scholarly journals PANDAA-monium: Intentional violations of conventional qPCR design enables rapid, HIV-1 subtype-independent drug resistance SNP detection

2019 ◽  
Author(s):  
Iain J. MacLeod ◽  
Christopher F. Rowley ◽  
M. Essex
Keyword(s):  
Drug Resistance ◽  
Large Scale ◽  
Clinical Decision Making ◽  
Site Directed Mutagenesis ◽  
Resistance Mutations ◽  
Degenerate Primers ◽  
Genotypic Resistance ◽  
Infected People ◽  
The Impact ◽  
Hiv 1

ABSTRACTGlobal efforts to ensure that 90% of all HIV-infected people receiving antiretroviral therapy (ART) will be virally suppressed by 2020 could be crippled by increases in acquired and transmitted HIV drug resistance (HIVDR), which challenge ART efficacy. The long-term sustainability of ART treatment programs is contingent on effective HIVDR monitoring yet current Sanger sequencing genotypic resistance tests are inadequate for large-scale implementation in low- and middle-income countries (LMICs). A simple, rapid, affordable HIVDR diagnostic would radically improve the treatment paradigm in LMICs by facilitating informed clinical decision-making upon ART failure. Although point mutation assays can be broadly deployed in this context, the primary challenge arises from extensive sequence variation surrounding targeted drug resistance mutations (DRMs). Here, we systematically and intentionally violate the canonical principles of qPCR design to develop a novel assay, Pan-Degenerate Amplification and Adaptation (PANDAA), that mitigates the impact of DRM-proximal secondary polymorphisms on probe-based qPCR performance to enable subtype-independent, focused resistance genotyping. Using extremely degenerate primers with 3’ termini overlapping the probe-binding site, the HIV-1 genome is adapted through site-directed mutagenesis to replace secondary polymorphisms flanking the target DRM during the initial qPCR cycles. We show that PANDAA can quantify key HIV DRMs present at ≥5% and has diagnostic sensitivity and specificity of 96.9% and 97.5%, respectively, to detect DRMs associated with ART failure. PANDAA is an innovative solution for HIVDR genotyping and is an advancement in qPCR technology that could be applicable to any scenario where target-proximal genetic variability has been a roadblock in diagnostic development.

scholarly journals MiDRMpol: A High-Throughput Multiplexed Amplicon Sequencing Workflow to Quantify HIV-1 Drug Resistance Mutations against Protease, Reverse Transcriptase, and Integrase Inhibitors

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 806
Author(s):  
Shambhu G. Aralaguppe ◽  
Anoop T. Ambikan ◽  
Manickam Ashokkumar ◽  
Milner M. Kumar ◽  
Luke Elizabeth Hanna ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Throughput ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Polymorphism Analysis ◽  
Resistance Mutations ◽  
Subtype C ◽  
Bioinformatics Pipeline ◽  
Drug Resistance Mutations ◽  
Hiv 1

The detection of drug resistance mutations (DRMs) in minor viral populations is of potential clinical importance. However, sophisticated computational infrastructure and competence for analysis of high-throughput sequencing (HTS) data lack at most diagnostic laboratories. Thus, we have proposed a new pipeline, MiDRMpol, to quantify DRM from the HIV-1 pol region. The gag-vpu region of 87 plasma samples from HIV-infected individuals from three cohorts was amplified and sequenced by Illumina HiSeq2500. The sequence reads were adapter-trimmed, followed by analysis using in-house scripts. Samples from Swedish and Ethiopian cohorts were also sequenced by Sanger sequencing. The pipeline was validated against the online tool PASeq (Polymorphism Analysis by Sequencing). Based on an error rate of <1%, a value of >1% was set as reliable to consider a minor variant. Both pipelines detected the mutations in the dominant viral populations, while discrepancies were observed in minor viral populations. In five HIV-1 subtype C samples, minor mutations were detected at the <5% level by MiDRMpol but not by PASeq. MiDRMpol is a computationally as well as labor efficient bioinformatics pipeline for the detection of DRM from HTS data. It identifies minor viral populations (<20%) of DRMs. Our method can be incorporated into large-scale surveillance of HIV-1 DRM.

Prevalence of integrase strand transfer inhibitor resistance mutations in antiretroviral-naive HIV-1-infected individuals in Cameroon

2020 ◽  
Vol 76 (1) ◽  
pp. 124-129
Author(s):  
Benjamin M Wenk ◽  
Herbert A Mbunkah ◽  
Ndi N Nsanwe ◽  
Eyongetah T Mbu ◽  
Lydia M Besong ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Universal Primers ◽  
Polymorphism Analysis ◽  
Strand Transfer ◽  
Resistance Mutations ◽  
Transfer Inhibitor ◽  
Study Sites ◽  
Inhibitor Resistance ◽  
The Impact ◽  
Hiv 1

Abstract Objectives In Cameroon, the integrase (IN) strand transfer inhibitor (INSTI) dolutegravir was recently introduced for the treatment of HIV-1 infection. Since pretreatment HIV-1 drug resistance can jeopardize the success of ART, and considering the high heterogeneity of circulating HIV-1 subtypes in Cameroon, we investigated the prevalence of pretreatment HIV-1 resistance to INSTIs. Methods Fingerprick dried blood spot samples were collected from 339 newly diagnosed HIV-1-infected individuals between 2015 and 2016 in four hospitals in Cameroon. Universal primers were designed to amplify the HIV-1 IN region from amino acid 1 to 276. Amplicons were sequenced with Illumina next-generation sequencing and analysed with the Polymorphism Analysis Sequencing (PASeq) platform, using the Stanford HIV Drug Resistance Database to interpret HIV-1 drug resistance mutations (DRMs). Results The amplification/sequencing success rate was 75.2% with 255/339 sequences obtained. Applying a cut-off of 1%, major DRMs to INSTIs were detected in 13 (5.1%) individuals, but only 1 individual harboured an INSTI DRM (E92G) at a nucleotide frequency ≥15%. However, 140/255 (54.9%) individuals harboured polymorphic accessory INSTI DRMs, mainly at high frequencies. In line with that observation, HIV-1 subtype diversity among individuals was high. Conclusions Pretreatment HIV-1 resistance to INSTIs was low in the study sites, which supports the use of INSTIs in Cameroon. Nevertheless, further studies are necessary to assess the impact of polymorphic accessory INSTI DRMs on INSTI-based ART regimens.

scholarly journals Impact of pretreatment low-abundance HIV-1 drug-resistant variants on virological failure among HIV-1/TB-co-infected individuals

2020 ◽  
Vol 75 (11) ◽  
pp. 3319-3326
Author(s):  
Benjamin Chimukangara ◽  
Jennifer Giandhari ◽  
Richard Lessells ◽  
Nonhlanhla Yende-Zuma ◽  
Benn Sartorius ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Virological Failure ◽  
Statistical Significance ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
The Impact ◽  
Hiv 1 ◽  
Control Study

Abstract Objectives To determine the impact of pretreatment low-abundance HIV-1 drug-resistant variants (LA-DRVs) on virological failure (VF) among HIV-1/TB-co-infected individuals treated with NNRTI first-line ART. Methods We conducted a case–control study of 170 adults with HIV-1/TB co-infection. Cases had at least one viral load (VL) ≥1000 RNA copies/mL after ≥6 months on NNRTI-based ART, and controls had sustained VLs &lt;1000 copies/mL. We sequenced plasma viruses by Sanger and MiSeq next-generation sequencing (NGS). We assessed drug resistance mutations (DRMs) using the Stanford drug resistance database, and analysed NGS data for DRMs at ≥20%, 10%, 5% and 2% thresholds. We assessed the effect of pretreatment drug resistance (PDR) on VF. Results We analysed sequences from 45 cases and 125 controls. Overall prevalence of PDR detected at a ≥20% threshold was 4.7% (8/170) and was higher in cases than in controls (8.9% versus 3.2%), P = 0.210. Participants with PDR at ≥20% had almost 4-fold higher odds of VF (adjusted OR 3.7, 95% CI 0.8–18.3) compared with those without, P = 0.104. PDR prevalence increased to 18.2% (31/170) when LA-DRVs at ≥2% were included. Participants with pretreatment LA-DRVs only had 1.6-fold higher odds of VF (adjusted OR 1.6, 95% CI 0.6–4.3) compared with those without, P = 0.398. Conclusions Pretreatment DRMs and LA-DRVs increased the odds of developing VF on NNRTI-based ART, although without statistical significance. NGS increased detection of DRMs but provided no additional benefit in identifying participants at risk of VF at lower thresholds. More studies assessing mutation thresholds predictive of VF are required to inform use of NGS in treatment decisions.

scholarly journals Impact of Novel Resistance Profiles in HIV-1 Reverse Transcriptase on Phenotypic Resistance to NVP

2012 ◽  
Vol 2012 ◽  
pp. 1-8
Author(s):  
Liyan Jiao ◽  
Hanping Li ◽  
Lin Li ◽  
Daomin Zhuang ◽  
Yongjian Liu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Site Directed Mutagenesis ◽  
Drug Resistant ◽  
Wild Type ◽  
Phenotypic Resistance ◽  
Drug Resistant Mutation ◽  
The Impact ◽  
Hiv 1 ◽  
Resistant Mutation ◽  
Phenotypic Drug Resistance

Objective. To clarify the impact of H221Y mutation on drug resistance to NVP.Methods. 646 bp HIV-1polgene fragments (from 592 to 1237 nucleotide) with different NNRTIs mutation profiles from AIDS patients receiving antiretroviral therapy containing NVP regimens were introduced into pNL4-3 backbone plasmid. H221Y and (or) Y181C mutations were reverted to wild type amino acids by site-directed mutagenesis, then strains containing various mutation patterns were packaged. Phenotypic drug resistance was analyzed on TZM-bl cells.Results. 12 strains containing different drug-resistant mutation profiles were constructed, including the K101Q series (K101Q/Y181C/H221Y, K101Q/Y181C, K101Q/H221Y, and K101Q), the V179D series (V179D/Y181C/H221Y, V179D/Y181C, V179D/H221Y, and V179D), and the K103N series (K103N/Y181C/H221Y, K103N/Y181C, K103N/H221Y, K103N). For strains containing the mutation profiles (K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, and K103N), the presence of H221Y reduced NVP susceptibility by2.1±0.5to3.6±0.5fold. To the mutation profiles K101Q/H221Y, K101Q, V179D/H221Y, V179D, K103N/H221Y, and K103N, the presence of Y181C reduced NVP susceptibility by41.9±8.4to1297.0±289.1fold. For the strains containing K101Q, V179D, and K103N, the presence of Y181C/H221Y combination decreased NVP susceptibility by100.6±32.5to3444.6±834.5fold.Conclusion. On the bases of various NNRTIs mutation profiles, Y181C remarkably improved the IC50to NVP, although H221Ymutation alone just increases 2.1 ∼ 3.6-fold resistance to NVP, the mutation could improve 100.6 ∼ 3444.6-fold resistance to NVP when it copresent with Y181C, the phenotypic drug resistance fold was improved extremely. For strains containing the mutation profiles (K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, and K103N), the presence of H221Y reduced NVP susceptibility by2.1±0.5to3.6±0.5fold.

scholarly journals Natural presence of the V179D and K103R/V179D mutations associated with resistance to nonnucleoside reverse transcriptase inhibitors in HIV-1 CRF65_cpx strains

2020 ◽  
Author(s):  
Yongjian Liu ◽  
Yu Zhang ◽  
Hanping Li ◽  
Xiaolin Wang ◽  
Lei Jia ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Geographic Distance ◽  
Sequence Database ◽  
Genotypic Resistance ◽  
Nonnucleoside Reverse Transcriptase Inhibitors ◽  
Treatment Naïve ◽  
Relationship Of ◽  
The Impact ◽  
Search Program ◽  
Hiv 1

Abstract Background: There is increasing evidence that HIV-1 genetic diversity can have an impact on drug resistance. The aim of this study is to investigate the epidemiological situation of CRF65_cpx and the impact of natural polymorphisms of this variant on genotypic resistance. Methods: We used the BLAST search program followed by phylogenetic analysis to identify additional CRF65_cpx pol sequences from the Los Alamos HIV Sequence Database. Maximum likelihood phylogeny was estimated to clarify the epidemiological relationship of CRF65_cpx strains. Genotypic resistance was determined by submitting sequences to the Stanford HIV Drug Resistance Database. Results: A total of 32 CRF65_cpx pol sequences were obtained. The CRF65_cpx strains were detected in seven provinces with large geographic distance. Yunnan CRF65_cpx sequences were mainly derived from a heterosexual risk group, whereas the CRF65_cpx sequences in other provinces were almost exclusively derived from an MSM population. With one exception of V179E, the other 31 strains harbored V179D mutation. The combination of V179D and K103R, conferring intermediate resistance to EFV and NVP, was detected in seven treatment-naive MSM patients. Conclusions: This study confirmed the expansion CRF65_cpx in China. Furthermore, we found the natural presence of the V179D and K103R/V179D mutations associated with resistance to NNRTIs in HIV-1 CRF65_cpx. Our findings highlight the contribution of polymorphic mutations to drug resistance and underscore the challenges in treating patients harboring CRF65_cpx strains.

scholarly journals An evolutionary-based approach to quantify the genetic barrier to drug resistance in fast-evolving viruses: an application to HIV-1 subtypes and integrase inhibitors

2019 ◽  
Author(s):  
Kristof Theys ◽  
Pieter Libin ◽  
Kristel Van Laethem ◽  
Ana B Abecasis
Keyword(s):  
Drug Resistance ◽  
Antiviral Treatment ◽  
Integrase Inhibitors ◽  
Resistance Mutations ◽  
Genetic Barrier ◽  
Viral Pathogens ◽  
Subtype B ◽  
Genetic Potential ◽  
The Impact ◽  
Hiv 1

AbstractViral pathogens causing global disease burdens are often characterised by high rates of evolutionary changes, facilitating escape from therapeutic or immune selective pressure. Extensive viral diversity at baseline can shorten the time to resistance emergence and alter mutational pathways, but the impact of genotypic background on the genetic barrier can be difficult to capture, in particular for antivirals in experimental stages, recently approved or expanded into new settings. We developed an evolutionary-based counting method to quantify the population genetic potential to resistance and assess differences between populations. We demonstrate its applicability to HIV-1 integrase inhibitors, as their increasing use globally contrasts with limited availability of non-B subtype resistant sequences and corresponding knowledge gap on drug resistance. A large sequence dataset encompassing most prevailing subtypes and resistance mutations of first- and second-generation inhibitors were investigated. A varying genetic potential for resistance across HIV-1 subtypes was detected for 15 mutations at 12 positions, with notably 140S in subtype B, while 140C was discarded to vary across subtypes. An additional analysis for HIV-1 reverse transcriptase inhibitors identified a higher potential for 65R in subtype C, on the basis of a differential codon usage not reported before. The evolutionary interpretation of genomic differences for antiviral treatment remains challenging. Our framework advances existing counting methods with an increased sensitivity that identified novel subtype dependencies as well as rejected previous statements. Future applications include novel HIV-1 drug classes as well as other viral pathogens.

scholarly journals 1283. Pretreatment HIV-1 Drug Resistance in Transmission Clusters of the Cologne-Bonn Region, Germany

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S391-S392 ◽  
Author(s):  
Melanie Stecher ◽  
Martin Hoenigl ◽  
Anna-Maria Eis-Huebinger ◽  
Clara Lehmann ◽  
Gerd Fätkenheuer ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Network Analysis ◽  
Risk Groups ◽  
Transcriptase Inhibitor ◽  
Area Network ◽  
Resistance Mutations ◽  
University Hospitals ◽  
Genotypic Resistance ◽  
Network Analyses ◽  
Hiv 1

Abstract Background In Germany, previous reports have demonstrated transmitted HIV-1 drug resistance mutations (DRM) in 10% of newly diagnosed individuals, affecting treatment failure and the choice of antiretroviral therapy (ART). Here, we sought to understand the molecular epidemiology of HIV DRM transmission throughout the Cologne-Bonn region, an area with one of the highest rate of new HIV infections in Europe (13.7 per 100,000 habitants). Methods We analyzed 714 HIV-1 ART naïve infected individuals diagnosed at the University Hospitals Cologne and Bonn between 2001 and 2016. Screening for DRM was performed according to the Stanford University Genotypic Resistance Interpretation. Shared DRM were defined as any DRM present in genetically linked individuals (&lt;1.5% genetic distance). Phylogenetic and network analyses were performed to infer putative relationships and shared DRMs. Results We detected 123 DRMs in our study population (17.2% of all sequences). Prevalence of any DRM was comparable among risk groups and was highest among people from an endemic area (i.e., country with HIV prevalence &gt;1%) (11/51, 21.6%). Nucleoside-and non-nucleoside reverse transcriptase inhibitor (NRTI/NNNRTI) resistance mutations were detected in 49 (7%) and 97 (13.6%) individuals, with the E138A in 29 (4.1%) and K103N in 11 (1.5%) being the most frequent. Frequency of DRM was comparable in clustering and not clustering individuals (17.1% vs. 17.5%). Transmission network analysis indicated that the frequency of DRM in clustering individuals was the highest in PWID (3/7, 42.9%) (Figure 1A). Genetically linked individuals harboring shared DRMs were more likely to live in suburban areas than in Central Cologne (18.8% vs. 8% of clustering sequences with DRM; Figure 1B). Conclusion The rate of DRMs was exceptionally high in the Cologne/Bonn area. Network analysis elucidated frequent cases of shared DRMs among genetically linked individuals, revealing the potential spread of DRMs and the need to prevent onward transmission of DRM in the Cologne-Bonn area. Disclosures M. Hoenigl, Gilead, Basilea, Merck: Speaker’s Bureau, Research grant and Speaker honorarium.

scholarly journals HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing

PLoS ONE ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. e0145772 ◽  
Author(s):  
Soo-Yon Rhee ◽  
Michael R. Jordan ◽  
Elliot Raizes ◽  
Arlene Chua ◽  
Neil Parkin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Point Of Care ◽  
Resistance Testing ◽  
Resistance Mutations ◽  
Genotypic Resistance ◽  
Drug Resistance Mutations ◽  
Genotypic Resistance Testing ◽  
Potential Applications ◽  
Hiv 1

The frequency of HIV-I drug resistance mutations among treatment-naïve individuals at a tertiary care centre in south India

2009 ◽  
Vol 20 (8) ◽  
pp. 522-526 ◽  
Author(s):  
A J Kandathil ◽  
R Kannangai ◽  
O C Abraham ◽  
P Rupali ◽  
S A Pulimood ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Tertiary Care ◽  
Resistance Mutations ◽  
Tertiary Care Centre ◽  
Genotypic Resistance ◽  
Drug Resistance Mutations ◽  
Clade B ◽  
Clade C ◽  
Treatment Naïve ◽  
Hiv 1

Antiretroviral treatment (ART) use in India requires information on baseline drug resistance mutations and polymorphisms in the protease (Pr) and reverse transcriptase (RT) genes of HIV-1 strains from treatment-naïve individuals. We report resistance predictor mutations and polymorphisms in the Pr and the RT sequence of non-clade B HIV-1 strains from ART naïve individuals. The genotypic resistance assay was done on 93 treatment-naïve individuals. The sequences were analysed by Stanford HIV drug resistance data for genotypic drug resistance analysis and REGA HIV-1 subtyping tool. Phylogenetic tree was generated with MEGA 4 for quality control. Ninety-two strains belonged to clade C and one to clade A (A1). Amino acid substitutions were seen at positions associated with drug resistance in Pr gene – 10, 24, 74 (each 3%) and position 82 (11%). Substitutions were seen at positions 41 (1%), 100 (1%), 101 (6%), 103 (2%), 179 (6%) and 181 (1%) of the RT sequence known to confer drug resistance in clade B. Polymorphisms in HIV-1 pol gene among treatment-naïve individuals were similar when compared with previous data. One strain each had Y181C substitution, T74S and E35G substitutions in the Pr and one had A98G, K101R and L210FL substitutions in RT.

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