Next generation sequencing based in-house HIV genotyping method: validation report

Ayitewala, Alisen; Ssewanyana, Isaac; Kiyaga, Charles

doi:10.1186/s12981-021-00390-8

Cited by 4 publications

(1 citation statement)

References 22 publications

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“…In conclusion, our study complements previous studies [ 23 , 24 , 33 , 46 , 47 , 48 , 49 ] that support the notion that the population sequencing and high-throughput sequencing are well correlated. Moreover, using HTS-GRT, we can detect minority DRMs that have previously been reported to be associated with poor virologic suppression.…”

Section: Discussionsupporting

confidence: 89%

Towards Next-Generation Sequencing for HIV-1 Drug Resistance Testing in a Clinical Setting

Teo

Norhisham

Lee³

et al. 2022

Viruses

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The HIV genotypic resistance test (GRT) is a standard of care for the clinical management of HIV/AIDS patients. In recent decades, population or Sanger sequencing has been the foundation for drug resistance monitoring in clinical settings. However, the advent of high-throughput or next-generation sequencing has caused a paradigm shift towards the detection and characterization of low-abundance covert mutations that would otherwise be missed by population sequencing. This is clinically significant, as these mutations can potentially compromise the efficacy of antiretroviral therapy, causing poor virologic suppression. Therefore, it is important to develop a more sensitive method so as to reliably detect clinically actionable drug-resistant mutations (DRMs). Here, we evaluated the diagnostic performance of a laboratory-developed, high-throughput, sequencing-based GRT using 103 archived clinical samples that were previously tested for drug resistance using population sequencing. As expected, high-throughput sequencing found all the DRMs that were detectable by population sequencing. Significantly, 78 additional DRMs were identified only by high-throughput sequencing, which is statistically significant based on McNemar’s test. Overall, our results complement previous studies, supporting the notion that the two methods are well correlated, and the high-throughput sequencing method appears to be an excellent alternative for drug resistance testing in a clinical setting.

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Section: Discussionsupporting

confidence: 89%

Towards Next-Generation Sequencing for HIV-1 Drug Resistance Testing in a Clinical Setting

Teo

Norhisham

Lee³

et al. 2022

Viruses

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The relevance of ultradeep sequencing for low HIV‐1 viral loads and proviruses in the clinical setting

Foury,

Saunier,

Taverniers

et al. 2024

Journal of Medical Virology

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Improving the therapeutic management of HIV‐positive persons is a major public health issue and includes better detection of drug resistance mutations (DRMs). The aim of this study was (i) to explore DRMs in HIV‐1‐positive persons presenting a blood viral load (VL) < 1000 genomes copies (gc)/mL, including the analyze of cerebrospinal fluid (CSF) and HIV‐DNA from peripheral blood mononuclear cells using ultradeep sequencing (UDS) and (ii), to evaluate how these DRMs could influence the clinical practices. For each patient (n = 12), including five low‐VL patients (i.e., <1000 gc/mL), HIV‐1 UDS targeting the protease, reverse transcriptase and integrase genes was performed on plasma, proviral DNA, and CSF when available. Sequencing discordances or failures were mostly found in samples from low‐VL patients. A 5% UDS cut‐off allowed to increase the sensitivity to detect DRMs in different compartments, excepted in CSF. Patients with the highest viral quasispecies heterogeneity were naïve of treatment or presented a medical history suggesting low selection pressure or virological failures. When analyzing compartmentalization and following‐up patients: low‐frequency variants (LFVs) were responsible for 47% (n = 8) and 76% (n = 13) of changes in drug resistance interpretation, respectively. In such cases, we conclude that UDS is a robust technique, which still could be improved by increase the RNA and/or DNA extraction in low‐VL samples to detect LFVs. Further studies are needed to define the impact of LFVs on antiretroviral treatments. At last, when considering a DRM, the use of mutational load would probably be more suitable than frequencies.

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Next Generation Sequencing Reveals a High Prevalence of HIV-1 Minority Variants and an Expanded Drug Resistance Profile Among Individuals Initiated to Antiretroviral Therapy in a Resource-constrained Setting

Nannyonjo

Omooja

Bugembe

et al. 2023

Preprint

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Introduction Because next-generation sequencing (NGS) can detect minority mutations that have been linked to treatment failure but are missed by population Sanger sequencing, it has the potential to enhance HIV treatment monitoring. Comparing NGS based on the Illumina platform to Sanger sequencing, we assessed the clinical importance of NGS in HIV-1 medication resistance testing. Methods In this retrospective case-control study, 167 people with matched Sanger sequencing data underwent HIV genotypic analysis using an Illumina-Miseq. These included 122 time-matched controls from the same cohort who had viral suppression at 12 months, and 45 patients with virologic failure at 12 months. Results NGS identified all major HIV drug resistance mutations detected by Sanger sequencing and revealed additional major mutations M184V and K65R that increased the resistance profile to antiretroviral therapy used in this cohort. Abacavir's HIV drug resistance score increased 60-fold, Zidovudine's by 25-fold and to Emtricitabine/Lamivudine by 90-fold. Overall, 108/167 (64.7%) of our subjects had minority DRMs at baseline. K70E, M184V, Y115F, and K70R were among the NRTI minority SDRMs discovered, whereas K103N, Y181C, and K101E were NNRTI minority SDRMs. PI minority SDRMs were also detected in 4 individuals. Being female (p = 0.005) and having a CD4 < 250 cells/mL (p = 0.029) were associated with minority mutations. Minority surveillance DRMs expanded the HIV drug resistance profiles of individuals. A higher frequency of baseline minority mutations correlated with a higher viral load count at end point (p < 0.005). Conclusions NGS identified pre-treatment minority variants linked to increased viral load count and enhanced resistance to NNRTIs and NRTIs, and it detected more major mutations than Sanger sequencing. Being female and having low CD4 count were associated with presence of minority mutations. NGS could be used to create drug resistance profiles for people receiving HIV-1 ART, allowing clinicians to use both major and minor mutation profiles to inform treatment choices and so increase the effectiveness of the currently available antiretroviral medication. This may be crucial if the UNAIDS 95-95-95 targets are to be met and if we are to eliminate HIV/AIDS as a public health issue by 2030.

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Next generation sequencing based in-house HIV genotyping method: validation report

Cited by 4 publications

References 22 publications

Towards Next-Generation Sequencing for HIV-1 Drug Resistance Testing in a Clinical Setting

Towards Next-Generation Sequencing for HIV-1 Drug Resistance Testing in a Clinical Setting

The relevance of ultradeep sequencing for low HIV‐1 viral loads and proviruses in the clinical setting

Next Generation Sequencing Reveals a High Prevalence of HIV-1 Minority Variants and an Expanded Drug Resistance Profile Among Individuals Initiated to Antiretroviral Therapy in a Resource-constrained Setting

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