Lapointe H R, Dong W, Lee G Q, Bangsberg D R, Martin J N, Mocello A R, Boum Y, Karakas A, Kirkby D, Poon A F Y, Harrigan P R, Brumme C J
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada.
Harvard School of Public Health, Boston, Massachusetts, USA Massachusetts General Hospital, Boston, Massachusetts, USA Mbarara University of Science of Technology, Mbarara, Uganda.
Antimicrob Agents Chemother. 2015 Nov;59(11):6824-33. doi: 10.1128/AAC.01490-15. Epub 2015 Aug 17.
Limited access to HIV drug resistance testing in low- and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1-infected individuals in such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n = 759) and from a Ugandan study cohort (n = 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing methods. Sequences were evaluated for nucleotide concordance between methods, using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of <3.0 log10 HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex "wide" sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.
在低收入和中等收入国家，获得艾滋病毒耐药性检测的机会有限，这阻碍了个体患者层面的临床决策。必须建立一个有效的方案来解决这一问题，以尽量减少此类环境中艾滋病毒-1感染者的负面治疗结果。这是一项观察性研究，旨在确定新型基因组测序平台（如Illumina MiSeq仪器）同时为数百个样本提供准确的艾滋病毒耐药基因型的潜力。在常规耐药性检测期间从加拿大患者（n = 759）和乌干达研究队列（n = 349）中采集血浆样本。使用MiSeq测序和传统的桑格测序方法对跨越艾滋病毒逆转录酶密码子90至234的扩增子进行测序。使用覆盖度和混合参数进行质量控制，评估两种方法之间序列的核苷酸一致性。还分析了共识序列在耐药性突变鉴定方面的差异。桑格测序和MiSeq测序分别成功检测了881个样本（80%）和892个样本（81%），其中832个样本两种方法都有结果。大多数失败案例是病毒载量<3.0 log10艾滋病毒RNA拷贝/ml的样本。总体而言，两种方法之间观察到99.3%的核苷酸一致性。MiSeq测序在检测桑格测序鉴定的耐药性突变方面达到了97.4%的灵敏度和99.3%的特异性。研究结果表明，Illumina MiSeq平台可以通过高多重“宽”测序方法产生高质量数据。这种策略可用于多种艾滋病毒亚型，证明了在资源有限的环境中进行广泛个体检测和年度人群监测的潜力。
