{
  "abstract": "Background In the era of fully suppressive antiretroviral therapy, HIV-1 RNA is frequently undetectable and viral persistence resides in integrated proviral DNA. When historical resistance data are unavailable, sequencing of proviral HIV-1 DNA may recover archived resistance-associated mutations (RAMs) and support clinical decision-making. Whole-genome sequencing (WGS) extends this approach beyond pol, enabling broader RAMs assessment and genome integrity. We evaluated the feasibility and analytical performance of proviral WGS (WGS-DNA) and its association with quantitative HIV-DNA levels and markers of proviral defectiveness.Materials and Methods Proviral HIV-1 DNA was extracted from 200uL of frozen whole blood and quantified using the HIV-1 DNA assay (Diatheva) on automated platform (Elitech Ingenious). DNAs were amplified using the AD4Seq HIV Whole Genome (Arrow Diagnostics) and sequenced on the Illumina iSeq100 platform. FASTQs were processed through an in-house pipeline for coverage analysis. Resistance-associated mutations (RAMs) in the pol region were interpreted using the Stanford HIV Drug Resistance Database, as well as proviral defectiveness, assessed by evaluating APOBEC-associated hypermutation patterns, premature stop codons and unusual mutations.Results HIV-DNA levels ranged from 34 to 830 copies/10^6 cells, reflecting substantial inter-individual variability. All samples were subtype B. WGS-DNA amplification was successful in 91.3% of samples (21/23). Among these, median genome coverage at 100× was 69.5% (IQR 51.4–78.9). High genome recovery (>80%) was achieved in 17.4% of samples at 100× and 48% at 30× depth. Coverage was heterogeneous across genomic regions ( figure 1), while amplicon-level analysis revealed dropout patterns (figure 2). No correlation was observed between HIV-DNA levels and genome coverage (Spearman ρ=0.09, p=0.71). Markers of proviral defectiveness were also detected: APOBEC-related mutations in 28.6% of samples, stop codons in 23.8%, and unusual mutations in 19.0%. At least one pol RAM was identified in 61.9% of samples (detailed in table 1). Stratification by HIV-DNA level suggested a higher relative abundance of defectiveness markers and pol RAMs in samples with <100 cp/10^6 cells compared with the other groups.Conclusions HIV-1 WGS-DNA is technically feasible and demonstrates reliable analytical performance in the pol region, supporting resistance interpretation in suppressed individuals. Genome recovery across the full provirus is heterogeneous and not predicted by quantitative HIV-DNA levels alone, indicating that template integrity likely plays a major role. These findings support the use of proviral WGS for resistance assessment, while emphasizing the need to account for genome completeness and defectiveness when interpreting results. Ongoing analyses integrating additional clinical and virological parameters aim to refine predictors of sequencing success and define the clinical role of proviral WGS.Abstract P3 Figure 1Genome-wide and gene-level median coverage with at least 100 reads across the HIV-1 proviral genome. The pol and gag regions demonstrated near-complete recovery, whereas structural and accessory genes such as env, rev and tat showed lower median coverage, reflecting heterogeneous genome recovery in proviral whole-genome sequencingAbstract P3 Figure 2Amplicon-level coverage across the HIV-1 proviral genome. Each horizontal track represents a sample aligned to the reference genome. Successful amplification rates were: Amplicon 1 (16/21), Amplicon 2 (14/21), Amplicon 3 (15/21), and Amplicon 4 (14/21). Genome incompleteness corresponds to selective amplicon dropout, as expected in an amplicon-based amplification strategy. Complete absence of coverage across an amplicon likely reflects amplification failure due to primer-binding site disruption, large internal deletions, or low template availability; while amplicons displaying reduced but detectable coverage suggest heterogeneous proviral populations containing both amplifiable and non-amplifiable templatesAbstract P3 Table 1Stratification of proviral WGS-DNA performance by HIV-DNA level group. HIV-DNA groups were defined according to proviral DNA quantification, measured as copies per 10^6 cells. Estimated proviral copies were calculated as (HIV-DNA/10^6 cells x number of quantified cells), representing the theoretical number of proviral templates available for amplification. Abbreviations: pol cov: coverage of pol gene with at least 100 reads; APOBEC: Apolipoprotein B mRNA Editing Catalytic Polypeptide-like; PI: Protease Inhibitors; NRTE Nucleoside/Nucleotide Transcriptase Inhibitors; NNRTI: Non-Nucleoside Reverse Transcriptase Inhibitors; INSTI: Integrase Strand Transfer Inhibitors. “n*” refers to number of samples in which that variable was detected, within the HIV-DNA group.",
  "authors": [
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "F Stefanelli"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "N Randazzo"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "N Nigro"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "M Lucente"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "G Garzillo"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "M Ferraris"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "V Chessa"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "R Qosja"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "L Taramasso"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "V Ricucci"
    },
    {
      "affiliations": [
        "IRCCS Azienda Ospedaliera Metropolitana San Martino Hospital, Genoa, Italy"
      ],
      "name": "B Bruzzone"
    }
  ],
  "title": "P3 Evaluation of whole genome sequencing of HIV-1 proviral DNA (WGS-DNA) for drug resistance testing",
  "uid": "0bab7a48-750a-5d09-9e49-5c00d5414af1"
}