# Large-scale evaluation of HIV-1 DNA drug resistance testing as a robust tool for clinical decision-making: A nationwide study in China

## TL;DR

This study shows that HIV-1 DNA drug resistance testing is reliable and effective, even in patients with low virus levels, supporting its use in clinical decisions.

## Contribution

Demonstrates the clinical utility of HIV-1 DNA drug resistance testing through a large-scale study and introduces a dominant sequence threshold for reliable results.

## Key findings

- EP-HIV co-isolation significantly improves RNA amplification success in low-level viremia cases.
- HIV-1 DNA DRT shows high reproducibility and agreement with RNA DRT across various viral load states.
- A dominant sequence threshold of 24.6% for HIV-1 DNA Sanger sequencing is experimentally defined.

## Abstract

Human immunodeficiency virus type 1 (HIV-1) drug resistance remains a major challenge in HIV/AIDS management, particularly in individuals with low-level viremia (LLV) where RNA-based drug resistance testing (DRT) often fails. Although HIV-1 DNA DRT represents a promising alternative, its clinical utility has been constrained by insufficient evidence. This nationwide study in China enrolled 9,428 people living with HIV (PLWH), analyzing 10,903 samples spanning a wide viral load (VL) spectrum. To improve RNA detection, an optimized primer design combined with an extracellular particle (EP)-HIV co-isolation technique was developed. We then evaluated the reproducibility of drug resistance mutation (DRM) profiles between paired RNA and DNA DRTs using Sanger sequencing (SS), with single-molecule sequencing employed to establish a dominant sequence threshold. Our findings demonstrated that primer optimization and EP-co-isolation significantly enhanced RNA amplification success. DRMs were prevalent across all VL strata. The combined concordance and degeneracy rates (C/D rates) (where multiple DNA DRMs included all RNA-derived DRMs) between RNA and DNA DRTs ranged from 90.4% to 100% in different gene regions, with higher discordance rates observed in the nucleoside reverse transcriptase inhibitor (NRTI) and non-NRTI (NNRTI) regions. Based on Stanford penalty scores across 25 antiretroviral drugs, the degeneracy group showed a 98.3% ± 1.7% interpretation agreement. Even within the discordance group, mean agreement remained high (89.5% ± 5.0%), with only four NNRTIs exhibiting agreement below 85%. The dominant sequence proportion threshold for HIV-1 DNA was determined to be 24.6%. This study provides strong evidence supporting the integration of HIV-1 DNA DRT into clinical practice for reliable drug resistance surveillance and treatment monitoring.

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•10,903 blood samples tested to justify clinical significance of HIV DNA DRT.•EP-HIV co-isolation remarkably increases LLV DRT sensitivity.•HIV RNA/DNA DRTs are highly reproducible across diverse VL states.•Dominant threshold of 24.6 % for HIV-1 DNA SS is defined experimentally.•DNA DRT can be employed as a cost-effective alternative to RNA DRT.

10,903 blood samples tested to justify clinical significance of HIV DNA DRT.

EP-HIV co-isolation remarkably increases LLV DRT sensitivity.

HIV RNA/DNA DRTs are highly reproducible across diverse VL states.

Dominant threshold of 24.6 % for HIV-1 DNA SS is defined experimentally.

DNA DRT can be employed as a cost-effective alternative to RNA DRT.