{
  "abstract": "Introduction The long-acting injectable dual therapy cabotegravir + rilpivirine improves quality of life and adherence in virologically suppressed person living with HIV (PLWH). However, risks for virological failure include HIV subtype and resistance mutations in HIV RNA or DNA. When subtype and genotypic history are incomplete, HIV DNA sequencing is recommended and increasingly performed in antiretroviral therapy (ART) experienced individuals. While plasma HIV RNA viremia predicts sequencing success, the relationship between HIV DNA levels and next generation sequencing (NGS) amplification performance remains unclear.Objective To evaluate a homebrew NGS genotypic resistance test (GRT) for detecting drug resistance mutations (DRMs) in protease (PR), reverse transcriptase (RT), and integrase (INT) regions of proviral HIV-1 DNA.Materials and Methods We analyzed 142 PBMC samples from 142 PLWH using an in-house NGS assay. PR, RT, and INT regions were amplified by nested PCR and sequenced using DNA Library Prep on the iSeq100 instrument (Illumina). DRMs and susceptibility were interpreted with Stanford HIVdb v9.8 at 10% and 20% detection thresholds. Total HIV-1 DNA was quantified in 55 samples using the HIV-1 DNA Test PRO (Diatheva) processed with the Elite InGenius platform (Elitech). Historical plasma GRT was available for 54 cases.Results HIV1 subtype was assigned in 122/142 samples: 62.3% were subtype B and 37.7% non B. Sequencing success rates were 84% for PR, 84% for RT, and 75% for INT. HIV-1 DNA levels strongly correlated with sequencing outcome: mean 365 copies/10 6 cells in successful samples vs 37.3 copies/106 in failed ones (p = 0.0002). ROC analysis identified a success threshold of 55 copies/106 PBMC. Major DRMs were detected in 29/122 (23,8%) individuals: 26 at frequencies above 20% and 3 at frequencies below 20%. Comparison of GRT DNA with historical RNA genotypes showed 70.4% of concordance (38/54). Among 16 discordant cases, 6 were linked to APOBEC related changes, 6 to higher DRM detection in HIV DNA, and 4 to higher DRM detection in historical RNA.Conclusions The NGS-based GRT-DNA assay provided reliable resistance profiles, with sequencing success of 84% (PR/RT) and 75% (INT). Sequencing performance correlated with total HIV-1 DNA, with a threshold of 55 copies/10 6 PBMC. The assay effectively detected both major and low frequency DRMs. Discordances with historical plasma genotypes were largely due to APOBEC editing or low frequency DRMs identified only with GRT-DNA assay. GRT-DNA is a valuable tool for resistance assessment in PLWH, lacking complete genotypic histories.",
  "authors": [
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "T Allice"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "F Cerutti"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "G Gregori"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "MG Milia"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "E Burdino"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "B Simoncelli"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "S Monteleone"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "M Cazzadore"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "A Bottoni"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "E Scuccimarra"
    },
    {
      "affiliations": [
        "Laboratory of Microbiology and Virology, ASL Città di Torino, Torino, Italy"
      ],
      "name": "V Ghisetti"
    }
  ],
  "title": "OC47 Evaluation of a NGS Assay for detecting HIV1 proviral drug resistance mutations: association with total HIV-1 DNA levels and with historical genotype resistance test",
  "uid": "b0359f69-f93e-56a0-b236-209ce0067735"
}