测到与病毒学失败相关，而 RT 位置 215（AZT 耐药）的突变与病毒学失败无关。
在本队列最终引入结构化 ART 时，基线时的严重免疫抑制和以前未经监督的短周期 ART 暴露显著影响了治疗结果。在这群儿童中，母亲短暂接触 AZT +/- NVP 进行母婴传播预防并未影响治疗结果。从冷冻 PBMC 中提取的 DNA-OLA 提供了一种高度特异的工具来检测已储存的耐药性。从干血斑中进行 RNA 共识基因分型和从血浆中进行 RNA-OLA 可以一致地检测到耐药突变，但仅与病毒学失败相关。
