HIV-1 genotypic resistance testing using single molecule real-time sequencing

J Clin Virol. 2024 Oct:174:105717. doi: 10.1016/j.jcv.2024.105717. Epub 2024 Jul 24.

Abstract

Background: HIV-1 resistance testing is recommended in clinical management and next-generation sequencing (NGS) methods are now available in many virology laboratories.

Objectives: To evaluate the diagnostic performance of Long-Read Single Molecule Real-time (SMRT) sequencing (Sequel, PacBio) for HIV-1 polymerase genotyping.

Study design: 111 prospective clinical samples (83 plasma and 28 leukocyte-enriched blood fraction) were analyzed for routine HIV-1 resistance genotyping using Sanger sequencing, Vela NGS, and SMRT sequencing. We developed a SMRT sequencing protocol and a bio-informatics pipeline to infer antiretroviral resistance on both haplotype and variant calling approaches.

Results: The polymerase was successfully sequenced by the three platforms in 98 % of plasma RNA samples for viral loads above 4 log copies/mL. The success rate decreased to 83 % using Sanger or Vela sequencing and to 67 % using SMRT sequencing for viral loads of 3 to 4 log copies/mL. Sensitivities of 50 %, 54 % and 61 % were obtained using SMRT, Vela, and Sanger sequencing, respectively, in cellular DNA from patients with prolonged undetectable plasma HIV-1 RNA. Ninety-eight percent of resistance-associated mutations (RAMs) identified with Sanger sequencing were detected using SMRT sequencing. Furthermore, 91 % of RAMs (> 5 % threshold) identified with Vela NGS were detected using SMRT sequencing. RAM quantification using Vela and SMRT sequencing was well correlated (Spearman correlation ρ = 0.82; P < 0.0001).

Conclusions: SMRT sequencing of the full-length HIV-1 polymerase appeared performant for characterizing HIV-1 genotypic resistance on both RNA and DNA clinical samples. Long-read sequencing is a new tool for mutation haplotyping and resistance analysis.

Keywords: HIV-1 polymerase; Haplotypes; Long reads; Next-generation sequencing.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-HIV Agents / pharmacology
  • Anti-HIV Agents / therapeutic use
  • Drug Resistance, Viral* / genetics
  • Genotype*
  • Genotyping Techniques / methods
  • HIV Infections* / virology
  • HIV-1* / drug effects
  • HIV-1* / genetics
  • High-Throughput Nucleotide Sequencing* / methods
  • Humans
  • Microbial Sensitivity Tests / methods
  • Mutation
  • Prospective Studies
  • RNA, Viral / genetics
  • Sensitivity and Specificity
  • Sequence Analysis, DNA
  • Viral Load*

Substances

  • RNA, Viral
  • Anti-HIV Agents