Nishizawa Masako, Matsuda Masakazu, Hattori Junko, Shiino Teiichiro, Matano Tetsuro, Heneine Walid, Johnson Jeffrey A, Sugiura Wataru
AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan.
PLoS One. 2015 Sep 11;10(9):e0135941. doi: 10.1371/journal.pone.0135941. eCollection 2015.
Drug-resistant HIV are more prevalent and persist longer than previously demonstrated by bulk sequencing due to the ability to detect low-frequency variants. To clarify a clinical benefit to monitoring minority-level drug resistance populations as a guide to select active drugs for salvage therapy, we retrospectively analyzed the dynamics of low-frequency drug-resistant population in antiretroviral (ARV)-exposed drug resistant individuals.
Six HIV-infected individuals treated with ARV for more than five years were analyzed. These individuals had difficulty in controlling viremia, and treatment regimens were switched multiple times guided by standard drug resistance testing using bulk sequencing. To detect minority variant populations with drug resistance, we used a highly sensitive allele-specific PCR (AS-PCR) with detection thresholds of 0.3-2%. According to ARV used in these individuals, we focused on the following seven reverse transcriptase inhibitor-resistant mutations: M41L, K65R, K70R, K103N, Y181C, M184V, and T215F/Y. Results of AS-PCR were compared with bulk sequencing data for concordance and presence of additional mutations. To clarify the genetic relationship between low-frequency and high-frequency populations, AS-PCR amplicon sequences were compared with bulk sequences in phylogenetic analysis.
The use of AS-PCR enabled detection of the drug-resistant mutations, M41L, K103N, Y181C, M184V and T215Y, present as low-frequency populations in five of the six individuals. These drug resistant variants persisted for several years without ARV pressure. Phylogenetic analysis indicated that pre-existing K103N and T215I variants had close genetic relationships with high-frequency K103N and T215I observed during treatment.
Our results demonstrate the long-term persistence of drug-resistant viruses in the absence of drug pressure. The rapid virologic failures with pre-existing mutant viruses detectable by AS-PCR highlight the clinical importance of low-frequency drug-resistant viruses. Thus, our results highlight the usefulness of AS-PCR and support its expanded evaluation in ART clinical management.
由于能够检测低频变异，耐药性HIV比之前通过群体测序所显示的更为普遍且持续时间更长。为了阐明监测少数水平耐药群体作为选择挽救治疗活性药物指南的临床益处，我们回顾性分析了接受抗逆转录病毒（ARV）治疗的耐药个体中低频耐药群体的动态变化。
分析了6名接受ARV治疗超过5年的HIV感染者。这些个体在控制病毒血症方面存在困难，并且治疗方案在使用群体测序的标准耐药性检测指导下多次更换。为了检测具有耐药性的少数变异群体，我们使用了检测阈值为0.3 - 2%的高灵敏度等位基因特异性PCR（AS-PCR）。根据这些个体所使用的ARV，我们重点关注以下七种对逆转录酶抑制剂耐药的突变：M41L、K65R、K70R、K103N、Y181C、M184V和T215F/Y。将AS-PCR的结果与群体测序数据进行比较，以确定一致性和是否存在其他突变。为了阐明低频和高频群体之间的遗传关系，在系统发育分析中将AS-PCR扩增子序列与群体序列进行比较。
使用AS-PCR能够检测到6名个体中有5名存在作为低频群体的耐药突变M41L、K103N、Y181C、M184V和T215Y。这些耐药变异在没有ARV压力的情况下持续了数年。系统发育分析表明，预先存在的K103N和T215I变异与治疗期间观察到的高频K103N和T215I具有密切的遗传关系。
我们的结果证明了耐药病毒在没有药物压力的情况下长期持续存在。通过AS-PCR可检测到的预先存在的突变病毒导致的快速病毒学失败突出了低频耐药病毒的临床重要性。因此，我们的结果突出了AS-PCR的有用性，并支持在抗逆转录病毒治疗临床管理中对其进行扩大评估。
