{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T14:20:18Z","timestamp":1784298018255,"version":"3.55.0"},"reference-count":30,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2024,9,6]],"date-time":"2024-09-06T00:00:00Z","timestamp":1725580800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Background: Next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). This work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting. Methods: Routine NGS-GRT data were generated from viral RNA at 11 Italian laboratories with the AD4SEQ HIV-1 Solution v2 commercial kit. NGS results were interpreted by the SmartVir system provided by the kit and by two online tools (HyDRA Web and Stanford HIVdb). NGS-GRT was considered valid when the coverage was &gt;100 reads (100\u00d7) at each PR\/RT\/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm. Results: Among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to SmartVir, HyDRA Web, and HIVdb. Considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. The proportion of valid samples was affected by viremia &lt;10,000\u20131000 copies\/mL and non-B subtypes. Mutations at an NGS frequency &gt;10% showed fair concordance among different interpretation tools. Conclusion: This Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. Within the current routine method for NGS-GRT, only mutations with frequency &gt;10% seem reliably detected across different interpretation tools.<\/jats:p>","DOI":"10.3390\/v16091422","type":"journal-article","created":{"date-parts":[[2024,9,6]],"date-time":"2024-09-06T05:02:28Z","timestamp":1725598948000},"page":"1422","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Comparison of Different HIV-1 Resistance Interpretation Tools for Next-Generation Sequencing in Italy"],"prefix":"10.3390","volume":"16","author":[{"given":"Daniele","family":"Armenia","sequence":"first","affiliation":[{"name":"Departmental Faculty, UniCamillus, Saint Camillus International University of Health Sciences, 00131 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1713-8682","authenticated-orcid":false,"given":"Luca","family":"Carioti","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine, University of Rome \u201cTor Vergata\u201d, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5629-3875","authenticated-orcid":false,"given":"Valeria","family":"Micheli","sequence":"additional","affiliation":[{"name":"Laboratory of Clinical Microbiology, Virology and Bioemergencies, ASST Fatebenefratelli Sacco-University of Milan, 20157 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabella","family":"Bon","sequence":"additional","affiliation":[{"name":"Microbiology Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, 40138 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tiziano","family":"Allice","sequence":"additional","affiliation":[{"name":"Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, 10149 Turin, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Celestino","family":"Bonura","sequence":"additional","affiliation":[{"name":"Dipartimento di Promozione della Salute, Materno-Infantile, di Medicina Interna e Specialistica di Eccellenza \u201cG. D\u2019Alessandro\u201d (PROSAMI), Azienda Ospedaliera Universitaria Policlinico \u201cP. Giaccone\u201d-University of Palermo, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bianca","family":"Bruzzone","sequence":"additional","affiliation":[{"name":"Hygiene Unit, Ospedale Policlinico San Martino, 16132 Genoa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fiorenza","family":"Bracchitta","sequence":"additional","affiliation":[{"name":"Laboratory of Clinical Microbiology, Virology and Bioemergencies, ASST Fatebenefratelli Sacco-University of Milan, 20157 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0480-8296","authenticated-orcid":false,"given":"Francesco","family":"Cerutti","sequence":"additional","affiliation":[{"name":"Laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, 10149 Turin, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7874-6289","authenticated-orcid":false,"given":"Giovanni Maurizio","family":"Giammanco","sequence":"additional","affiliation":[{"name":"Dipartimento di Promozione della Salute, Materno-Infantile, di Medicina Interna e Specialistica di Eccellenza \u201cG. D\u2019Alessandro\u201d (PROSAMI), Azienda Ospedaliera Universitaria Policlinico \u201cP. Giaccone\u201d-University of Palermo, 90127 Palermo, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Federica","family":"Stefanelli","sequence":"additional","affiliation":[{"name":"Hygiene Unit, Ospedale Policlinico San Martino, 16132 Genoa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9923-8267","authenticated-orcid":false,"given":"Maria Addolorata","family":"Bonifacio","sequence":"additional","affiliation":[{"name":"Section of Experimental and Clinical Pathology, Department of Precision and Regenerative Medicine and Jonic Area, University of Bari, 70121 Bari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ada","family":"Bertoli","sequence":"additional","affiliation":[{"name":"Virology Unit, Polyclinic of \u201cTor Vergata\u201d, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marialinda","family":"Vatteroni","sequence":"additional","affiliation":[{"name":"Virology Unit, AOU Pisana, 56126 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriele","family":"Ibba","sequence":"additional","affiliation":[{"name":"Microbiology and Virology Unit, Diagnostic Department, AOU Sassari, 07100 Sassari, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2741-8840","authenticated-orcid":false,"given":"Federica","family":"Novazzi","sequence":"additional","affiliation":[{"name":"Department of Medicine and Technological Innovation, University of Insubria, 21100 Varese, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Rosaria","family":"Lipsi","sequence":"additional","affiliation":[{"name":"Microbiology and Virology Unit, Policlinico Riuniti Foggia Hospital, 71121 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nunzia","family":"Cuomo","sequence":"additional","affiliation":[{"name":"U.O.C. Microbiologia e Virologia, P.O. \u201cD.Cotugno\u201d-AO dei Colli, 80100 Napoli, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4306-2960","authenticated-orcid":false,"given":"Ilaria","family":"Vicenti","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Ceccherini-Silberstein","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine, University of Rome \u201cTor Vergata\u201d, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1659-9241","authenticated-orcid":false,"given":"Barbara","family":"Rossetti","sequence":"additional","affiliation":[{"name":"Infectious Disease Department, USL SUDEST, Toscana, Misericordia Hospital, 58100 Grosseto, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9921-3684","authenticated-orcid":false,"given":"Antonia","family":"Bezenchek","sequence":"additional","affiliation":[{"name":"IPRO-InformaPRO S.r.l., 00152 Rome, Italy"},{"name":"EuResist Network GEIE, 00152 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9934-377X","authenticated-orcid":false,"given":"Francesco","family":"Saladini","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0344-6281","authenticated-orcid":false,"given":"Maurizio","family":"Zazzi","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6228-1114","authenticated-orcid":false,"given":"Maria Mercedes","family":"Santoro","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine, University of Rome \u201cTor Vergata\u201d, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,9,6]]},"reference":[{"key":"ref_1","unstructured":"European AIDS Clinical Society (2024, July 31). Guidelines\u2014Version 12.0. Available online: https:\/\/www.eacsociety.org\/media\/guidelines-12.0.pdf."},{"key":"ref_2","unstructured":"(2024, July 31). Panel on Antiretroviral Guidelines for Adults and Adolescents, Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents with HIV, Department of Health and Human Services, Available online: https:\/\/clinicalinfo.hiv.gov\/sites\/default\/files\/guidelines\/documents\/adult-adolescent-arv\/guidelines-adult-adolescent-arv.pdf."},{"key":"ref_3","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_4","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1016\/j.ijid.2020.02.004","article-title":"Shall I Trust the Report? Variable Performance of Sanger Sequencing Revealed by Deep Sequencing on HIV Drug Resistance Mutation Detection","volume":"93","author":"Chen","year":"2020","journal-title":"Int. J. Infect. Dis."},{"key":"ref_5","first-page":"226","article-title":"PRESTIGIO RING: \u201cA 59-year-old HIV-1 positive, highly treatment-experienced woman failing darunavir\/ritonavir plus raltegravir\u201d","volume":"46","author":"Labate","year":"2023","journal-title":"New Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1146\/annurev-genom-091212-153406","article-title":"Deep Sequencing of HIV: Clinical and Research Applications","volume":"15","author":"Chabria","year":"2014","journal-title":"Annu. Rev. Genom. Hum. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"\u00c1vila-R\u00edos, S., Parkin, N., Swanstrom, R., Paredes, R., Shafer, R., Ji, H., and Kantor, R. (2020). Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations. Viruses, 12.","DOI":"10.3390\/v12060617"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e00105-18","DOI":"10.1128\/JCM.00105-18","article-title":"Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing","volume":"56","author":"Tzou","year":"2018","journal-title":"J. Clin. Microbiol."},{"key":"ref_9","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_10","doi-asserted-by":"crossref","first-page":"1486","DOI":"10.1002\/jmv.25224","article-title":"Performance Comparison of Deep Sequencing Platforms for Detecting HIV-1 Variants in the Pol Gene","volume":"90","author":"Nicot","year":"2018","journal-title":"J. Med. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1186\/s12977-014-0122-8","article-title":"Cross-Clade Simultaneous HIV Drug Resistance Genotyping for Reverse Transcriptase, Protease, and Integrase Inhibitor Mutations by Illumina MiSeq","volume":"11","author":"Dudley","year":"2014","journal-title":"Retrovirology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.jviromet.2014.11.003","article-title":"Evaluation of GS Junior and MiSeq Next-Generation Sequencing Technologies as an Alternative to Trugene Population Sequencing in the Clinical HIV Laboratory","volume":"212","author":"Ram","year":"2015","journal-title":"J. Virol. Methods"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Taylor, T., Lee, E.R., Nykoluk, M., Enns, E., Liang, B., Capina, R., Gauthier, M.K., Van Domselaar, G., Sandstrom, P., and Brooks, J. (2019). A MiSeq-HyDRA Platform for Enhanced HIV Drug Resistance Genotyping and Surveillance. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-45328-3"},{"key":"ref_14","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"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.virusres.2016.12.008","article-title":"Promises and Pitfalls of Illumina Sequencing for HIV Resistance Genotyping","volume":"239","author":"Brumme","year":"2017","journal-title":"Virus Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.virusres.2016.10.019","article-title":"Deep Sequencing for HIV-1 Clinical Management","volume":"239","author":"Paredes","year":"2017","journal-title":"Virus Res."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Lee, E.R., Parkin, N., Jennings, C., Brumme, C.J., Enns, E., Casadell\u00e0, M., Howison, M., Coetzer, M., Avila-Rios, S., and Capina, R. (2020). Performance Comparison of next Generation Sequencing Analysis Pipelines for HIV-1 Drug Resistance Testing. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-58544-z"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Noguera-Julian, M., Lee, E.R., Shafer, R.W., Kantor, R., and Ji, H. (2020). Dry Panels Supporting External Quality Assessment Programs for Next Generation Sequencing-Based HIV Drug Resistance Testing. Viruses, 12.","DOI":"10.3390\/v12060666"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Tzou, P.L., Kosakovsky Pond, S.L., Avila-Rios, S., Holmes, S.P., Kantor, R., and Shafer, R.W. (2020). Analysis of Unusual and Signature APOBEC-Mutations in HIV-1 Pol Next-Generation Sequences. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0225352"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00253-22","DOI":"10.1128\/jcm.00253-22","article-title":"HIV-1 Drug Resistance Assay Using Ion Torrent Next Generation Sequencing and On-Instrument End-to-End Analysis Software","volume":"60","author":"Pyne","year":"2022","journal-title":"J. Clin. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1093\/jac\/dkac430","article-title":"Frequency Matters: Comparison of Drug Resistance Mutation Detection by Sanger and Next-Generation Sequencing in HIV-1","volume":"78","author":"Balakrishna","year":"2023","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"961","DOI":"10.1016\/j.jmoldx.2019.06.003","article-title":"Prospective Evaluation of the Vela Diagnostics Next-Generation Sequencing Platform for HIV-1 Genotypic Resistance Testing","volume":"21","author":"Weber","year":"2019","journal-title":"J. Mol. Diagn."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Mohamed, S., Boulm\u00e9, R., and Sayada, C. (2023). From Capillary Electrophoresis to Deep Sequencing: An Improved HIV-1 Drug Resistance Assessment Solution Using In Vitro Diagnostic (IVD) Assays and Software. Viruses, 15.","DOI":"10.3390\/v15020571"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1165","DOI":"10.1093\/cid\/ciu019","article-title":"Performance of HIV-1 drug resistance testing at low-level viremia and its ability to predict future virologic outcomes and viral evolution in treatment-naive individuals","volume":"58","author":"Min","year":"2014","journal-title":"Clin. Infect. Dis."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1156","DOI":"10.1093\/cid\/ciu020","article-title":"Reliability and clinical relevance of the HIV-1 drug resistance test in patients with low viremia levels","volume":"58","author":"Santoro","year":"2014","journal-title":"Clin. Infect. Dis."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1125","DOI":"10.1097\/QAD.0000000000000203","article-title":"HIV drug resistance detected during low-level viraemia is associated with subsequent virologic failure","volume":"28","author":"Swenson","year":"2014","journal-title":"AIDS"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1583","DOI":"10.1093\/infdis\/jiad327","article-title":"Geographic and Population Distributions of Human Immunodeficiency Virus (HIV)-1 and HIV-2 Circulating Subtypes: A Systematic Literature Review and Meta-Analysis (2010\u20132021)","volume":"228","author":"Williams","year":"2023","journal-title":"J. Infect. Dis."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Fabeni, L., Armenia, D., Abbate, I., Gagliardini, R., Mazzotta, V., Bertoli, A., Gennari, W., Forbici, F., Berno, G., and Piermatteo, L. (2024). HIV-1 Transmitted Drug Resistance in Newly Diagnosed Individuals in Italy over the Period 2015\u201321. J. Antimicrob. Chemother., dkae189.","DOI":"10.1093\/jac\/dkae189"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1097\/COH.0000000000000534","article-title":"HIV Subtype Diversity Worldwide","volume":"14","author":"Bbosa","year":"2019","journal-title":"Curr. Opin. HIV AIDS"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1007\/s11904-023-00674-w","article-title":"Learning from Persistent Viremia: Mechanisms and Implications for Clinical Care and HIV-1 Cure","volume":"20","author":"Wu","year":"2023","journal-title":"Curr. HIV\/AIDS Rep."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/9\/1422\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:49:37Z","timestamp":1760111377000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/9\/1422"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,9,6]]},"references-count":30,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2024,9]]}},"alternative-id":["v16091422"],"URL":"https:\/\/doi.org\/10.3390\/v16091422","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,9,6]]}}}