Fonager Jannik, Larsson Jonas T, Hussing Christian, Neess Engsig Frederik, Nielsen Claus, Fischer Thea Kølsen
Department of Microbiological Diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark.
Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark.
J Clin Virol. 2015 Dec;73:95-100. doi: 10.1016/j.jcv.2015.11.009. Epub 2015 Nov 10.
The current widely applied standard method to screen for HIV-1 genotypic resistance is based on Sanger population sequencing (Sseq), which does not allow for the identification of minority variants (MVs) below the limit of detection for the Sseq-method in patients receiving integrase strand-transfer inhibitors (INSTI). Next generation sequencing (NGS) has facilitated the detection of MVs at a much deeper level than Sseq.
Here, we compared Illumina MiSeq and Sseq approaches to evaluate the detection of MVs involved in resistance to the three commonly used INSTI: raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG).
NGS and Sseq were used to analyze RT-PCR products of the HIV-1 integrase coding region from six patients and in serial samples from two patients. NGS sequences were assembled and analyzed using the low frequency variant detection (LFVDT) tool in CLC genomic workbench.
Sseq detected INSTI resistance and accessory mutations in three of the patients (called INSTI Res+), while no resistance or accessory mutations were detected in the remaining three patients (called INSTI Res-). Additional INSTI resistance and/or accessory mutations were detected by NGS analysis of integrase sequences from all three INSTI Res+ and one INSTI Res- patient.
Our observations suggested that NGS demonstrated a higher sensitivity than sSEQ in the identification of INSTI relevant MVs both in patients at treatment baseline and in patients receiving INSTI therapy. Thus NGS can be a valuable tool in monitoring of antiretroviral minority resistance in patients receiving INSTI therapy.
目前广泛应用的筛查HIV-1基因型耐药性的标准方法基于桑格群体测序（Sseq），该方法无法识别接受整合酶链转移抑制剂（INSTI）治疗患者中低于Sseq方法检测下限的少数变异（MVs）。下一代测序（NGS）有助于在比Sseq更深的水平上检测MVs。
在此，我们比较了Illumina MiSeq和Sseq方法，以评估对三种常用INSTI（拉替拉韦（RAL）、埃替拉韦（EVG）和多替拉韦（DTG））耐药相关MVs的检测情况。
使用NGS和Sseq分析6名患者HIV-1整合酶编码区的RT-PCR产物以及2名患者的系列样本。使用CLC基因组工作台中的低频变异检测（LFVDT）工具对NGS序列进行组装和分析。
Sseq在3名患者中检测到INSTI耐药性和辅助突变（称为INSTI Res+），而在其余3名患者中未检测到耐药性或辅助突变（称为INSTI Res-）。通过对所有3名INSTI Res+患者和1名INSTI Res-患者的整合酶序列进行NGS分析，检测到了额外的INSTI耐药性和/或辅助突变。
我们的观察结果表明，在治疗基线患者和接受INSTI治疗的患者中，NGS在识别与INSTI相关的MVs方面表现出比Sseq更高的灵敏度。因此，NGS可成为监测接受INSTI治疗患者抗逆转录病毒少数耐药性的有价值工具。
