Full-spectrum HIV drug resistance mutation detection by high-resolution complete pol gene sequencing

Background: Drug resistance mutation testing is a key element for HIV clinical management, informing effective treatment regimens. However, resistance screening in current clinical practice is limited in reporting linked cross-class resistance mutations and minority variants, both of which may increase the risk of virological failure.

Methods: To address these limitations, we obtained 358 full-length pol gene sequences from 52 specimens of 20 HIV infected individuals by combining microdroplet amplification, unique molecular identifier (UMI) labeling, and long-read high-throughput sequencing.

Results: We conducted a rigorous assessment of the accuracy of our pipeline for precision drug resistance mutation detection, verifying that a sequencing depth of 35 high-throughput reads achieved complete, error-free pol gene sequencing. We detected 26 distinct drug resistance mutations to Protease Inhibitors (PIs), Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), and Integrase Strand Transfer Inhibitors (INSTIs). We detected linked cross-class drug resistance mutations (PI+NRTI, PI+NNRTI, and NRTI+NNRTI) that confer cross-resistance to multiple drugs in different classes. Fourteen different types of minority mutations were also detected with frequencies ranging from 3.2% to 19%, and the presence of these mutations was verified by Sanger reference sequencing. We detected a putative transmitted drug resistance mutation (TDRM) in one individual that persisted for over seven months from the first sample collected at the acute stage of infection prior to seroconversion.

Conclusions: Our comprehensive drug resistance mutation profiling can advance clinical practice by reporting mutation linkage and minority variants to better guide antiretroviral therapy options.