Sensitive Oligonucleotide Ligation Assay for Low-Level Detection of Nevirapine Resistance Mutations in Human Immunodeficiency Virus Type 1 Quasispecies

Lalonde, Matthew S.; Troyer, Ryan M.; Syed, Aslam; Bulime, Stanley; Demers, Korey; Bajunirwe, Francis; Arts, Eric J.

doi:10.1128/jcm.00431-07

Cited by 25 publications

(27 citation statements)

References 51 publications

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“…The gp120 region was amplified from cellular DNA using F_gp120/B_gp120 as described above. Amplicons were probed for sequence identity at gp120 residues 117 and 425 using OLA as described previously (31,32). Frequency of wild-type or mutant residue identity versus total signal was used to determine viral fitness.…”

Section: Methodsmentioning

confidence: 99%

HIV-1 Resistance to Maraviroc Conferred by a CD4 Binding Site Mutation in the Envelope Glycoprotein gp120

Ratcliff

Shi

Arts

2013

J Virol

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Maraviroc (MVC) is a CCR5 antagonist that inhibits HIV-1 entry by binding to the coreceptor and inducing structural alterations in the extracellular loops. In this study, we isolated MVC-resistant variants from an HIV-1 primary isolate that arose after 21 weeks of tissue culture passage in the presence of inhibitor. gp120 sequences from passage control and MVC-resistant cultures were cloned into NL4-3 via yeast-based recombination followed by sequencing and drug susceptibility testing. Using 140 clones, three mutations were linked to MVC resistance, but none appeared in the V3 loop as was the case with previous HIV-1 strains resistant to CCR5 antagonists. Rather, resistance was dependent upon a single mutation in the C4 region of gp120. Chimeric clones bearing this N425K mutation replicated at high MVC concentrations and displayed significant shifts in 50% inhibitory concentrations (IC 50 s), characteristic of resistance to all other antiretroviral drugs but not typical of MVC resistance. Previous reports on MVC resistance describe an ability to use a drug-bound form of the receptor, leading to reduction in maximal drug inhibition. In contrast, our structural models on K425 gp120 suggest that this resistant mutation impacts CD4 interactions and highlights a novel pathway for MVC resistance.

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Section: Methodsmentioning

confidence: 99%

HIV-1 Resistance to Maraviroc Conferred by a CD4 Binding Site Mutation in the Envelope Glycoprotein gp120

Ratcliff

Shi

Arts

2013

J Virol

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“…The ability to detect and identify low levels of analytes is of growing importance in medicine and nanotechnology [1][2][3][4][5][6][7]. Diagnostic monitoring of drug delivery nanoparticle levels [8][9][10], identification of cell-free circulating (cfc) DNA/RNA and other nanoparticulate biomarkers [11][12][13][14][15], and detection of pathogens in clinical and environmental samples [16] are examples where low level detection of analytes are of extreme importance.…”

Section: Introductionmentioning

confidence: 99%

Low level epifluorescent detection of nanoparticles and DNA on dielectrophoretic microarrays

McCanna¹,

Sonnenberg²,

Heller³

2013

Journal of Biophotonics

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Common epifluorescent microscopy lacks the sensitivity to detect low levels of analytes directly in clinical samples, such as drug delivery nanoparticles or disease related DNA biomarkers. Advanced systems such as confocal microscopes may improve detection, but several factors limit their applications. This study now demonstrates that combining an epifluorescent microscope with a dielectrophoretic (DEP) microelectrode array device enables the detection of nanoparticles and DNA biomarkers at clinically relevant levels. Using DEP microarray devices, nanoparticles and DNA biomarkers are rapidly isolated and concentrated onto specific microscopic locations where they are easily detected by epifluorescent microscopy. In this study, 40nm nanoparticles were detected down to 2–3 × 103/ul levels and DNA was detected down to the 200 pg/ml level. The synergy of epifluorescent microscopy and DEP microarray devices provides a new paradigm for DNA biomarker diagnostics and the monitoring of drug delivery nanoparticle concentrations. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…OLAs utilize the specificity of ligase to join allele-specific probes to distinguish single nucleotide polymorphisms. OLA has been adapted to detect single nucleotide mutations associated with HIV-1 drug resistance (11) and, with modifications, has been shown to be able to detect as little as 0.4% mutant virus (18). HIV-1 drug resistance genotypes were determined using the OLA as described previously (3) with slight variations for subtype and to optimize the optical density (OD) signal.…”

Section: Methodsmentioning

confidence: 99%

Detection of HIV-1 Drug Resistance in Women Following Administration of a Single Dose of Nevirapine: Comparison of Plasma RNA to Cellular DNA by Consensus Sequencing and by Oligonucleotide Ligation Assay

et al. 2010

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A single dose of nevirapine (sdNVP) to prevent mother-to-child transmission of HIV-1 increases the risk of failure of subsequent NVP-containing antiretroviral therapy (ART), especially when initiated within 6 months of sdNVP administration, emphasizing the importance of understanding the decay of nevirapine-resistant mutants. Nevirapine-resistant HIV-1 genotypes (with the mutations K103N, Y181C, and/or G190A) from 21 women were evaluated 10 days and 6 weeks after sdNVP administration and at the initiation of ART. Resistance was assayed by consensus sequencing and by a more sensitive assay (oligonucleotide ligation assay [OLA]) using plasma-derived HIV-1 RNA and cell-associated HIV-1 DNA. OLA detected nevirapine resistance in more specimens than consensus sequencing did (63% versus 33%, P < 0.01). When resistance was detected only by OLA (n ‫؍‬ 45), the median mutant concentration was 18%, compared to 61% when detected by both sequencing and OLA (n ‫؍‬ 51) (P < 0.0001). The proportion of women whose nevirapine resistance was detected by OLA 10 days after sdNVP administration was higher when we tested their HIV-1 RNA (95%) than when we tested their HIV-1 DNA (88%), whereas at 6 weeks after sdNVP therapy, the proportion was greater with DNA (85%) than with RNA (67%) and remained higher with DNA (33%) than with RNA (11%) at the initiation of antiretroviral treatment (median, 45 weeks after sdNVP therapy). Fourteen women started NVP-ART more than 6 months after sdNVP therapy; resistance was detected by OLA in 14% of the women but only in their DNA. HIV-1 resistance to NVP following sdNVP therapy persists longer in cellular DNA than in plasma RNA, as determined by a sensitive assay using sufficient copies of virus, suggesting that DNA may be superior to RNA for detecting resistance at the initiation of ART.A single dose of nevirapine (sdNVP) reduces mother-tochild transmission (MTCT) of HIV-1 by 47% to 58% (12,17) but is associated with the selection of mutant viruses resistant to nonnucleoside reverse transcriptase inhibitors (NNRTI) (7,15) and may jeopardize the effectiveness of subsequent nevirapine (NVP)-based antiretroviral treatment (ART) (5, 15, 21). However, the adverse impact of sdNVP therapy on NVP-ART efficacy seems to decrease with longer time intervals between the administration of sdNVP and the initiation of NVP-ART (ART start) (21, 29).In 21 to 32% of sdNVP-treated mothers, consensus sequencing of HIV-1 RNA detects NNRTI mutations, which decay to undetectable levels over 1 year (7,8,15). More sensitive assays detect NNRTI resistance mutations in 51 to 87% of postpartum sdNVPexposed mothers (9,22), and these mutations remain detectable in 8 to 33% of women after a year (5,8,10). Comparative analyses of the decay of NVP resistance in HIV-1 DNA and RNA in blood using sensitive methods found a lower proportion of women with resistance in their HIV-1 DNA (5, 22), although the input of HIV-1 DNA was not directly determined in these studies, precluding estimations of the sensitivity limits of the assays.We inv...

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Sensitive Oligonucleotide Ligation Assay for Low-Level Detection of Nevirapine Resistance Mutations in Human Immunodeficiency Virus Type 1 Quasispecies

Cited by 25 publications

References 51 publications

HIV-1 Resistance to Maraviroc Conferred by a CD4 Binding Site Mutation in the Envelope Glycoprotein gp120

HIV-1 Resistance to Maraviroc Conferred by a CD4 Binding Site Mutation in the Envelope Glycoprotein gp120

Low level epifluorescent detection of nanoparticles and DNA on dielectrophoretic microarrays

Detection of HIV-1 Drug Resistance in Women Following Administration of a Single Dose of Nevirapine: Comparison of Plasma RNA to Cellular DNA by Consensus Sequencing and by Oligonucleotide Ligation Assay

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