Application Value of In-House HIV-1 DNA Genotypic Resistance Testing for Individuals with Low-Level Viremia

Background: To provide evidence for HIV-1 DNA genotypic resistance testing (GRT) strategies in low-level viremia (LLV) populations by comparing the amplification efficiency and resistance mutation profiles between HIV-1 DNA GRT and RNA GRT. (2) Methods: Convenience sampling was used for participant enrolment. Plasma HIV-1 RNA and peripheral blood monocyte (PBMC) DNA were extracted separately. The protease–reverse transcriptase (PR-RT) region was amplified via an in-house protocol, followed by Sanger and Nanopore sequencing. The exact McNemar’s test and Fisher’s test were applied to compare amplification success rates. Univariate and multivariable logistic regression analyses implemented in R were conducted to identify factors associated with the detection of HIV-1 drug-resistant strains. BEAST-based molecular clock analysis was performed to calculate the divergence time between RNA-derived and DNA-derived sequences. (3) Results: A total of 209 samples were enrolled, and valid sequences were obtained from 166 samples. Within the viral load range of 50–200 copies/mL, HIV-1 DNA GRT yielded significantly higher amplification success rates than RNA GRT (p < 0.05). The overall resistance detection rate for DNA GRT (20.8%) was lower than that for RNA GRT (33.7%), with discrepancies mainly involving nucleoside reverse transcriptase inhibitor (NRTI)- and non-nucleoside reverse transcriptase inhibitor (NNRTI)-associated mutations. Individuals receiving antiretroviral therapy (ART) for 7–18 months exhibited higher odds of detecting drug-resistant strains using either DNA GRT or RNA GRT. The median evolutionary divergence time between matched RNA- and DNA-derived sequences was 3.13 years. By contrast, the median evolutionary divergence time between paired DNA generated by Sanger and Nanopore sequencing was 0.84 years. (4) Conclusions: DNA GRT serves as a valuable supplementary screening tool in public health scenarios of LLV with failed RNA GRT amplification. However, clinical interpretation should combine RNA GRT findings, longitudinal viral load monitoring and treatment history, rather than relying solely on DNA GRT to modify antiretroviral regimens.