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Proviral DNA sequencing may provide access to archived viral variants and improve understanding of long-term resistance and clinical evolution. In this retrospective longitudinal study, 27 individuals with HIV-2, both ART-experienced and ART-na\u00efve, followed at a hospital in Lisbon, were analyzed. The HIV-2 protease gene was amplified from peripheral blood mononuclear cell-derived proviral DNA, cloned, and sequenced (Sanger sequencing) at baseline and, for ART-treated participants, after eight years of follow-up. Resistance profiles were interpreted using the Stanford HIVdb, HIV-2EU, and Rega algorithms. Clinical data, including ART history, CD4 counts, and plasma viral load, were collected longitudinally. Amino acid diversity was assessed using Shannon entropy, and longitudinal CD4 dynamics were evaluated using mixed-effects models with time-varying ART exposure. Sensitivity analyses were performed using generalized estimating equations (GEE). A total of 222 clonal HIV-2 protease sequences clustered within group A. Major PI resistance mutations were detected in 21.4% of ART-experienced and 23.1% of ART-na\u00efve individuals at baseline. Longitudinal resistance trajectories varied across participants, including persistence, apparent emergence, and non-detection of previously identified mutations. Mixed-effects modeling revealed substantial inter-individual variability in CD4 trajectories, with no statistically significant associations observed between CD4 evolution and ART status, time, or their interaction. GEE analyses yielded consistent results, supporting robustness across modeling frameworks. Entropy analysis identified localized sequence diversity changes restricted to a small number of protease residues, with positions 60 and 75 differing between groups at baseline and position 21 showing longitudinal variation among treated participants. This study demonstrates that proviral DNA sequencing captures archived HIV-2 protease diversity and reveals persistent and dynamic resistance patterns within the viral reservoir. While no population-level association between ART exposure and CD4 trajectory was observed, marked inter-individual variability highlights the complexity of longitudinal immune recovery in HIV-2 infection. These findings support the value of proviral sequencing as a complementary research tool for characterizing long-term viral evolution in settings where plasma-based genotyping is limited.<\/jats:p>","DOI":"10.3390\/ijms27125183","type":"journal-article","created":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T10:04:14Z","timestamp":1780913054000},"page":"5183","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Longitudinal Analysis of HIV-2 Proviral DNA Reveals Archived Protease Inhibitor Resistance and Reservoir Evolution over Eight Years"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1009-6715","authenticated-orcid":false,"given":"Paloma","family":"Gon\u00e7alves","sequence":"first","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"In\u00eas","family":"Lopes","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreia","family":"Martins","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Filipa","family":"Maia","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9599-7805","authenticated-orcid":false,"given":"Francisco","family":"Martin","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pedro","family":"Borrego","sequence":"additional","affiliation":[{"name":"Centro de Administra\u00e7\u00e3o e Pol\u00edticas P\u00fablicas (CAPP), Instituto Superior de Ci\u00eancias Sociais e Pol\u00edticas, Universidade de Lisboa, 1300-663 Lisbon, Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7932-1154","authenticated-orcid":false,"given":"Francisco","family":"Antunes","sequence":"additional","affiliation":[{"name":"Instituto de Sa\u00fade Ambiental, Faculdade de Medicina da Universidade de Lisboa and Laborat\u00f3rio 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AIDS","volume":"233","author":"Clavel","year":"1986","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1038\/s41586-019-1200-9","article-title":"Mapping HIV prevalence in sub-Saharan Africa between 2000 and 2017","volume":"570","author":"Cork","year":"2019","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"veab024","DOI":"10.1093\/ve\/veab024","article-title":"HIV-2 diversity displays two clades within group A with distinct geographical distribution and evolution","volume":"7","author":"Visseaux","year":"2021","journal-title":"Virus Evol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"889","DOI":"10.1099\/vir.0.038638-0","article-title":"Phylogeographical footprint of colonial history in the global dispersal of human immunodeficiency virus type 2 group A","volume":"93","author":"Faria","year":"2012","journal-title":"J. Gen. Virol."},{"key":"ref_5","unstructured":"(2024). Infe\u00e7\u00e3o por VIH em Portugal\u20142024, Dire\u00e7\u00e3o-Geral da Sa\u00fade. (In Portuguese)."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.meegid.2016.08.010","article-title":"Hiv-2 molecular epidemiology","volume":"46","author":"Visseaux","year":"2016","journal-title":"Infect. Genet. Evol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1002\/(SICI)1096-9071(200005)61:1<111::AID-JMV18>3.0.CO;2-W","article-title":"Human immunodeficiency virus type 2 (HIV-2) in Portugal: Clinical spectrum, circulating subtypes, virus isolation, and plasma viral load","volume":"61","author":"Soriano","year":"2000","journal-title":"J. Med. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1116","DOI":"10.1086\/315010","article-title":"Lower human immunodeficiency virus (HIV) type 2 viral load reflects the difference in pathogenicity of HIV-1 and HIV-2","volume":"180","author":"Popper","year":"1999","journal-title":"J. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Ceccarelli, G., Giovanetti, M., Sagnelli, C., Ciccozzi, A., d\u2019Ettorre, G., Angeletti, S., Borsetti, A., and Ciccozzi, M. (2021). Human Immunodeficiency Virus Type 2: The Neglected Threat. Pathogens, 10.","DOI":"10.3390\/pathogens10111377"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1097\/COH.0000000000000299","article-title":"Mortality and survival patterns of people living with HIV-2","volume":"11","author":"Tchounga","year":"2016","journal-title":"Curr. Opin. HIV AIDS"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e25","DOI":"10.1016\/S2352-3018(18)30254-6","article-title":"Long-term follow-up of HIV-2-related AIDS and mortality in Guinea-Bissau: A prospective open cohort study","volume":"6","author":"Esbjornsson","year":"2019","journal-title":"Lancet HIV"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Moranguinho, I., Taveira, N., and Bartolo, I. (2023). Antiretroviral Treatment of HIV-2 Infection: Available Drugs, Resistance Pathways, and Promising New Compounds. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24065905"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1290","DOI":"10.1093\/infdis\/jiad562","article-title":"The University of Washington\u2013Senegal HIV-2 Study Group. Antiviral Activity of Lenacapavir Against Human Immunodeficiency Virus Type 2 (HIV-2) Isolates and Drug-Resistant HIV-2 Mutants","volume":"229","author":"Smith","year":"2024","journal-title":"J. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1002\/cpt.3447","article-title":"Lenacapavir: Playing the Long Game in the New Era of Antiretrovirals","volume":"117","author":"Neverette","year":"2025","journal-title":"Clin. Pharmacol. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1323","DOI":"10.1086\/597802","article-title":"Antiretroviral drug resistance in HIV-2: Three amino acid changes are sufficient for classwide nucleoside analogue resistance","volume":"199","author":"Smith","year":"2009","journal-title":"J. Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"740","DOI":"10.1093\/cid\/ciad339","article-title":"Safety and Efficacy of Triple Therapy With Dolutegravir Plus 2 Nucleoside Reverse Transcriptase Inhibitors in Treatment-Naive Human Immunodeficiency Virus Type 2 Patients: Results from a 48-Week Phase 2 Study","volume":"77","author":"Pacheco","year":"2023","journal-title":"Clin. Infect. Dis."},{"key":"ref_17","first-page":"221","article-title":"Implementing HIV-1 genotypic resistance testing in antiretroviral therapy programs in Africa: Needs, opportunities, and challenges","volume":"15","author":"Lessells","year":"2013","journal-title":"AIDS Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/s12981-020-0259-6","article-title":"HIV treatment in Guinea-Bissau: Room for improvement and time for new treatment options","volume":"17","author":"Jespersen","year":"2020","journal-title":"AIDS Res. Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"931","DOI":"10.1186\/s13063-021-05902-5","article-title":"Resource and infrastructure challenges on the RESIST-2 Trial: An implementation study of drug resistance genotype-based algorithmic ART switches in HIV-2-infected adults in Senegal","volume":"22","author":"Raugi","year":"2021","journal-title":"Trials"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"117860","DOI":"10.1016\/j.bmc.2024.117860","article-title":"Current status of the small molecule anti-HIV drugs in the pipeline or recently approved","volume":"111","author":"Umumararungu","year":"2024","journal-title":"Bioorg. Med. Chem."},{"key":"ref_21","unstructured":"WHO (2026, March 01). HIV Drug Resistance Strategy, 2021 Update. Available online: https:\/\/www.who.int\/publications\/i\/item\/9789240030565."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e3404","DOI":"10.21769\/BioProtoc.3404","article-title":"Quantification of HIV-2 DNA in Whole Blood","volume":"9","author":"Szojka","year":"2019","journal-title":"Bio-protocol"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1128\/JCM.02303-20","article-title":"HIV-2 Drug Resistance Genotyping from Dried Blood Spots","volume":"59","author":"Raugi","year":"2020","journal-title":"J. Clin. Microbiol."},{"key":"ref_24","unstructured":"WHO (2020). WHO Manual for HIV Drug Resistance Testing Using Dried Blood Spot Specimens, WHO."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1178","DOI":"10.1089\/aid.2006.22.1178","article-title":"Natural polymorphisms of HIV type 2 pol sequences from drug-naive individuals","volume":"22","author":"Parreira","year":"2006","journal-title":"AIDS Res. Hum. Retroviruses"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"857","DOI":"10.1089\/aid.2008.0015","article-title":"HIV type 2 protease, reverse transcriptase, and envelope viral variation in the PBMC and genital tract of ARV-naive women in Senegal","volume":"24","author":"Gottlieb","year":"2008","journal-title":"AIDS Res. Hum. Retroviruses"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2751","DOI":"10.1128\/AAC.00405-13","article-title":"Complex patterns of protease inhibitor resistance among antiretroviral treatment-experienced HIV-2 patients from Senegal: Implications for second-line therapy","volume":"57","author":"Raugi","year":"2013","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1093\/cid\/ciaf110","article-title":"HIV-2EU\u2014Supporting Standardized HIV-2 Drug Resistance Interpretation: An Update","volume":"81","author":"Charpentier","year":"2025","journal-title":"Clin. Infect. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1093\/aje\/kwi268","article-title":"Individual variation in CD4 cell count trajectory among human immunodeficiency virus-infected men and women on long-term highly active antiretroviral therapy: An application using a Bayesian random change-point model","volume":"162","author":"Chu","year":"2005","journal-title":"Am. J. Epidemiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2869","DOI":"10.1093\/jac\/dkx210","article-title":"CD4 cell count response to first-line combination ART in HIV-2+ patients compared with HIV-1+ patients: A multinational, multicohort European study","volume":"72","author":"Wittkop","year":"2017","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1962","DOI":"10.1093\/infdis\/jiaa026","article-title":"Expanded Spectrum of Antiretroviral-Selected Mutations in Human Immunodeficiency Virus Type 2","volume":"221","author":"Tzou","year":"2020","journal-title":"J. Infect. Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1173","DOI":"10.1093\/jac\/dkx530","article-title":"Minority resistant variants are also present in HIV-2-infected antiretroviral-naive patients","volume":"73","author":"Storto","year":"2018","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"de Pina-Araujo, I.I.M., Guimaraes, M.L., Bello, G., Vicente, A.C., and Morgado, M.G. (2014). Profile of the HIV epidemic in Cape Verde: Molecular epidemiology and drug resistance mutations among HIV-1 and HIV-2 infected patients from distinct islands of the archipelago. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0096201"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1002\/jmv.20296","article-title":"Polymorphism and drug-selected mutations in the reverse transcriptase gene of HIV-2 from patients living in southeastern France","volume":"75","author":"Colson","year":"2005","journal-title":"J. Med. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1186\/1742-4690-7-98","article-title":"Polymorphisms of HIV-2 integrase and selection of resistance to raltegravir","volume":"7","author":"Bercoff","year":"2010","journal-title":"Retrovirology"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1093\/jac\/dks363","article-title":"Mutations selected in HIV-2-infected patients failing a regimen including atazanavir","volume":"68","author":"Aleixo","year":"2013","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"ofz034","DOI":"10.1093\/ofid\/ofz034","article-title":"Persistence of Human Immunodeficiency Virus-1 Drug Resistance Mutations in Proviral Deoxyribonucleic Acid After Virologic Failure of Efavirenz-Containing Antiretroviral Regimens","volume":"6","author":"Vardhanbhuti","year":"2019","journal-title":"Open Forum Infect. Dis."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1713","DOI":"10.1080\/21505594.2022.2129353","article-title":"High frequency of CD8 escape mutations in elite controllers as new obstacle for HIV cure","volume":"13","author":"Ramos","year":"2022","journal-title":"Virulence"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"101301","DOI":"10.1016\/j.coviro.2023.101301","article-title":"HIV persistence: Silence or resistance?","volume":"59","author":"Pasternak","year":"2023","journal-title":"Curr. Opin. Virol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Avila-Rios, 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_41","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_42","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1097\/COH.0000000000000737","article-title":"Technologies for HIV-1 drug resistance testing: Inventory and needs","volume":"17","author":"Metzner","year":"2022","journal-title":"Curr. Opin. HIV AIDS"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1634","DOI":"10.1038\/s41598-020-58544-z","article-title":"Performance comparison of next generation sequencing analysis pipelines for HIV-1 drug resistance testing","volume":"10","author":"Lee","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"115112","DOI":"10.1016\/j.jviromet.2025.115112","article-title":"Hybrid next-generation sequencing protocol for testing HIV-2 drug resistance","volume":"334","author":"Goncalves","year":"2025","journal-title":"J. Virol. Methods"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Ouyang, F., Yuan, D., Zhai, W., Liu, S., Zhou, Y., and Yang, H. (2024). HIV-1 Drug Resistance Detected by Next-Generation Sequencing among ART-Naive Individuals: A Systematic Review and Meta-Analysis. Viruses, 16.","DOI":"10.3390\/v16020239"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1186\/s12977-022-00601-8","article-title":"Defective HIV-1 genomes and their potential impact on HIV pathogenesis","volume":"19","author":"Kuniholm","year":"2022","journal-title":"Retrovirology"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1093\/jac\/dkt326","article-title":"Defective HIV-1 quasispecies in the form of multiply drug-resistant proviral DNA within cells can be rescued by superinfection with different subtype variants of HIV-1 and by HIV-2 and SIV","volume":"69","author":"Quan","year":"2014","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"105261","DOI":"10.1016\/j.idnow.2026.105261","article-title":"The HIV-2 reservoir: Lessons from HIV-1 and knowledge gaps","volume":"56","author":"Melandri","year":"2026","journal-title":"Infect. Dis. Now."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e0005222","DOI":"10.1128\/cmr.00052-22","article-title":"Genotypic Resistance Testing of HIV-1 DNA in Peripheral Blood Mononuclear Cells","volume":"35","author":"Chu","year":"2022","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Liang, B., Luo, M., Scott-Herridge, J., Semeniuk, C., Mendoza, M., Capina, R., Sheardown, B., Ji, H., Kimani, J., and Ball, B.T. (2011). A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0026745"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"veaf099","DOI":"10.1093\/ve\/veaf099","article-title":"Single genome amplification and molecular cloning of HIV-1 populations in acute HIV-1 infection: Implications for studies on HIV-1 diversity and evolutionary rate","volume":"12","author":"Hsieh","year":"2026","journal-title":"Virus Evol."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Auld, A.F., Ekra, K.A., Shiraishi, R.W., Tuho, M.Z., Kouakou, J.S., Mohamed, F., Ettiegne-Traore, V., Sabatier, J., Essombo, J., and Adjorlolo-Johnson, G. (2014). Temporal trends in treatment outcomes for HIV-1 and HIV-2-infected adults enrolled in Cote d\u2019Ivoire\u2019s national antiretroviral therapy program. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0098183"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"2947","DOI":"10.1093\/bioinformatics\/btm404","article-title":"Clustal W and Clustal X version 2.0","volume":"23","author":"Larkin","year":"2007","journal-title":"Bioinformatics"},{"key":"ref_54","unstructured":"Nicholas, H.B., and Deerfield, D.W.I. (1997). GeneDoc: Analysis and visualization of genetic variation. Embnet News, CiNii."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"vbaf025","DOI":"10.1093\/bioadv\/vbaf025","article-title":"Hypermut 3: Identifying specific mutational patterns in a defined nucleotide context that allows multistate characters","volume":"5","author":"Lapp","year":"2025","journal-title":"Bioinform. Adv."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1093\/molbev\/msu300","article-title":"IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies","volume":"32","author":"Nguyen","year":"2015","journal-title":"Mol. Biol. Evol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1038\/nmeth.4285","article-title":"ModelFinder: Fast model selection for accurate phylogenetic estimates","volume":"14","author":"Kalyaanamoorthy","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"W293","DOI":"10.1093\/nar\/gkab301","article-title":"Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation","volume":"49","author":"Letunic","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1111\/j.2517-6161.1995.tb02031.x","article-title":"Controlling the false discovery rate: A practical and powerful approach to multiple testing","volume":"57","author":"Benjamini","year":"1995","journal-title":"J. R. Stat. Soc. Ser. B (Methodol.)"},{"key":"ref_60","unstructured":"R Core Team (2026). R: A Language and Environment for Statistical Computing, R Core Team."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"1","DOI":"10.18637\/jss.v067.i01","article-title":"Fitting Linear Mixed-Effects Models Using lme4","volume":"67","author":"Bates","year":"2015","journal-title":"J. Stat. Softw."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"1","DOI":"10.18637\/jss.v082.i13","article-title":"lmerTest Package: Tests in Linear Mixed Effects Models","volume":"82","author":"Kuznetsova","year":"2017","journal-title":"J. Stat. Softw."},{"key":"ref_63","unstructured":"Pinheiro, J., Bates, D., and R Core Team (2026, April 24). nlme: Linear and Nonlinear Mixed Effects Models. Available online: https:\/\/CRAN.R-project.org\/package=nlme."},{"key":"ref_64","doi-asserted-by":"crossref","unstructured":"Pinheiro, J.C., and Bates, D.M. (2000). 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