A Genotypic Test for HIV-1 Tropism Combining Sanger Sequencing with Ultradeep Sequencing Predicts Virologic Response in Treatment-Experienced Patients

A tropism test is required prior to initiation of CCR5 antagonist therapy in HIV-1 infected individuals, as these agents are not effective in patients harboring CXCR4 (X4) coreceptor-using viral variants. We developed a clinical laboratory-based genotypic tropism test for detection of CCR5-using (R5) or X4 variants that utilizes triplicate population sequencing (TPS) followed by ultradeep sequencing (UDS) for samples classified as R5. Tropism was inferred using the bioinformatic algorithms geno2pheno[coreceptor] and PSSMx4r5. Virologic response as a function of tropism readout was retrospectively assessed using blinded samples from treatment-experienced subjects who received maraviroc (N = 327) in the MOTIVATE and A4001029 clinical trials. MOTIVATE patients were classified as R5 and A4001029 patients were classified as non-R5 by the original Trofile test. Virologic response was compared between the R5 and non-R5 groups determined by TPS, UDS alone, the reflex strategy and the Trofile Enhanced Sensitivity (TF-ES) test. UDS had greater sensitivity than TPS to detect minority non-R5 variants. The median log10 viral load change at week 8 was −2.4 for R5 subjects, regardless of the method used for classification; for subjects with non-R5 virus, median changes were −1.2 for TF-ES or the Reflex Test and −1.0 for UDS. The differences between R5 and non-R5 groups were highly significant in all 3 cases (p针对HIV-1感染者，在启动CCR5拮抗剂疗法前需开展嗜性检测，因为此类药物对携带使用CXCR4（X4）共受体的病毒变异株的感染者无效。本研究开发了一款基于临床实验室的基因型嗜性检测方法，用于识别使用CCR5（R5）或X4共受体的病毒变异株：该方法先采用三重群体测序（triplicate population sequencing, TPS）进行初步分类，对归类为R5型的样本则进一步实施超深度测序（ultradeep sequencing, UDS）。嗜性推断采用生物信息学算法geno2pheno[coreceptor]与PSSMx4r5。本研究对病毒学应答随嗜性检测结果的变化关系进行了回顾性评估，分析样本来自两项临床试验MOTIVATE及A4001029中接受马拉韦罗（maraviroc，N=327）的经治受试者的盲法样本。原始Trofile检测将MOTIVATE受试者归类为R5型，将A4001029受试者归类为非R5型。研究分别对比了通过TPS、单独UDS、溯源策略及Trofile增强敏感性（TF-ES）检测划定的R5与非R5组的病毒学应答情况。结果显示，UDS检测罕见非R5变异株的敏感性优于TPS。无论采用何种分类方法，R5型受试者在第8周的log10病毒载量变化中位数为-2.4；对于携带非R5型病毒的受试者，TF-ES检测或溯源测试的病毒载量变化中位数为-1.2，UDS检测则为-1.0。在三种检测策略下，R5与非R5组间的差异均具有高度统计学显著性（p