Yang Xiaojie, Yan Chang, Li Mengying, Liang Xuelei, Sun Rui, Lv Wenhao, Zhang Hanxi, Yu Fengting, Zhang Fujie
Beijing Ditan Hospital, Capital Medical University, Beijing, China.
Clinical Center for HIV/AIDS, Capital Medical University, Beijing, China.
Open Forum Infect Dis. 2025 May 12;12(6):ofaf286. doi: 10.1093/ofid/ofaf286. eCollection 2025 Jun.
HIV drug resistance is a crucial factor in treatment failure. Sanger sequencing, the current gold standard for resistance testing, cannot detect low-abundance variants constituting <20% of the virus population. Although next-generation sequencing (NGS) offers higher sensitivity, it is susceptible to biases during the amplification stage, further reducing the proportion of low-abundance mutant sequences. This study aimed to evaluate the application of droplet digital polymerase chain reaction (ddPCR) combined with Sanger sequencing and NGS in resistance monitoring, seeking a more efficient method for drug-resistant mutation detection.
Samples from 106 participants with treatment failure were subjected to amplification by ddPCR and bulk polymerase chain reaction (PCR). Amplification was successful with both methods in 103 participants, and Sanger sequencing was subsequently performed on their samples. NGS was performed on 16 of these participants. The success rates of genotyping were compared across viral loads. Additionally, we analyzed the consistency of Sanger sequencing results, explored differences in NGS results, and examined the relationship between low-frequency drug-resistant mutations and patient prognosis.
The amplification success rate of ddPCR is 97.2% (103/106) and increases with higher viral loads. The phylogenetic analysis results indicate that the 2 amplification methods exhibit good consistency, with 99% (102/103) of the gene sequences from the same sample clustering. The overall prevalence of HIV DRMs was 50.5% (52/103), and both methods yielded the same resistance results. NGS of 16 participants identified low-frequency resistance mutations in 15 individuals. The bulk PCR detected 31 mutations, while ddPCR detected 39 mutations.
The combination of ddPCR and NGS detects more low-frequency variants and offers greater accuracy than conventional bulk PCR. Our ddPCR technology provides a more efficient method for HIV drug resistance testing.
HIV耐药性是治疗失败的关键因素。桑格测序作为目前耐药性检测的金标准，无法检测到占病毒群体不到20%的低丰度变异体。尽管下一代测序（NGS）具有更高的灵敏度，但它在扩增阶段容易出现偏差，进一步降低了低丰度突变序列的比例。本研究旨在评估液滴数字聚合酶链反应（ddPCR）结合桑格测序和NGS在耐药性监测中的应用，寻找一种更有效的耐药突变检测方法。
对106例治疗失败的参与者的样本进行ddPCR和常规聚合酶链反应（PCR）扩增。103例参与者的样本通过两种方法均成功扩增，随后对其样本进行桑格测序。对其中16例参与者进行NGS。比较不同病毒载量下基因分型的成功率。此外，我们分析了桑格测序结果的一致性，探讨了NGS结果的差异，并研究了低频耐药突变与患者预后的关系。
ddPCR的扩增成功率为97.2%（103/106），且随着病毒载量的升高而增加。系统发育分析结果表明，两种扩增方法具有良好的一致性，来自同一样本的基因序列有99%（102/103）聚类。HIV耐药性相关突变（DRMs）的总体流行率为50.5%（52/103），两种方法产生的耐药结果相同。对16例参与者进行NGS，在15例个体中检测到低频耐药突变。常规PCR检测到31个突变，而ddPCR检测到39个突变。
ddPCR与NGS相结合可检测到更多低频变异体，比传统的常规PCR具有更高的准确性。我们的ddPCR技术为HIV耐药性检测提供了一种更有效的方法。
