scholarly journals A new sensitive and robust next-generation sequencing platform for HIV-1 drug resistance mutations testing

2021 ◽  
Author(s):  
Bin Yu ◽  
Changzhong Jin ◽  
Zixuan Ma ◽  
Ziwei Cai ◽  
Tingsen Li ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Sanger Sequencing ◽  
Resistance Mutations ◽  
Altman Plot ◽  
Blood Samples ◽  
Bland Altman Plot ◽  
Drug Resistance Mutations ◽  
Minority Variants ◽  
Generation Sequencing ◽  
Hiv 1

Next-generation sequencing (NGS) is a trending new platform which allows cheap, quantitative, high-throughput, parallel sequencing for minority variants with frequencies less than 20% of the HIV-1 quasi-species. In clinical setting, these advantages are crucial for choosing antiretroviral drugs with low genetic barriers and will potentially benefit treatment outcomes. In this investigation, we implemented the Boxin HIV-1 NGS platform for genotyping the drug-resistance-associated variants in PR/RT regions. Plasmids with known mutations were used to analyze the accuracy, reproducibility, and reliability of the Boxin NGS assay. Variant frequencies reported by Boxin NGS and the theoretical value were highly concordant. The Bland-Altman plot and the coefficient of variation (7%) suggested that the method has excellent reproducibility and reliability. Sanger sequencing confirmed the existence of these known variants with frequencies equal or above 20%. 78 blood samples were obtained from AIDS patients and underwent PR/RT region genotyping by Sanger sequencing and Boxin NGS. 33 additional drug resistance mutations were identified by Boxin NGS, 23/33 mutations were minority variants with frequencies below 20%. 15 blood samples obtained from AIDS patients underwent PR/RT region genotyping by Sanger sequencing, Boxin NGS, and Vela NGS. The Bland-Altman plot suggested that the variant frequencies detected by Boxin and Vela were highly concordant. Moreover, Boxin NGS assay detected five more minority variants with frequencies ranged from 1% to 20%. In a series of samples collected from 2016 to 2017, Boxin NGS reported a M184V mutation with a frequency of 4.92%, 3 months earlier than this mutation was firstly detected by Vela NGS and Sanger sequencing. In conclusion, Boxin NGS had good accuracy, reproducibility, and reliability. Boxin NGS was highly concordant with Sanger sequencing and Vela NGS. In terms of genotyping HIV-1 variants in PR/RT regions, Boxin NGS was more cost-efficient and appeared to have increased sensitivity without compromising sequence accuracy.

scholarly journals Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments

2018 ◽  
Vol 15 (8) ◽  
pp. 1697 ◽  
Author(s):  
Andrea Arias ◽  
Pablo López ◽  
Raphael Sánchez ◽  
Yasuhiro Yamamura ◽  
Vanessa Rivera-Amill
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
T Cell ◽  
Sanger Sequencing ◽  
Response To Therapy ◽  
Next Generation ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Generation Sequencing ◽  
Hiv 1

The implementation of antiretroviral treatment combined with the monitoring of drug resistance mutations improves the quality of life of HIV-1 positive patients. The drug resistance mutation patterns and viral genotypes are currently analyzed by DNA sequencing of the virus in the plasma of patients. However, the virus compartmentalizes, and different T cell subsets may harbor distinct viral subsets. In this study, we compared the patterns of HIV distribution in cell-free (blood plasma) and cell-associated viruses (peripheral blood mononuclear cells, PBMCs) derived from ART-treated patients by using Sanger sequencing- and Next-Generation sequencing-based HIV assay. CD4+CD45RA−RO+ memory T-cells were isolated from PBMCs using a BD FACSAria instrument. HIV pol (protease and reverse transcriptase) was RT-PCR or PCR amplified from the plasma and the T-cell subset, respectively. Sequences were obtained using Sanger sequencing and Next-Generation Sequencing (NGS). Sanger sequences were aligned and edited using RECall software (beta v3.03). The Stanford HIV database was used to evaluate drug resistance mutations. Illumina MiSeq platform and HyDRA Web were used to generate and analyze NGS data, respectively. Our results show a high correlation between Sanger sequencing and NGS results. However, some major and minor drug resistance mutations were only observed by NGS, albeit at different frequencies. Analysis of low-frequency drugs resistance mutations and virus distribution in the blood compartments may provide information to allow a more sustainable response to therapy and better disease management.

scholarly journals Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure

2016 ◽  
Vol 60 (6) ◽  
pp. 3380-3397 ◽  
Author(s):  
Fred Kyeyune ◽  
Richard M. Gibson ◽  
Immaculate Nankya ◽  
Colin Venner ◽  
Samar Metha ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Treatment Failure ◽  
Sanger Sequencing ◽  
Antiretroviral Treatment ◽  
Low Frequency ◽  
Drug Resistant ◽  
Viral Quasispecies ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Hiv 1

Most patients failing antiretroviral treatment in Uganda continue to fail their treatment regimen even if a dominant drug-resistant HIV-1 genotype is not detected. In a recent retrospective study, we observed that approximately 30% of HIV-infected individuals in the Joint Clinical Research Centre (Kampala, Uganda) experienced virologic failure with a susceptible HIV-1 genotype based on standard Sanger sequencing. Selection of minority drug-resistant HIV-1 variants (not detectable by Sanger sequencing) under antiretroviral therapy pressure can lead to a shift in the viral quasispecies distribution, becoming dominant members of the virus population and eventually causing treatment failure. Here, we used a novel HIV-1 genotyping assay based on deep sequencing (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drug-resistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more sensitive methods to detect minority HIV-1 variants.

scholarly journals Correction: First evaluation of the Next-Generation Sequencing platform for the detection of HIV-1 drug resistance mutations in Belgium

PLoS ONE ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. e0211587
Author(s):  
Géraldine Dessilly ◽  
Léonie Goeminne ◽  
Anne-thérèse Vandenbroucke ◽  
François E. Dufrasne ◽  
Anandi Martin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Resistance Mutations ◽  
Sequencing Platform ◽  
Drug Resistance Mutations ◽  
Next Generation Sequencing Platform ◽  
Generation Sequencing ◽  
Hiv 1

scholarly journals A16 Next-generation sequencing to detect transmitted drug resistance mutations in Romanian people who inject drugs

2019 ◽  
Vol 5 (Supplement_1) ◽  
Author(s):  
Marius Surleac ◽  
Simona Paraschiv ◽  
Ionelia Nicolae ◽  
Leontina Banica ◽  
Ovidiu Vlaicu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
People Who Inject Drugs ◽  
Transmitted Drug Resistance ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Generation Sequencing ◽  
Tropism Prediction ◽  
Higher Sensitivity

Abstract Romania has faced an HIV outbreak among people who inject drugs (PWID) since 2011. The introduction of so-called ‘legal highs’ (amphetamine-type stimulants) on the drug market a few years prior contributed substantially to this outbreak. Next-generation sequencing (NGS) provides the possibility to detect drug resistance mutations with higher sensitivity than Sanger sequencing. The aim of this study was to search for transmitted drug resistance (TDR) mutations in strains from PWID recently diagnosed with HIV infection by parallel use of Sanger sequencing and NGS. The study was conducted on strains from 34 PWID diagnosed with HIV infection between 2016 and 2017. Sequencing was performed for the pol (PR, RT, and INT) and env (V2-V3 loop) regions. Sanger sequencing was performed with the commercial ViroseqTMHIV-1 Genotyping system (Abbott Laboratories) and with an in-house protocol for the env gene. NGS was performed in the same genomic regions using Nextera DNA Library Preparation Kit (Illumina) and the Miseq instrument (Illumina). NGS data were processed for error correction, read mapping, and detection of drug resistance mutations with HIV-1 Deepchek analysis software. Geno2pheno algorithm was used for viral tropism prediction and the WHO 2009 list for TDRM analysis. By using NGS, we detected seven cases (20.6%) of TDR in PWID and only two cases (5.8%) with standard sequencing. The TDR mutations detected by NGS were K103N, K101EN, Y181C, T215S in RT gene, I54V and M46L in PR, and none in INT. Two NNRTI mutations (K103N and K101EN) were detected in the same sample. Most of the TDR identified were present in the minority population (between 1% and 2% of the total reads) explaining the higher sensitivity of NGS method compared with standard sequencing. No significant differences were observed between these two methods when tropism prediction was analyzed. The majority of the viruses circulating in this group were R5-tropic. All strains showed more resistance mutations when analyzed by deep sequencing than by Sanger sequencing and more than previously observed in other risk groups. NGS proved to be a sensitive tool to detect TDR in newly infected PWID.

scholarly journals First evaluation of the Next-Generation Sequencing platform for the detection of HIV-1 drug resistance mutations in Belgium

PLoS ONE ◽  
2018 ◽  
Vol 13 (12) ◽  
pp. e0209561 ◽  
Author(s):  
Géraldine Dessilly ◽  
Léonie Goeminne ◽  
Anne-thérèse Vandenbroucke ◽  
Francois E. Dufrasne ◽  
Anandi Martin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Resistance Mutations ◽  
Sequencing Platform ◽  
Drug Resistance Mutations ◽  
Next Generation Sequencing Platform ◽  
Generation Sequencing ◽  
Hiv 1

scholarly journals HIV-DRLink: A tool for detecting linked HIV-1 drug resistance mutations in next generation sequencing data

2019 ◽  
Author(s):  
Wei Shao ◽  
Valerie F. Boltz ◽  
Junko Hattori ◽  
Michael J. Bale ◽  
Frank Maldarelli ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Genome Sequencing ◽  
Research Tool ◽  
Next Generation ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Single Genome ◽  
Generation Sequencing ◽  
Hiv 1

AbstractThe prevalence of HIV-1 drug resistance is increasing worldwide and monitoring its emergence is important for the successful management of populations receiving combination antiretroviral therapy (cART). Using Ultrasensitive Single-Genome Sequencing (uSGS), a next-generation method that avoids PCR bias and PCR recombination, a recent report showed that pre-existing dual-class drug resistance mutations linked on the same viral genomes were predictive of treatment failure while unlinked mutations were not. Because of the large numbers of sequences generated by uSGS and other next-generation sequencing methods, it is difficult to assess each sequence individually for linked resistance mutations. Several software/programs exist to report the frequencies of individual mutations in large datasets but they provide no information on their linkage. Here, we report the HIV-DRLink program, a research tool that provides mutation frequencies in the total dataset as well as their linkage to other mutations conferring resistance to the same or different drug classes. The HIV-DRLink program should only be used on datasets generated by methods that eliminate artifacts due to PCR recombination, for example, standard Single-Genome Sequencing (SGS) or Ultrasensitive Single-Genome Sequencing (uSGS). HIV-DRLink is exclusively a research tool and is not intended to inform clinical decisions.

Sign in / Sign up

Export Citation Format

Share Document