Department of Experimental Medicine and Biochemical Sciences, University of Rome 'Tor Vergata', Rome, Italy.
J Antimicrob Chemother. 2011 Nov;66(11):2615-23. doi: 10.1093/jac/dkr354. Epub 2011 Sep 2.
This proof-of-concept study aimed to identify whether mutations considered not yet relevant for drug resistance (but located at key drug-resistance positions) can act as 'sentinels' of minority resistant variants in HIV-1 drug-naive patients.
We focused our attention on three reverse transcriptase (RT) mutations (T69S, L210M and K103R) easily detected by standard population sequencing [i.e. the genotypic resistance test (GRT)]. Ultra-deep pyrosequencing (UDPS) of HIV-1 RT was performed using GS-FLX Roche, on plasma RNA from 40 drug-naive patients infected with HIV-1 subtype B without primary resistance detected by GRT. Only RT drug resistance mutations detected at >0.1% in both forward and reverse directions were considered. Associations between GRT sentinel mutations and UDPS drug resistance were assessed using Fisher's exact test.
UDPS detected drug resistance mutations in 18/40 drug-naive patients. Patients carrying HIV-1 strains with T69S and L210M by GRT showed a trend to greater infection by minority drug-resistant variants than control patients infected by HIV-1 without these mutations (5/10 and 7/10 versus 3/10; P = not significant). No association was found for K103R by GRT. Notably, T69S and L210M (but not K103R or control viruses) were associated with GRT minority drug-resistant variants with a prevalence >1% (3/10 and 4/10 versus 0/20 in K103R and controls; P = 0.03 and P = 0.008, respectively). Moreover, the presence of L210M or T69S viruses by GRT significantly correlated with that of minority thymidine analogue mutations by UDPS (6/20 patients carrying HIV-1 strains with T69S/L210M versus 0/20 patients carrying HIV-1 having K103R or none of these mutations; P = 0.03).
This proof-of-concept study suggests the existence of genetic markers, detectable by routine testing, potentially acting as sentinel mutations of minority drug resistance. Their identification may help in the selection of patients at high risk of resistance in reservoirs without the necessity of using UDPS.
本概念验证研究旨在确定那些被认为与耐药性无关（但位于关键耐药位置）的突变是否可以作为 HIV-1 初治患者中少数耐药变异的“哨兵”。
我们将注意力集中在三种逆转录酶（RT）突变（T69S、L210M 和 K103R）上，这些突变可通过标准的群体测序（即基因型耐药检测（GRT））轻松检测到。对 40 名未接受过药物治疗的 HIV-1 感染患者的血浆 RNA 进行了 HIV-1 RT 的超深度焦磷酸测序（UDPS），这些患者的 HIV-1 亚型 B 未通过 GRT 检测到原发性耐药。只有在正反两个方向上均以>0.1%的比例检测到的 RT 药物耐药突变才被认为是耐药突变。使用 Fisher 确切检验评估 GRT 前哨突变与 UDPS 耐药之间的相关性。
在 40 名初治患者中，UDPS 检测到了耐药突变。通过 GRT 携带 T69S 和 L210M 的 HIV-1 株的患者比感染未携带这些突变的 HIV-1 的对照患者更倾向于感染少数耐药变异株（5/10 和 7/10 比 3/10；P 值为非显著）。GRT 检测不到 K103R 突变的相关性。值得注意的是，T69S 和 L210M（但不是 K103R 或对照病毒）与 GRT 少数耐药变异株（>1%的流行率）相关（3/10 和 4/10 比 K103R 和对照病毒中的 0/20；P 值分别为 0.03 和 0.008）。此外，GRT 中存在 L210M 或 T69S 病毒与 UDPS 中存在少数胸腺嘧啶类似物突变显著相关（20 名携带 T69S/L210M HIV-1 株的患者中 6/20 例与 20 名携带 K103R 或未携带这些突变的 HIV-1 的患者中 0/20 例；P 值为 0.03）。
本概念验证研究表明，存在可通过常规检测检测到的遗传标志物，这些标志物可能作为少数耐药性的前哨突变。它们的鉴定可能有助于在无需使用 UDPS 的情况下，从耐药性储库中选择耐药风险较高的患者。
