From Capillary Electrophoresis to Deep Sequencing: An Improved HIV-1 Drug Resistance Assessment Solution Using In Vitro Diagnostic (IVD) Assays and Software

Background: Drug-resistance mutations were mostly detected using capillary electrophoresis sequencing, which does not detect minor variants with a frequency below 20%. Next-Generation Sequencing (NGS) can now detect additional mutations which can be useful for HIV-1 drug resistance interpretation. The objective of this study was to evaluate the performances of CE-IVD assays for HIV-1 drug-resistance assessment both for target-specific and whole-genome sequencing, using standardized end-to-end solution platforms. Methods: A total of 301 clinical samples were prepared, extracted, and amplified for the three HIV-1 genomic targets, Protease (PR), Reverse Transcriptase (RT), and Integrase (INT), using the CE-IVD DeepChek® Assays; and then 19 clinical samples, using the CE-IVD DeepChek® HIV Whole Genome Assay, were sequenced on the NGS iSeq100 and MiSeq (Illumina, San Diego, CA, USA). Sequences were compared to those obtained by capillary electrophoresis. Quality control for Molecular Diagnostics (QCMD) samples was added to validate the clinical accuracy of these in vitro diagnostics (IVDs). Nineteen clinical samples were then tested with the same sample collection, handling, and measurement procedure for evaluating the use of NGS for whole-genome HIV-1. Sequencing analyzer outputs were submitted to a downstream CE-IVD standalone software tailored for HIV-1 analysis and interpretation. Results: The limits of range detection were 1000 to 106 cp/mL for the HIV-1 target-specific sequencing. The median coverage per sample for the three amplicons (PR/RT and INT) was 13,237 reads. High analytical reproducibility and repeatability were evidenced by a positive percent agreement of 100%. Duplicated samples in two distinct NGS runs were 100% homologous. NGS detected all the mutations found by capillary electrophoresis and identified additional resistance variants. A perfect accuracy score with the QCMD panel detection of drug-resistance mutations was obtained. Conclusions: This study is the first evaluation of the DeepChek® Assays for targets specific (PR/RT and INT) and whole genome. A cutoff of 3% allowed for a better characterization of the viral population by identifying additional resistance mutations and improving the HIV-1 drug-resistance interpretation. The use of whole-genome sequencing is an additional and complementary tool to detect mutations in newly infected untreated patients and heavily experienced patients, both with higher HIV-1 viral-load profiles, to offer new insight and treatment strategies, especially using the new HIV-1 capsid/maturation inhibitors and to assess the potential clinical impact of mutations in the HIV-1 genome outside of the usual HIV-1 targets (RT/PR and INT)

Clinical impact of ultra deep versus Sanger sequencing detection of minority mutations on HIV-1 drug resistance genotype interpretation after virological failure

International audienc

Improved Prediction of Salvage Antiretroviral Therapy Outcomes Using Ultrasensitive HIV-1 Drug Resistance Testing

Background. The clinical relevance of ultrasensitive human immunodeficiency virus type 1 (HIV-1) genotypic resistance testing in antiretroviral treatment (ART)-experienced individuals remains unknown. Methods. This was a retrospective, multicentre, cohort study in ART-experienced, HIV-1-infected adults who initiated salvage ART including, at least 1 ritonavir-boosted protease inhibitor, raltegravir or etravirine. Presalvage ART Sanger and 454 sequencing of plasma HIV-1 were used to generate separate genotypic sensitivity scores (GSS) using the HIVdb, ANRS, and REGA algorithms. Virological failure (VF) was defined as 2 consecutive HIV-1 RNA levels ≥200 copies/mL at least 12 weeks after salvage ART initiation, whereas subjects remained on the same ART. The ability of Sanger and 454-GSS to predict VF was assessed by receiver operating characteristic (ROC) curves and survival analyses. Results. The study included 132 evaluable subjects; 28 (21%) developed VF. Using HIVdb, 454 predicted VF better than Sanger sequencing in the ROC curve analysis (area under the curve: 0.69 vs 0.60, Delong test P = .029). Time toVF was shorter for subjects with 454-GSS < 3 vs 454-GSS ≥ 3 (Log-rank P = .003) but not significantly different between Sanger-GSS < 3 and ≥3. Factors independently associated with increased risk of VF in multivariate Cox regression were a 454-GSS < 3 (HR = 4.6, 95 CI, [1.5, 14.0], P = .007), and the number of previous antiretrovirals received (HR = 1.2 per additional drug, 95 CI, [1.1, 1.3], P = .001). Equivalent findings were obtained with the ANRS and REGA algorithms. Conclusions. Ultrasensitive HIV-1 genotyping improves GSS-based predictions of virological outcomes of salvage ART relative to Sanger sequencing. Thismay improve the clinical management of ART-experienced subjects living with HIV-1. Clinical Trials Registration. NCT01346878. Keywords. HIV-1; antiretroviral drug resistance; deep sequencing; salvage antiretroviral therapy; genotypic susceptibility score