The Kirby Institute, University of New South Wales, Sydney, NSW, Australia.
J Clin Microbiol. 2013 Jul;51(7):2063-71. doi: 10.1128/JCM.00510-13. Epub 2013 Apr 17.
The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR). DNA sequences with low FPRs are more likely to be from CXCR4-using (X4) viruses. Phase 1 of the EQA involved chromatogram interpretation. Phases 2, 2/3, and 3 involved patient and clonal samples. Clinical samples used in these phases were from treatment-experienced HIV-infected volunteers; 18/20 had viral loads of <50 copies/ml, and 10/15 were CXCR4-tropic on prior phenotyping. All samples were tested in triplicate, and any replicate with a geno2pheno FPR of <10% was designated X4. Performance was deemed adequate if ≤2 R5 and ≤1 X4 specimens were miscalled. For several clinical samples in the EQA, triplicate testing revealed marked DNA variability (FPR range, 0 to 96.7%). Therefore, a consensus-based approach was employed for each sample, i.e., a median FPR across laboratories was used to define sample tropism. Further sequencing analysis showed mixed viral populations in the clinical samples, explaining the differences in tropism predictions. All laboratories passed the EQA after achieving predefined competence thresholds in either of the phase 2 rounds. The use of clinical samples from patients resembling those who were likely to be screened in the MARCH, coupled with triplicate testing, revealed inherent DNA variability that might have been missed if single or duplicate testing and/or clonal samples alone were used. These data highlight the importance of intensive EQA of tropism laboratories before embarking on clinical studies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01384682 [http://www.clinicaltrials.gov/ct2/show/study/NCT01384682?term=NCT01384682&rank=1].).
马拉维若韦切换协作研究（MARCH）是一项针对稳定接受抗逆转录病毒治疗的无病毒血症患者的研究，利用基于人群的前病毒 DNA 序列分析来确定 HIV 嗜性和对马拉维若韦的敏感性。为确保 MARCH 实验室进行临床样本嗜性评估的能力，实施了一项外部质量评估（EQA）计划（n=14）。MARCH EQA 有三个预研究阶段，评估使用生物信息算法 geno2pheno 的 V3 环测序和嗜性确定，该算法生成假阳性率（FPR）。FPR 较低的 DNA 序列更有可能来自 CXCR4 利用（X4）病毒。EQA 的第 1 阶段涉及色谱解释。第 2、2/3 和 3 阶段涉及患者和克隆样本。这些阶段使用的临床样本来自治疗经验丰富的 HIV 感染志愿者；18/20 例患者的病毒载量<50 拷贝/ml，10/15 例患者在先前表型测定中为 CXCR4 嗜性。所有样本均进行了三次重复检测，任何 geno2pheno FPR<10%的重复检测均被指定为 X4。如果≤2 个 R5 和≤1 个 X4 标本被误判，则认为性能合格。在 EQA 的几个临床样本中，三次重复检测显示出明显的 DNA 变异性（FPR 范围为 0 至 96.7%）。因此，对每个样本采用了基于共识的方法，即使用实验室之间的中位数 FPR 来定义样本嗜性。进一步的测序分析显示，临床样本中存在混合的病毒群体，这解释了嗜性预测的差异。所有实验室在达到第 2 轮中任一轮的预定能力阈值后都通过了 EQA。使用与 MARCH 中可能进行筛选的患者相似的患者的临床样本，并进行三次重复检测，揭示了潜在的 DNA 变异性，如果仅使用单次或重复检测和/或克隆样本，可能会忽略这些变异性。这些数据强调了在进行临床研究之前，对嗜性实验室进行强化 EQA 的重要性。（本研究已在 ClinicalTrials.gov 注册，注册号为 NCT01384682 [http://www.clinicaltrials.gov/ct2/show/study/NCT01384682?term=NCT01384682&rank=1]）。
