Characterizing Recent HIV Infection by High-Fidelity Multiplexed Primer ID-Based Next-Generation Sequencing among Newly Diagnosed Individuals in Zhuhai, China: A Cross-Sectional Study.

Abstract

Background: Differentiating recent from chronic HIV infections is critical for interrupting transmission chains. However, conventional Next-Generation Sequencing (NGS) is confounded by technical errors that inflate genetic diversity, blurring this distinction and compromising downstream molecular epidemiological analyses.

Methods: We applied Multiplexed Primer ID-Based Next-Generation Sequencing (MPID-NGS) to plasma from 153 antiretroviral therapy (ART)‑naïve newly diagnosed with HIV individuals in Zhuhai, China (2022‑2024) to generate error‑corrected template consensus sequences, from which infection recency was classified using genetic diversity thresholds. We then benchmarked this approach against standard NGS and serological Limiting Antigen Avidity Enzyme Immunoassay (LAg-EIA), and further assessed selection pressures and reconstructed molecular transmission networks.

Results: MPID‑NGS removed >93% of Reverse Transcriptase (RT) and 69% of Variable Regions 1 to 3 (V1V3) low‑frequency artifacts. Primer ID (PID)‑corrected V1V3 diversity showed the strongest correlation with LAg‑EIA Normalized Optical Density (ODn) (ρ = 0.52), while other PID-corrected measures also showed significant but weaker associations, supporting the utility of error-corrected genetic diversity as a staging metric. We classified 15.7% as recent and 76.5% as chronic, with serological concordance of 87.5% and 86.4%, respectively. Despite overall purifying selection in RT, recent infections had significantly higher ω than chronic ones (p < 0.001), reflecting stage‑specific adaptive evolution. Men who have sex with men (MSM) showed higher RT ω than non‑MSM (p < 0.001) and independently predicted transmission clustering (aOR = 3.247, p = 0.003). An acute case exhibited transmitted/founder virus (T/F virus) hallmarks: ultra‑low diversity, star‑like phylogeny, reduced Potential N-Linked Glycosylation Sites (PNGS), and estimated infection time of only 19.5 days.

Conclusions: MPID-NGS integrates high-fidelity staging, evolutionary profiling, and network analysis, revealing that recent MSM infections are strongly associated with local molecular clusters, elevated clustering risk, and distinct adaptive evolution, thus offering a robust tool for HIV-1 surveillance and intervention.