Univ Lille, Faculté de Médecine, CHU Lille, Laboratoire de Virologie EA3610, F-59000 Lille, France.
Service Universitaire des Maladies Infectieuses, CH Dron, Tourcoing, France.
J Antimicrob Chemother. 2017 Oct 1;72(10):2823-2830. doi: 10.1093/jac/dkx232.
Sanger sequencing of plasma RNA is the standard method for HIV-1 drug resistance testing in treatment-naive patients, but is limited by the non-detection of resistance-associated mutations (RAMs) with prevalence below approximately 20%.
We compared RNA and DNA Sanger sequencing (RSS and DSS) with RNA next-generation sequencing (NGS) for RAM detection in HIV-1 reverse transcriptase (RT), protease (PR) and integrase (IN) genes.
Sanger sequencing was performed on RNA and DNA, following the recommendations of the French Agency for AIDS Research (ANRS). NGS was performed on RNA using the HIV-1 Drug Resistance Assay, v. 3.0 (Roche) on the 454 GS Junior sequencer. The IAS-USA list was used to identify RAMs. ANRS, Rega and Stanford algorithms were used for drug resistance interpretation.
The study included 48 ART-naive patients. The number of patients with at least one major RAM was 3, 3, 4 and 8 when using RSS, DSS, NGS 20% and NGS 5%, respectively. Numerous minor mutations were detected in patients, especially in the protease gene. None of the methods detected any major mutation in the integrase gene. Overall, the mutation detection rate was similar between RSS and DSS, and higher with NGS 20%. Differences in drug resistance interpretation were found between algorithms. No impact of the minority RAMs detected by NGS was found on the short-term treatment outcome.
DSS does not clearly improve the detection of RAMs in ART-naive patients, as compared with RSS. NGS allows detection of additional minority RAMs; however, their clinical relevance requires further investigation.
桑格测序血浆 RNA 是用于检测未经治疗的患者 HIV-1 耐药性的标准方法，但由于检测不到耐药相关突变（RAMs），其检测率低于约 20%。
我们比较了 RNA 和 DNA 桑格测序（RSS 和 DSS）与 RNA 下一代测序（NGS）在 HIV-1 逆转录酶（RT）、蛋白酶（PR）和整合酶（IN）基因中检测 RAM 的性能。
按照法国艾滋病研究机构（ANRS）的建议，对 RNA 和 DNA 进行 Sanger 测序。使用 HIV-1 耐药性测定，v. 3.0（罗氏）在 454 GS 初级测序仪上进行 RNA NGS。IAS-USA 列表用于识别 RAMs。使用 ANRS、Rega 和斯坦福算法进行耐药性解释。
本研究纳入了 48 名未接受 ART 的患者。使用 RSS、DSS、NGS 20%和 NGS 5%时，至少有一种主要 RAM 的患者人数分别为 3、3、4 和 8。患者中检测到大量的次要突变，尤其是在蛋白酶基因中。整合酶基因中未检测到任何主要突变。总体而言，RSS 和 DSS 之间的突变检测率相似，而 NGS 20%时更高。不同算法之间存在耐药性解释的差异。未发现 NGS 检测到的少数 RAMs 对短期治疗结果有影响。
与 RSS 相比，DSS 并未明显提高对未经治疗的患者 RAMs 的检测率。NGS 可以检测到额外的少数 RAMs，但需要进一步研究其临床相关性。
