Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis
Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis
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DOI：
10.1128/jcm.43.1.406-413.2005
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发表时间：
2005-01-01
影响因子：
9.4
通讯作者：
Coffin, JM
中科院分区：
文献类型：
--
作者：
Palmer, S;Kearney, M;Coffin, JM
为了研究标准基因分型方法遗漏耐药突变的程度，我们使用新开发的单基因组测序技术分析了26例疑似多重耐药人类免疫缺陷病毒1型患者的相同血浆样本，并将其与标准基因型分析进行比较。血浆样本来自既往暴露于至少两种抗逆转录病毒药物类别且抗逆转录病毒方案失败的患者。通过逆转录酶（RT）-PCR和批量PCR产物测序获得标准基因型。对于单基因组测序，将来源于血浆RNA的cDNA连续稀释至每个反应1个拷贝，并扩增和测序包含p6、蛋白酶和部分RT的区域。从每个血浆样品获得15至46个单个病毒基因组的序列。单基因组测序鉴定的耐药突变在26例研究患者中的24例中未被标准基因型分析检测到。在标准基因型中几乎从未检测到存在于少于10%的单基因组中的突变（86个中的1个）。类似地，在标准基因型中，仅25%的时间检测到存在于10%至35%的单基因组中的突变。例如，在一名患者中，通过单基因组测序鉴定的10个突变，并赋予对蛋白酶抑制剂（PI），核苷类似物逆转录酶抑制剂和非核苷逆转录酶抑制剂（NNRTI）的耐药性，未被标准基因分型方法检测到。这些突变中的每一种都存在于所分析的20个基因组中的5%至20%中;该样品中15%的基因组含有连锁的PI突变，其中没有一个存在于标准基因型中。在另一个患者样本中，33%的基因组含有5个连锁的NNRTI耐药突变，其中没有一个被标准基因型分析检测到。这些发现说明了标准基因型检测低频耐药突变的不足。除了具有更高的灵敏度外，单基因组测序还可以识别赋予高水平耐药性的连锁突变。这种连锁不能通过标准的基因型分析来检测。
To investigate the extent to which drug resistance mutations are missed by standard genotyping methods, we analyzed the same plasma samples from 26 patients with suspected multidrug-resistant human immunodeficiency virus type 1 by using a newly developed single-genome sequencing technique and compared it to standard genotype analysis. Plasma samples were obtained from patients with prior exposure to at least two antiretroviral drug classes and who were on a failing antiretroviral regimen. Standard genotypes were obtained by reverse transcriptase (RT)-PCR and sequencing of the bulk PCR product. For single-genome sequencing, cDNA derived from plasma RNA was serially diluted to 1 copy per reaction, and a region encompassing p6, protease, and a portion of RT was amplified and sequenced. Sequences from 15 to 46 single viral genomes were obtained from each plasma sample. Drug resistance mutations identified by single-genome sequencing were not detected by standard genotype analysis in 24 of the 26 patients studied. Mutations present in less than 10% of single genomes were almost never detected in standard genotypes (1 of 86). Similarly, mutations present in 10 to 35% of single genomes were detected only 25% of the time in standard genotypes. For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to protease inhibitors (PIs), nucleoside analog reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors (NNRTIs) were not detected by standard genotyping methods. Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations, none of which were present in the standard genotype. In another patient sample, 33% of genomes contained five linked NNRTI resistance mutations, none of which were detected by standard genotype analysis. These findings illustrate the inadequacy of the standard genotype for detecting low-frequency drug resistance mutations. In addition to having greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance. Such linkage cannot be detected by standard genotype analysis.