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

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.

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

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph15081697 ◽

2018 ◽

Vol 15 (8) ◽

pp. 1697 ◽

Cited By ~ 8

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.

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FDA authorizes marketing of first next-generation sequencing test for detecting HIV-1 drug resistance mutations

Case Medical Research ◽

10.31525/cmr-2106c4c ◽

2019 ◽

Author(s):

Keyword(s):

Drug Resistance ◽

Next Generation Sequencing ◽

Next Generation ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Generation Sequencing ◽

Hiv 1

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HIV Drug Resistance Mutations Detection by Next-Generation Sequencing during Antiretroviral Therapy Interruption in China

Pathogens ◽

10.3390/pathogens10030264 ◽

2021 ◽

Vol 10 (3) ◽

pp. 264

Author(s):

Miaomiao Li ◽

Shujia Liang ◽

Chao Zhou ◽

Min Chen ◽

Shu Liang ◽

...

Keyword(s):

Drug Resistance ◽

Next Generation Sequencing ◽

Antiretroviral Therapy ◽

Detection Threshold ◽

Next Generation ◽

Resistance Mutations ◽

Hiv Drug Resistance ◽

Drug Resistance Mutations ◽

Therapy Interruption ◽

Generation Sequencing

Patients with antiretroviral therapy interruption have a high risk of virological failure when re-initiating antiretroviral therapy (ART), especially those with HIV drug resistance. Next-generation sequencing may provide close scrutiny on their minority drug resistance variant. A cross-sectional study was conducted in patients with ART interruption in five regions in China in 2016. Through Sanger and next-generation sequencing in parallel, HIV drug resistance was genotyped on their plasma samples. Rates of HIV drug resistance were compared by the McNemar tests. In total, 174 patients were included in this study, with a median 12 (interquartile range (IQR), 6–24) months of ART interruption. Most (86.2%) of them had received efavirenz (EFV)/nevirapine (NVP)-based first-line therapy for a median 16 (IQR, 7–26) months before ART interruption. Sixty-one (35.1%) patients had CRF07_BC HIV-1 strains, 58 (33.3%) CRF08_BC and 35 (20.1%) CRF01_AE. Thirty-four (19.5%) of the 174 patients were detected to harbor HIV drug-resistant variants on Sanger sequencing. Thirty-six (20.7%), 37 (21.3%), 42 (24.1%), 79 (45.4%) and 139 (79.9) patients were identified to have HIV drug resistance by next-generation sequencing at 20% (v.s. Sanger, p = 0.317), 10% (v.s. Sanger, p = 0.180), 5% (v.s. Sanger, p = 0.011), 2% (v.s. Sanger, p < 0.001) and 1% (v.s. Sanger, p < 0.001) of detection thresholds, respectively. K65R was the most common minority mutation, of 95.1% (58/61) and 93.1% (54/58) in CRF07_BC and CRF08_BC, respectively, when compared with 5.7% (2/35) in CRF01_AE (p < 0.001). In 49 patients that followed-up a median 10 months later, HIV drug resistance mutations at >20% frequency such as K103N, M184VI and P225H still existed, but with decreased frequencies. The prevalence of HIV drug resistance in ART interruption was higher than 15% in the survey. Next-generation sequencing was able to detect more minority drug resistance variants than Sanger. There was a sharp increase in minority drug resistance variants when the detection threshold was below 5%.

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P2 Clinical evaluation of the next-generation sequencing (NGS) for detection of drug-resistance mutations in HIV-1

Journal of Virus Eradication ◽

10.1016/s2055-6640(20)30827-x ◽

2017 ◽

Vol 3 ◽

pp. 4

Author(s):

G. Dessilly ◽

L. Goeminne ◽

A. Duquenne ◽

A.T. Vandenbroucke ◽

E. André ◽

...

Keyword(s):

Drug Resistance ◽

Next Generation Sequencing ◽

Clinical Evaluation ◽

Next Generation ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Hiv 1

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

PLoS ONE ◽

10.1371/journal.pone.0211587 ◽

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

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Coupling chemical mutagenesis to next generation sequencing for the identification of drug resistance mutations in Leishmania

Nature Communications ◽

10.1038/s41467-019-13344-6 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 11

Author(s):

Arijit Bhattacharya ◽

Philippe Leprohon ◽

Sophia Bigot ◽

Prasad Kottayil Padmanabhan ◽

Angana Mukherjee ◽

...

Keyword(s):

Drug Resistance ◽

Next Generation Sequencing ◽

Chemical Mutagenesis ◽

Next Generation ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Paromomycin Resistance ◽

Allelic Replacement ◽

Genome Wide ◽

Generation Sequencing

AbstractCurrent genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging. Here, we use chemical mutagenesis, drug selection and next generation sequencing to characterize miltefosine and paromomycin resistant clones of the parasite Leishmania. We highlight several genes involved in drug resistance by sequencing the genomes of 41 resistant clones and by concentrating on recurrent SNVs. We associate genes linked to lipid metabolism or to ribosome/translation functions with miltefosine or paromomycin resistance, respectively. We prove by allelic replacement and CRISPR-Cas9 gene-editing that the essential protein kinase CDPK1 is crucial for paromomycin resistance. We have linked CDPK1 in translation by functional interactome analysis, and provide evidence that CDPK1 contributes to antimonial resistance in the parasite. This screen is powerful in exploring networks of drug resistance in an organism with diploid to mosaic aneuploid genome, hence widening the scope of its applicability.

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