Improved Detection of HIV-1 Drug Resistance With Droplet Digital Polymerase Chain Reaction: A Comparative Study and Its Implications

Xiaojie Yang; Chang Yan; Mengying Li; Xuelei Liang; Rui Sun; Wenhao Lv; Hanxi Zhang; Fengting Yu; Fujie Zhang

doi:10.1093/ofid/ofaf286

Improved Detection of HIV-1 Drug Resistance With Droplet Digital Polymerase Chain Reaction: A Comparative Study and Its Implications

Open Forum Infect Dis. 2025 May 12;12(6):ofaf286. doi: 10.1093/ofid/ofaf286. eCollection 2025 Jun.

Authors

Xiaojie Yang¹, Chang Yan¹, Mengying Li^{1

2

3}, Xuelei Liang¹, Rui Sun¹, Wenhao Lv¹, Hanxi Zhang¹, Fengting Yu^{1

2

3}, Fujie Zhang^{1

2}

Affiliations

¹ Beijing Ditan Hospital, Capital Medical University, Beijing, China.
² Clinical Center for HIV/AIDS, Capital Medical University, Beijing, China.
³ Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China.

Abstract

Background: 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.

Methods: 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.

Results: 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 pol 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.

Conclusions: 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.

Keywords: HIV; droplet digital PCR; drug resistance mutations; low frequency.