Division of Medical Virology, Stellenbosch University, Stellenbosch, South Africa.
J Virol. 2012 Jun;86(11):6231-7. doi: 10.1128/JVI.06541-11. Epub 2012 Mar 28.
Standard genotypic antiretroviral resistance testing, performed by bulk sequencing, does not readily detect variants that comprise <20% of the circulating HIV-1 RNA population. Nevertheless, it is valuable in selecting an antiretroviral regimen after antiretroviral failure. In patients with poor adherence, resistant variants may not reach this threshold. Therefore, deep sequencing would be potentially valuable for detecting minority resistant variants. We compared bulk sequencing and deep sequencing to detect HIV-1 drug resistance at the time of a second-line protease inhibitor (PI)-based antiretroviral regimen failure. Eligibility criteria were virologic failure (HIV-1 RNA load of >500 copies/ml) of a first-line nonnucleoside reverse transcriptase inhibitor-based regimen, with at least the M184V mutation (lamivudine resistance), and second-line failure of a lopinavir/ritonavir (LPV/r)-based regimen. An amplicon-sequencing approach on the Roche 454 system was used. Six patients with viral loads of >90,000 copies/ml and one patient with a viral load of 520 copies/ml were included. Mutations not detectable by bulk sequencing during first- and second-line failure were detected by deep sequencing during second-line failure. Low-frequency variants (>0.5% of the sequence population) harboring major protease inhibitor resistance mutations were found in 5 of 7 patients despite poor adherence to the LPV/r-based regimen. In patients with intermittent adherence to a boosted PI regimen, deep sequencing may detect minority PI-resistant variants, which likely represent early events in resistance selection. In patients with poor or intermittent adherence, there may be low evolutionary impetus for such variants to reach fixation, explaining the low prevalence of PI resistance.
标准的基因耐药性检测（通过批量测序进行）无法轻易检测到构成 HIV-1 RNA 群体<20%的变异体。然而，在抗逆转录病毒治疗失败后，它在选择抗逆转录病毒治疗方案方面具有重要价值。在依从性差的患者中，耐药变异体可能无法达到这个阈值。因此，深度测序可能有助于检测少数耐药变异体。我们比较了批量测序和深度测序，以检测二线蛋白酶抑制剂（PI）为基础的抗逆转录病毒治疗方案失败时的 HIV-1 耐药性。入选标准为：一线非核苷类逆转录酶抑制剂为基础的方案出现病毒学失败（HIV-1 RNA 载量>500 拷贝/ml），至少存在 M184V 突变（拉米夫定耐药），二线以洛匹那韦/利托那韦（LPV/r）为基础的方案失败。使用罗氏 454 系统的扩增子测序方法。共纳入 6 例病毒载量>90000 拷贝/ml的患者和 1 例病毒载量为 520 拷贝/ml的患者。在二线治疗失败时，通过深度测序检测到了在一线和二线治疗失败时通过批量测序无法检测到的突变。尽管 LPV/r 为基础的方案依从性差，但在 7 例患者中的 5 例中发现了低频率（>0.5%的序列群体）的主要蛋白酶抑制剂耐药突变。在间歇性服用强化 PI 方案的患者中，深度测序可能会检测到少数 PI 耐药变异体，这可能代表耐药选择的早期事件。在依从性差或间歇性服用的患者中，这些变异体可能没有足够的进化动力达到固定状态，这解释了 PI 耐药的低流行率。
