Optimization of the Oligonucleotide Ligation Assay, a Rapid and Inexpensive Test for Detection of HIV-1 Drug Resistance Mutations, for Non-North American Variants

Beck, Ingrid; Crowell, Claudia S.; Kittoe, Robin; Bredell, Helba; Machaba, Molefe; Willamson, Carolyn; Janssens, Wouter; Jallow, Sabelle; Groen, G van der; Shao, Yiming; Jacob, Mini; Samuel, N; Lorenzana, Ivette; Ngo‐Giang‐Huong, Nicole; Cassol, Sharon; Alemnji, George; Frenkel, Lisa M.

doi:10.1097/qai.0b013e31817ed7d7

Cited by 39 publications

(44 citation statements)

References 31 publications

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“…These usually produce an indeterminate OLA result, i.e., both the mutant and wild-type reactions are negative, thus reducing the possibility of false-negative genotypes. Because genetic polymorphisms in HIV variants are common, it has been difficult to design universal OLA probes that can effectively evaluate viruses from all HIV subtypes (18). In this study, optimization of the OLA probes for subtypes prevalent in Mozambique and Kenya, as well as the use of mixed bases at sites of frequent genetic polymorphisms, significantly reduced the rate of indeterminate results, in comparison with previous reports (18,33).…”

Section: Discussioncontrasting

confidence: 41%

“…However, assay failure may occur when polymorphisms are located within the 4 nucleotides flanking the ligation site, and occasionally when multiple target mismatches are in the region complementary to one of the probes (16)(17)(18). These usually produce an indeterminate OLA result, i.e., both the mutant and wild-type reactions are negative, thus reducing the possibility of false-negative genotypes.…”

Section: Discussionmentioning

confidence: 99%

“…Because genetic polymorphisms in HIV variants are common, it has been difficult to design universal OLA probes that can effectively evaluate viruses from all HIV subtypes (18). In this study, optimization of the OLA probes for subtypes prevalent in Mozambique and Kenya, as well as the use of mixed bases at sites of frequent genetic polymorphisms, significantly reduced the rate of indeterminate results, in comparison with previous reports (18,33). More importantly, HIV sequence variations did not adversely affect quantification of mutant concentrations by OLA, as demonstrated by the high level of correlation between OLA and pyrosequencing results among Kenyan specimens that included HIV subtypes A, C, D, and AE.…”

Section: Discussionmentioning

confidence: 99%

“…The oligonucleotide ligation assay (OLA) is a sensitive and specific point-mutation assay for detection of HIV drug resistance to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs), and NNRTIs (16)(17)(18)(19). Because mutant-and wildtype-specific probes are hybridized and ligated at a relatively low temperature, the OLA accommodates viral polymorphisms in the regions of the probes to a greater degree than do allele-specific PCR assays (20,21), and the assay is technically less complex than the LigAmp assay (22).…”

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confidence: 99%

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Validation of an Oligonucleotide Ligation Assay for Quantification of Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by Use of Massively Parallel Sequencing

Beck¹,

Deng

Payant³

et al. 2014

J Clin Microbiol

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Global HIV treatment programs need sensitive and affordable tests to monitor HIV drug resistance. We compared mutant detection by the oligonucleotide ligation assay (OLA), an economical and simple test, to massively parallel sequencing. Nonnucleoside reverse transcriptase inhibitor (K103N, V106M, Y181C, and G190A) and lamivudine (M184V) resistance mutations were quantified in blood-derived plasma RNA and cell DNA specimens by OLA and 454 pyrosequencing. A median of 1,000 HIV DNA or RNA templates (range, 163 to 1,874 templates) from blood specimens collected in Mozambique (n ‫؍‬ 60) and Kenya (n ‫؍‬ 51) were analyzed at 4 codons in each sample (n ‫؍‬ 441 codons assessed). Mutations were detected at 75 (17%) codons by OLA sensitive to 2.0%, at 71 codons (16%; P ‫؍‬ 0.78) by pyrosequencing using a cutoff value of >2.0%, and at 125 codons (28%; P < 0.0001) by pyrosequencing sensitive to 0.1%. Discrepancies between the assays included 15 codons with mutant concentrations of ϳ2%, one at 8.8% by pyrosequencing and not detected by OLA, and one at 69% by OLA and not detected by pyrosequencing. The latter two cases were associated with genetic polymorphisms in the regions critical for ligation of the OLA probes and pyrosequencing primers, respectively. Overall, mutant concentrations quantified by the two methods correlated well across the codons tested (R 2 > 0.8). Repeat pyrosequencing of 13 specimens showed reproducible detection of 5/24 mutations at <2% and 6/6 at >2%. In conclusion, the OLA and pyrosequencing performed similarly in the quantification of nonnucleoside reverse transcriptase inhibitor and lamivudine mutations present at >2% of the viral population in clinical specimens. While pyrosequencing was more sensitive, detection of mutants below 2% was not reproducible.

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Section: Discussioncontrasting

confidence: 41%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Validation of an Oligonucleotide Ligation Assay for Quantification of Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by Use of Massively Parallel Sequencing

Beck¹,

Deng

Payant³

et al. 2014

J Clin Microbiol

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“…HIV-1 drug resistance mutations were detected using an oligonucleotide ligation assay (OLA) 20,21 (http://depts .washington.edu/idimmweb/faculty/frenkel/OLAmanual 1305april04.pdf). Specimens were tested for select mutations conferring high-level resistance to NVP (K103N, Y181C, G190A), 3TC (M184V), and zidovudine (ZDV)/d4T (T215Y/ F).…”

Section: Hiv-1 Genotypingmentioning

confidence: 99%

Monitoring of HIV Type 1 DNA Load and Drug Resistance in Peripheral Blood Mononuclear Cells During Suppressive Antiretroviral Therapy Does Not Predict Virologic Failure

Beck

Jang

McKernan-Mullin

et al. 2012

AIDS Research and Human Retroviruses

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Our objective was to determine whether monitoring HIV-1 DNA concentration or new resistance mutations in peripheral blood mononuclear cells (PBMCs) during effective antiretroviral therapy (ART) predicts virologic failure. A retrospective analysis used blood specimens and clinical data from three nevirapine containing arms of a four-arm, open-label, randomized trial comparing ART regimens in HIV-1-infected children who had failed mono-or dual-nucleoside therapy. Sensitive assays compared cell-associated HIV-1 DNA concentrations and nevirapine (NVP) and lamivudine (3TC) resistance mutations in children with plasma HIV-1 RNA < 400 copies(c)/ml who did or did not experience subsequent virologic failure. Forty-six children were analyzed through the last available follow-up specimen, collected at 48 (n = 16) or 96 (n = 30) weeks of ART. Thirty-five (76%) had sustained viral suppression and 11 (24%) had plasma viral rebound to ‡ 400 c/ml (virologic failure detected at a median of 36 weeks). HIV-1 DNA levels at baseline, 24, 48, and 96 weeks of ART were similar in children who did vs. did not experience virologic failure ( p = 0.82). HIV-1 DNA levels did not increase prior to viral rebound. NVP resistance mutations were detected in 91% of subjects in the failure group vs. 3% in the suppressed group ( p < 0.0001). Among nine evaluable children, NVP mutations were first detected prior to virologic failure in two (22%), at viral rebound in five (56%), and after failure in two (22%) children. HIV-1 DNA concentrations did not predict virologic failure in this cohort. New drug resistance mutations were detected in the PBMCs of a minority of virologically suppressed children who subsequently failed ART.

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HIV-1 genotypic drug resistance testing: digging deep, reaching wide?

Laethem

Theys

Vandamme

2015

Current Opinion in Virology

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For many years, population-based Sanger sequencing has been the golden standard for drug resistance testing within the routine follow-up of HIV-1 infected patients in resource-rich settings. Often, the data generated within this framework were subsequently used for research and surveillance purposes: to understand therapy response and to gain insights into epidemiological processes. Sanger sequencing was however ill suited for diagnostic and prognostic use in resource-limited settings (RLS) and therefore not broadly implemented. Next-generation sequencing (NGS) technologies provide high-throughput approaches by the rapid acquisition of thousands to millions of short nucleotide sequences. Depending on the experimental design, the roll-out of NGS drug resistance testing at a larger scale is feasible, providing better characterization and understanding of the evolving population of viral variants within a patient and potentially improving the prognostic value of drug resistance testing. Whether the same will become true for RLS will largely depend on affordability and sample logistics, and this may affect other mutation-specific approaches.

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Optimization of the Oligonucleotide Ligation Assay, a Rapid and Inexpensive Test for Detection of HIV-1 Drug Resistance Mutations, for Non-North American Variants

Cited by 39 publications

References 31 publications

Validation of an Oligonucleotide Ligation Assay for Quantification of Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by Use of Massively Parallel Sequencing

Validation of an Oligonucleotide Ligation Assay for Quantification of Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by Use of Massively Parallel Sequencing

Monitoring of HIV Type 1 DNA Load and Drug Resistance in Peripheral Blood Mononuclear Cells During Suppressive Antiretroviral Therapy Does Not Predict Virologic Failure

HIV-1 genotypic drug resistance testing: digging deep, reaching wide?

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