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首页> 外文期刊>Journal of Clinical Microbiology >Multiple, Linked Human Immunodeficiency Virus Type 1 Drug Resistance Mutations in Treatment-Experienced Patients Are Missed by Standard Genotype Analysis
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Multiple, Linked Human Immunodeficiency Virus Type 1 Drug Resistance Mutations in Treatment-Experienced Patients Are Missed by Standard Genotype Analysis

机译：标准基因型分析错过了治疗经验丰富的患者中的多种链接的人类免疫缺陷病毒1型耐药突变

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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.
机译：为了研究标准基因分型方法遗漏的耐药性突变的程度，我们使用新开发的单基因组测序技术分析了26名疑似多药耐药的人类免疫缺陷病毒1型患者的26份血浆样品，并将其与标准品进行了比较。基因型分析。血浆样本是从先前至少接触过两种抗逆转录病毒药物且抗逆转录病毒治疗方案无效的患者获得的。通过逆转录酶（RT）-PCR和对大量PCR产物进行测序，获得标准基因型。对于单基因组测序，每个反应将血浆RNA衍生的cDNA连续稀释至1个拷贝，并扩增包含p6，蛋白酶和一部分RT的区域并进行测序。从每个血浆样品中获得15至46个单一病毒基因组的序列。在标准的基因型分析中，在26位研究的患者中有24位未通过单基因组测序鉴定出耐药性突变。在不到10％的单一基因组中存在的突变几乎从未在标准基因型中检测到（86个中的1个）。同样，在标准基因型中，只有25％的时间检测到10％至35％的单个基因组中存在的突变。例如，在一名患者中，通过标准基因分型方法未检测到10个通过单基因组测序鉴定并赋予对蛋白酶抑制剂（PIs），核苷类似物逆转录酶抑制剂和非核苷逆转录酶抑制剂（NNRTIs）的抗性的突变。这些突变中的每一个都存在于所分析的20个基因组的5至20％中。该样本中15％的基因组包含连锁的PI突变，标准基因型中均不存在。在另一例患者样本中，33％的基因组包含五个连锁的NNRTI抗性突变，通过标准基因型分析均未检测到。这些发现说明标准基因型不足以检测低频耐药性突变。除了具有更高的敏感性外，单基因组测序还可以鉴定连锁突变，从而赋予高水平的耐药性。通过标准基因型分析不能检测到这种连锁。

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