{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T06:55:47Z","timestamp":1790664947041,"version":"4.1.0"},"reference-count":49,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2022,10,7]],"date-time":"2022-10-07T00:00:00Z","timestamp":1665100800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The HIV genotypic resistance test (GRT) is a standard of care for the clinical management of HIV\/AIDS patients. In recent decades, population or Sanger sequencing has been the foundation for drug resistance monitoring in clinical settings. However, the advent of high-throughput or next-generation sequencing has caused a paradigm shift towards the detection and characterization of low-abundance covert mutations that would otherwise be missed by population sequencing. This is clinically significant, as these mutations can potentially compromise the efficacy of antiretroviral therapy, causing poor virologic suppression. Therefore, it is important to develop a more sensitive method so as to reliably detect clinically actionable drug-resistant mutations (DRMs). Here, we evaluated the diagnostic performance of a laboratory-developed, high-throughput, sequencing-based GRT using 103 archived clinical samples that were previously tested for drug resistance using population sequencing. As expected, high-throughput sequencing found all the DRMs that were detectable by population sequencing. Significantly, 78 additional DRMs were identified only by high-throughput sequencing, which is statistically significant based on McNemar\u2019s test. Overall, our results complement previous studies, supporting the notion that the two methods are well correlated, and the high-throughput sequencing method appears to be an excellent alternative for drug resistance testing in a clinical setting.<\/jats:p>","DOI":"10.3390\/v14102208","type":"journal-article","created":{"date-parts":[[2022,10,12]],"date-time":"2022-10-12T02:10:27Z","timestamp":1665540627000},"page":"2208","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Towards Next-Generation Sequencing for HIV-1 Drug Resistance Testing in a Clinical Setting"],"prefix":"10.3390","volume":"14","author":[{"given":"Calesta Hui Yi","family":"Teo","sequence":"first","affiliation":[{"name":"Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore"},{"name":"School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore 599490, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nurul Hannah Binte","family":"Norhisham","sequence":"additional","affiliation":[{"name":"School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore 599490, Singapore"},{"name":"School of Social Sciences, Nanyang Technological University, Singapore 639818, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ogestelli Fabia","family":"Lee","sequence":"additional","affiliation":[{"name":"School of Life and Physical Sciences, PSB Academy, Singapore 039594, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siyu","family":"Png","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, National University Health System, Singapore 119228, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chean Nee","family":"Chai","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, National University Health System, Singapore 119228, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriel","family":"Yan","sequence":"additional","affiliation":[{"name":"Department of Medicine, National University Health System, Singapore 119228, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julian Wei-Tze","family":"Tang","sequence":"additional","affiliation":[{"name":"Respiratory Sciences, University of Leicester, Leicester LE1 7RH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6065-0000","authenticated-orcid":false,"given":"Chun Kiat","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, National University Health System, Singapore 119228, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,10,7]]},"reference":[{"key":"ref_1","unstructured":"(2022, August 20). UNAIDS Data 2021. Available online: https:\/\/www.unaids.org\/en\/resources\/documents\/2021\/2021_unaids_data\/."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1016\/S0140-6736(06)69157-5","article-title":"HIV\/AIDS epidemiology, pathogenesis, prevention, and treatment","volume":"368","author":"Simon","year":"2006","journal-title":"Lancet"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"F83","DOI":"10.1097\/00002030-200006160-00001","article-title":"A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS","volume":"14","author":"Baxter","year":"2000","journal-title":"AIDS"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1097\/00002030-200202150-00008","article-title":"Usefulness of monitoring HIV drug resistance and adherence in individuals failing highly active antiretroviral therapy: A randomized study (ARGENTA)","volume":"16","author":"Cingolani","year":"2002","journal-title":"AIDS"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1316","DOI":"10.1086\/496984","article-title":"Should Resistance Testing Be Performed for Treatment-Naive HIV-Infected Patients? A Cost-Effectiveness Analysis","volume":"41","author":"Sax","year":"2005","journal-title":"Clin. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Mbuagbaw, L., Ongolo-Zogo, C., Mendoza, O.C., Zani, B., Morfaw, F., Nyambi, A., Wang, A., Kiflen, M., El-Kechen, H., and Leenus, A. (2021). Guidelines are needed for studies of pre-treatment HIV drug resistance: A methodological study. BMC. Med. Res. Methodol., 21.","DOI":"10.1186\/s12874-021-01258-1"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1016\/S0140-6736(14)60164-1","article-title":"HIV infection: Epidemiology, pathogenesis, treatment, and prevention","volume":"384","author":"Maartens","year":"2014","journal-title":"Lancet"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1582","DOI":"10.1126\/science.271.5255.1582","article-title":"HIV-1 dynamics in vivo: Virion clearance rate, infected cell life-span, and viral generation time","volume":"271","author":"Perelson","year":"1996","journal-title":"Science"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"13910","DOI":"10.1073\/pnas.96.24.13910","article-title":"Mutation rates among RNA viruses","volume":"96","author":"Drake","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1038\/nrg2323","article-title":"Rates of evolutionary change in viruses: Patterns and determinants","volume":"9","author":"Duffy","year":"2008","journal-title":"Nat. Rev. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1097\/01.aids.0000216363.36786.2b","article-title":"HIV-1 pol mutation frequency by subtype and treatment experience: Extension of the HIVseq program to seven non-B subtypes","volume":"20","author":"Rhee","year":"2006","journal-title":"AIDS"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Johnson, J.A., Li, J., Wei, X., Lipscomb, J., Irlbeck, D., Craig, C., Smith, A., Bennett, D.E., Monsour, M., and Sandstrom, P. (2008). Minority HIV-1 drug resistance mutations are present in antiretroviral treatment-na\u00efve populations and associate with reduced treatment efficacy. PLoS Med., 5.","DOI":"10.1371\/journal.pmed.0050158"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4087","DOI":"10.1128\/JCM.01246-08","article-title":"Genotypic antiretroviral resistance testing for human immunodeficiency virus type 1 integrase inhibitors by use of the TruGene sequencing system","volume":"46","author":"Paar","year":"2008","journal-title":"J. Clin. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1693","DOI":"10.1086\/379773","article-title":"Progressive reversion of human immunodeficiency virus type 1 resistance mutations in vivo after transmission of a multiply drug-resistant virus","volume":"37","author":"Gandhi","year":"2003","journal-title":"Clin. Infect. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4095","DOI":"10.3390\/v6104095","article-title":"Current perspectives on HIV-1 antiretroviral drug resistance","volume":"6","author":"Iyidogan","year":"2014","journal-title":"Viruses"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/j.antiviral.2009.09.015","article-title":"Clinical management of HIV-1 resistance","volume":"85","author":"Paredes","year":"2010","journal-title":"Antivir. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1016\/j.coviro.2012.07.004","article-title":"Human viral diseases: What is next for antiviral drug discovery?","volume":"2","author":"Clercq","year":"2012","journal-title":"Curr. Opin. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e620","DOI":"10.1016\/S2352-3018(20)30172-7","article-title":"Virological failure, HIV-1 drug resistance, and early mortality in adults admitted to hospital in Malawi: An observational cohort study","volume":"7","author":"Fielding","year":"2020","journal-title":"Lancet HIV"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1016\/j.idc.2019.05.004","article-title":"Management of Virologic Failure and HIV Drug Resistance","volume":"33","author":"McCluskey","year":"2019","journal-title":"Infect. Dis. Clin. N. Am."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5463","DOI":"10.1073\/pnas.74.12.5463","article-title":"DNA sequencing with chain-terminating inhibitors","volume":"74","author":"Sanger","year":"1977","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1195","DOI":"10.1101\/gr.6468307","article-title":"Characterization of mutation spectra with ultra-deep pyrosequencing: Application to HIV-1 drug resistance","volume":"17","author":"Wang","year":"2007","journal-title":"Genome Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2417","DOI":"10.1001\/jama.283.18.2417","article-title":"Antiretroviral drug resistance testing in adult HIV-1 infection: Recommendations of an International AIDS Society-USA Panel","volume":"283","author":"Hirsch","year":"2000","journal-title":"JAMA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2167","DOI":"10.1128\/AAC.02710-13","article-title":"Sensitive deep-sequencing-based HIV-1 genotyping assay to simultaneously determine susceptibility to protease, reverse transcriptase, integrase, and maturation inhibitors, as well as HIV-1 coreceptor tropism","volume":"58","author":"Gibson","year":"2014","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2823","DOI":"10.1093\/jac\/dkx232","article-title":"RNA and DNA Sanger sequencing versus next-generation sequencing for HIV-1 drug resistance testing in treatment-naive patients","volume":"72","author":"Alidjinou","year":"2017","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1086\/596736","article-title":"Low-abundance drug-resistant viral variants in chronically HIV-infected, antiretroviral treatment-naive patients significantly impact treatment outcomes","volume":"199","author":"Simen","year":"2009","journal-title":"J. Infect. Dis."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1323","DOI":"10.1089\/aid.2010.0123","article-title":"HIV-1 integrase sequence variability in antiretroviral na\u00efve patients and in triple-class experienced patients subsequently treated with raltegravir","volume":"26","author":"Varghese","year":"2010","journal-title":"AIDS Res. Hum. Retrovir."},{"key":"ref_27","first-page":"111","article-title":"2019 update of the drug resistance mutations in HIV-1","volume":"27","author":"Wensing","year":"2019","journal-title":"Top. Antivir. Med."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"930","DOI":"10.1093\/jac\/dku426","article-title":"Low-frequency drug-resistant HIV-1 and risk of virological failure to first-line NNRTI-based ART: A multicohort European case-control study using centralized ultrasensitive 454 pyrosequencing","volume":"70","author":"Giallonardo","year":"2015","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.jcv.2014.10.020","article-title":"Minority resistant HIV-1 variants and the response to first-line NNRTI therapy","volume":"62","author":"Nicot","year":"2015","journal-title":"J. Clin. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"S847","DOI":"10.1093\/infdis\/jix430","article-title":"The Role of HIV-1 Drug-Resistant Minority Variants in Treatment Failure","volume":"216","author":"Arribas","year":"2017","journal-title":"J. Infect. Dis."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1667","DOI":"10.1093\/cid\/cit125","article-title":"Clinical implications of HIV-1 minority variants","volume":"56","author":"Li","year":"2013","journal-title":"Clin. Infect. Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e553","DOI":"10.1016\/S2352-3018(16)30151-5","article-title":"Next-generation sequencing and HIV drug resistance surveillance","volume":"3","author":"Hamers","year":"2016","journal-title":"Lancet HIV"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Li, M., Liang, S., Zhou, C., Chen, M., Liang, S., Liu, C., Zuo, Z., Liu, L., Feng, Y., and Song, C. (2021). HIV Drug Resistance Mutations Detection by Next-Generation Sequencing during Antiretroviral Therapy Interruption in China. Pathogens, 10.","DOI":"10.3390\/pathogens10030264"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"S829","DOI":"10.1093\/infdis\/jix397","article-title":"Next-Generation Human Immunodeficiency Virus Sequencing for Patient Management and Drug Resistance Surveillance","volume":"216","author":"Edgil","year":"2017","journal-title":"J. Infect. Dis."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1086\/655397","article-title":"Minority variants of drug-resistant HIV","volume":"202","author":"Gianella","year":"2010","journal-title":"J. Infect. Dis."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1002\/jmv.23202","article-title":"An in-house HIV genotyping assay for the detection of drug resistance mutations in Southeast Asian patients infected with HIV-1","volume":"84","author":"Lee","year":"2012","journal-title":"J. Med. Virol."},{"key":"ref_37","unstructured":"(2022, August 20). RECall (v2.30)\u2013Web Based Sequence Analysis. Available online: https:\/\/recall.bccfe.ca\/account\/login\/."},{"key":"ref_38","unstructured":"(2022, August 20). HIVdb Program: Sequence Analysis. Available online: https:\/\/hivdb.stanford.edu\/hivdb\/by-sequences\/."},{"key":"ref_39","unstructured":"(2022, August 20). HIVdb Program: Mutations Analysis. Available online: https:\/\/hivdb.stanford.edu\/hivdb\/by-patterns\/."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"lqab019","DOI":"10.1093\/nargab\/lqab019","article-title":"Sequencing error profiles of Illumina sequencing instruments","volume":"3","author":"Stoler","year":"2021","journal-title":"NAR Genom. Bioinform."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1086\/595703","article-title":"Minority quasispecies of drug-resistant HIV-1 that lead to early therapy failure in treatment-naive and -adherent patients","volume":"48","author":"Metzner","year":"2009","journal-title":"Clin. Infect. Dis."},{"key":"ref_42","first-page":"672","article-title":"Low frequency nonnucleoside reverse-transcriptase inhibitor-resistant variants contribute to failure of efavirenz-containing regimens in treatment- experienced patients","volume":"201","author":"Halvas","year":"2010","journal-title":"J. Infect. Dis."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Delobel, P., Saliou, A., Nicot, F., Dubois, M., Trancart, S., Tangre, P., Aboulker, J., Taburet, A., Molina, J., and Massip, P. (2011). Minor HIV-1 variants with the K103N resistance mutation during intermittent efavirenz-containing antiretroviral therapy and virological failure. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0021655"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"893","DOI":"10.1093\/infdis\/jis925","article-title":"Impact of minority nonnucleoside reverse transcriptase inhibitor resistance mutations on resistance genotype after virologic failure","volume":"207","author":"Li","year":"2013","journal-title":"J. Infect. Dis."},{"key":"ref_45","first-page":"662","article-title":"Pre-existing minority drug-resistant HIV-1 variants, adherence, and risk of antiretroviral treatment failure","volume":"201","author":"Paredes","year":"2010","journal-title":"J. Infect. Dis."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"104376","DOI":"10.1016\/j.jcv.2020.104376","article-title":"Evaluation of Vela Diagnostics HIV-1 genotyping assay on an automated next generation sequencing platform","volume":"127","author":"May","year":"2020","journal-title":"J. Clin. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Parkin, N.T., Avila-Rios, S., Bibby, D.F., Brumme, C.J., Eshleman, S.H., Harrigan, P.R., Howison, M., Hunt, G., Ji, H., and Kantor, R. (2020). Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping. Viruses, 12.","DOI":"10.3390\/v12070694"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1186\/s12981-021-00390-8","article-title":"Next generation sequencing based in-house HIV genotyping method: Validation report","volume":"18","author":"Ayitewala","year":"2021","journal-title":"AIDS Res. Ther."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Bonifacio, M.A., Genchi, C., Lagioia, A., Talamo, V., Volpe, A., and Mariggi\u00f2, M.A. (2022). Analytical Assessment of the Vela Diagnostics NGS Assay for HIV Genotyping and Resistance Testing: The Apulian Experience. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23052727"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/10\/2208\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:47:50Z","timestamp":1760143670000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/10\/2208"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,10,7]]},"references-count":49,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2022,10]]}},"alternative-id":["v14102208"],"URL":"https:\/\/doi.org\/10.3390\/v14102208","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,10,7]]}}}