ArticleInfection and drug resistance2025
Development and Evaluation of an Optimised Sanger-Based Assay for HIV-1 Drug Resistance Genotyping in Chinese Circulating Strains Across Subtypes and Viral Loads.
Article in Infection and drug resistance, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Purpose: HIV drug resistance is increasing globally, especially in resource-limited settings. NNRTIs, commonly used as first-line ART, have a low genetic barrier and are prone to resistance mutations. Lifelong ART contributes to the accumulation of drug resistance mutations (DRMs), compromising treatment efficacy. Genotypic resistance testing is essential before initiating or modifying ART. However, the Sanger sequencing method relies on successful PCR amplification, which is often suboptimal in low viral load (VL) samples, limiting sensitivity and coverage. Patients and Methods: We developed an optimized primer system targeting conserved regions in the protease (PR), reverse transcriptase (RT), and integrase (IN) genes, covering PR aa 1-99, RT aa 1-410 (including NNRTI resistance sites Y318 and N348), and IN aa 1-288. A total of 2,071 HIV-positive plasma samples collected in China (Jan 2023-Dec 2024) were analyzed using a PCR-Sanger sequencing method. Subtyping was performed using BLAST, COMET 2.4, and the HIV-1 Gene Sequences Database (China). Amplification success rates and mutation detection were evaluated across VL levels. Results: The overall amplification success rates were 87.40% (1,810/2,071) for the PR/RT region and 87.06% (1,803/2,071) for the IN region. In samples with VLs of 50-200 copies/mL, the success rates remained above 80% for PR/RT and 78.10% for IN. For samples with VL ≥1000 copies/mL, both regions achieved amplification rates above 99%. Among eight samples harboring Y318 or N348 mutations, all were successfully amplified at 1000, 400, 200, and 100 copies/mL. Three of them consistently yielded detectable mutations across all gradients. Subtyping revealed CRF01_AE and CRF07_BC as the predominant strains, consistent with national epidemiological trends. Conclusion: The optimized system improves amplification sensitivity and mutation coverage, especially in low-VL samples. It enables stable detection of key NNRTI resistance mutations and shows strong subtype compatibility, supporting its utility in clinical resistance surveillance and early detection.
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