Hauser Andrea, Kuecherer Claudia, Kunz Andrea, Dabrowski Piotr Wojtek, Radonić Aleksandar, Nitsche Andreas, Theuring Stefanie, Bannert Norbert, Sewangi Julius, Mbezi Paulina, Dugange Festo, Harms Gundel, Meixenberger Karolin
HIV and other Retroviruses, Robert Koch-Institute, Berlin, Germany.
Institute of Tropical Medicine and International Health, Charité - Universitätsmedizin Berlin, Berlin, Germany.
PLoS One. 2015 Oct 15;10(10):e0140809. doi: 10.1371/journal.pone.0140809. eCollection 2015.
Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT).
Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System.
Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2-3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage.
Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.
2006年世界卫生组织建议实施三联抗逆转录病毒药物预防措施后，对感染HIV的孕妇进行了HIV-1耐药性发展情况的筛查。该研究提供了一个机会，可比较基于扩增子的454超深度测序（UDS）和等位基因特异性实时PCR（ASPCR）用于检测HIV-1逆转录酶（RT）中耐药性微小变异的情况。
之前通过ASPCR对34名坦桑尼亚女性的血浆样本进行分析，检测由齐多夫定（AZT）、拉米夫定（3TC）和奈韦拉平（NVP）选择的病毒RT中的关键耐药突变（K70R、K103N、Y181C、M184V、T215Y/F）。在本研究中，使用454 GS FLX系统通过基于扩增子的UDS对相同样本的RT区域进行耐药突变研究。
通过UDS在69%（20/29）的女性中鉴定出耐药HIV变异体，通过ASPCR在45%（13/29）的女性中鉴定出耐药HIV变异体。UDS鉴定出的耐药突变绝对数量是ASPCR鉴定出数量的两倍（45个对24个）。通过UDS，在24个ASPCR检测到的耐药突变中有14个在相同位置被鉴定出来。UDS和ASPCR之间的总体一致性为61.0%（25/41）。UDS定量的变异体比例比ASPCR低约2至3倍。与ASPCR（检测限=650拷贝/ml）相比，UDS从病毒载量低于20,000拷贝/ml的样本中生成扩增子的失败频率更高，导致序列覆盖缺失或不足。
两种方法都能提供有关耐药性HIV-1微小变异体的有用信息。ASPCR比UDS具有更高的灵敏度，但仅限于单个耐药突变。相比之下，UDS受限于其对高病毒载量的要求以实现足够的序列覆盖，但序列信息揭示了所分析基因组区域内的完整耐药模式。正在对UDS的检测限进行改进，届时UDS将有助于监测HIV-1准种中的耐药性微小变异体。
