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Nicola Squillace,1 Elena Ricci,2 Barbara Menzaghi,3 Giuseppe Vittorio De Socio,4 Simone Passerini,5 Canio Martinelli,6 Maria Sabrina Mameli,7 Paolo Maggi,8 Katia Falasca,9 Laura Cordier,5 Benedetto Maurizio Celesia,10 Elena Salomoni,11 Antonio Di Biagio,12 Giovanni Francesco Pellicanò,13 Paolo Bonfanti1 On behalf of the CISAI Study Group1Infectious Diseases Unit ASST-MONZA, San Gerardo Hospital-University of Milano-Bicocca, Monza, Italy; 2“ASIA” Foundation ONLUS, Milan, Italy; 3Unit of Infectious Diseases, ASST della Valle Olona, Busto Arsizio, Italy; 4Department of Internal Medicine 2, Infectious Diseases Unit, “Santa Maria della Misericordia” General Hospital, Perugia, Italy; 5 1st Department of Infectious Diseases, ASST Fatebenefratelli Sacco, Milan, Italy; 6Infectious Diseases Unit, Careggi Hospital, Florence, Italy; 7Unit of Infectious Diseases, Department of Medical, Surgical and Experimental Sciences, University of Sassari, Sassari, Italy; 8University of Campania “Luigi Vanvitelli”, Napoli, Italy; 9Clinic of Infectious Diseases, Department of Medicine and Science of Aging, University “G. d’Annunzio” Chieti-Pescara, Chieti, Italy; 10Unit of Infectious Diseases, University of Catania, ARNAS Garibaldi, Catania, Italy; 11Infectious Diseases Unit, Santa Maria Annunziata Hospital, Usl centro, Florence, Italy; 12Infectious Diseases, San Martino Hospital Genoa, Genoa, Italy; 13Department of Human Pathology of the Adult and the Developmental Age “G. Barresi”, Unit of Infectious Diseases, University of Messina, Messina, ItalyCorrespondence: Nicola SquillaceInfectious Diseases Unit, Azienda Socio Sanitaria Territoriale di MONZA, San Gerardo Hospital-University of Milano-Bicocca, Via Pergolesi 33, Monza 20900, ItalyTel +390392339588Fax +390392339327Email nicolasquillace74@gmail.comObjective: We aimed to investigate the effect of switching from tenofovir disoproxil fumarate (TDF) to tenofovir alafenamide (TAF) on the hepatic safety and metabolic profile.Methods: Consecutive HIV patients, enrolled in the Surveillance Cohort Long-term Toxicity Antiretrovirals/Antivirals (SCOLTA) project, switching from TDF to TAF were included. Changes from baseline (T0) to 6-month follow-up (T1) were evaluated using paired t-test and signed rank test.Results: A total of 190 patients switched from TDF to TAF and had one 6-month follow-up visit. They were 80% male, 74.2% at CDC stage A–B, 93.7% with undetectable HIV-viral load. Mean age was 46.7± 10.7 years, body mass index was 25.0± 3.9 kg/m2, median CD4 cell count was 634 cell/μL (interquartile range [IQR]=439– 900), aspartate aminotransferase (AST) was 23 (IQR=19– 30) IU/L, and alanine aminotransferase (ALT) was 24 (IQR=17– 34) IU/L. At T1, both AST (median=− 1, IQR=− 5– 2 IU/L, P=0.004) and ALT (median=− 2, IQR=− 7– 3 IU/L, P=0.0004) showed a significant decrease. Among 28 patients with ALT > 40 at baseline, reduction was significant both clinically (− 17, IQR=− 32–− 1) and statistically (P=0.0003). Total cholesterol levels (TC) increased (+13.4± 3.8 mg/dL, P=0.0006), as well as HDL-cholesterol (HDL-C) (+3.8± 1.2 mg/dL, P=0.02), LDL Cholesterol (LDL-C) (+7.6± 3.4, P=0.03) and glucose (+4.0± 1.8 mg/dL, P=0.02). D:A:D: and Framingham risk score did not change at 6 months after switch.Conclusion: A significant reduction of liver enzymes was observed after switching from TDF to TAF, especially in subjects with initial level of ALT > 40 IU/L. Glucose, TC, HDL-C, and LDL-C increased, with no effect on cardiovascular risk scores.Keywords: HIV, TDF, TAF, liver enzymes

Paolo Bonfanti,1 Andrea De Vito,2 Elena Ricci,3 Barbara Menzaghi,4 Giancarlo Orofino,5 Nicola Squillace,1 Chiara Molteni,6 Giuseppe Vittorio De Socio,7 Elena Salomoni,8 Benedetto Maurizio Celesia,9 Chiara Dentone,10 Valeria Colombo,11 Giordano Madeddu2 1Infectious Diseases Unit ASST-MONZA, San Gerardo Hospital-University of Milano-Bicocca, Milan, Italy; 2Unit of Infectious Diseases, Department of Medical, Surgical and Experimental Sciences, University of Sassari, Sassari, Italy; 3Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 4Unit of Infectious Diseases, ASST della Valle Olona – Busto Arsizio (VA), Busto Arsizio, Italy; 5Division I of Infectious and Tropical Diseases, ASL Città di Torino, Turin, Italy; 6Unit of Infectious Diseases, A. Manzoni Hospital, Lecco, Italy; 7Department of Internal Medicine 2, Infectious Diseases Unit, Perugia “Santa Maria della Misericordia” General Hospital, Perugia, Italy; 8Infectious Diseases Unit 1, Santa Maria Annunziata Hospital, Azienda USL Toscana Centro, Florence, Italy; 9Infectious Diseases University of Catania ARNAS Garibaldi Catania, Catania, Italy; 10Division of Infectious Diseases, Department of Health Sciences, IRCCS Ospedale Policlinico San Martino, Genova, Italy; 11Infectious Disease Unit, DIBC “Luigi Sacco”, University of Milan, Milan, ItalyCorrespondence: Giordano MadedduUnit of Infectious Diseases, Department of Medical, Surgical and Experimental Sciences, University of Sassari, Viale San Pietro 43, Sassari 07100, ItalyTel +39 3403781734Fax +39 079217620Email giordano@uniss.itObjective: Few data exist about the effect of dolutegravir (DTG) on bone mineral density (BMD) in real life. The aim of this study was to determine rates of change in BMD over time in people living with HIV (PLWH) treated with DTG.Design: The SCOLTA project is a multicenter observational study enrolling HIV-infected people who start newly commercialized drugs prospectively, with the aim of identifying toxicities and adverse events (AE) in a real-life setting.Methods: Dual-energy X-ray absorptiometry at the femoral neck (FN) and lumbar spine (LS) was performed at study entry (baseline, BL) and after 96 weeks. Percentage BMD change from BL was evaluated using a general linear model, including factors potentially associated with bone loss.Results: One hundred and sixty PLWH were enrolled (26.3% female, mean age 49.9 ± 11.2 years) from April 2015 to April 2017. Overall, we could calculate BMD change from baseline, for at least one site, in 133 subjects (83.1%). After a median of 102 weeks (IQR: 90– 110), mean FN BMD increased, but not significantly, whereas LS BMD showed a significant mean increase of 13.1 (95% confidence interval, CI: 1.7– 24.6) mg/cm3 (+1.6%, 95% CI: 0.3%, 2.8%) after a median time of 102 weeks (IQR: 84– 110). As regards LS BMD, patients with osteopenia/osteoporosis at study entry experienced a high increase from baseline (20.6, 95% CI: 3.1, 38.1 mg/cm3), as well as experienced subjects (16.9, 95% CI: 4.7, 29.2 mg/cm3) and those on vitamin D supplementation (26.8, 95% CI: 7.7, 45.9 mg/cm3).Conclusion: Dolutegravir-containing regimens could reduce the negative impact of antiretroviral therapy on bone, especially in patients with low BMD.Keywords: HIV infection, dolutegravir, bone mineral density, real-life setting, adverse events, DXA scan

Paola Bagella,1 Nicola Squillace,2 Elena Ricci,3 Roberto Gulminetti,4 Giuseppe Vittorio De Socio,5 Lucia Taramasso,6 Giovanni Pellicanò,7 Barbara Menzaghi,8 Benedetto Maurizio Celesia,9 Chiara Dentone,10 Giancarlo Orofino,11 Paolo Bonfanti,12 Giordano Madeddu13On behalf of the C.I.S.A.I. Study Group, Italy1Unit of Post-acute Long Term Care, ATS Sardegna, Sassari, Italy; 2Infectious Diseases Unit, San Gerardo Hospital, University of Milano-Bicocca, Monza, Italy; 3Department of Infectious Diseases, ASST Fatebenefratelli Sacco, Milan, Italy; 4Unit of Infectious Diseases, IRCCS San Matteo Hospital, Pavia, Italy; 5Infectious Diseases Unit Department of Medicine, Azienda Ospedaliero-Universitaria di Perugia, Santa Maria Hospital, Perugia, Italy; 6Infectious Diseases Unit, Department of Internal Medicine, Fondazione IRCCS Ca‘ Granda Ospedale Maggiore Policlinico, Milan, Italy; 7Department of Human Pathology of the Adult and the Developmental Age ‘G. Barresi‘, Unit of Infectious Diseases, University of Messina, Messina, Italy; 8Unit of Infectious Diseases, ASST della Valle Olona, Busto Arsizio, Italy; 9Unit of Infectious Diseases, Garibaldi Hospital, Catania, Italy; 10Unit of Infectious Diseases, Sanremo Hospital, Sanremo, Italy; 11Unit of Infectious Diseases, Amedeo di Savoia Hospital, Turin, Italy; 12Unit of Infectious Diseases, A. Manzoni Hospital, Lecco, Italy; 13Unit of Infectious Diseases, Department of Clinical, Surgical and Experimental Medicine, University of Sassari, Sassari, ItalyIntroduction: Metabolic disorders are common amongst HIV-infected patients. Data from real-life setting on the impact of DTG/ABC/3TC in virologically suppressed HIV-infected patients are scarce.Methods: We investigated the modification of metabolic profile including fasting glucose, lipid profile and markers of insulin resistance (IR) in experienced patients switching from a boosted protease inhibitors (bPI) or a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen to DTG/ABC/3TC in a prospective, observational, multicenter study.Results: We enrolled 131 HIV-infected patients, of whom 91 (69.5%) males, mean age was 50.5±10.6 years. CDC stage was A in 66 (50.4%) patients, of whom 91 (69.5%) had acquired HIV through sexual contacts. The previous regimen was bPI-based in 79 patients (60.3%) and NNRTI-based in 52 (39.7%). Patients switching from NNRTI showed a significant reduction at week 24 in total cholesterol (TC) and low-density lipoprotein cholesterol (LDL). Triglycerides/high-density lipoprotein cholesterol (TG/HDL) ratio, HDL, median TG and TG/HDL ratio did not show significant modification during follow-up times. Among patients switching from a bPI, we observed a significant reduction in TC and LDL at both follow-up times and a slight increase in HDL. Triglycerides/HDL ratio, median TG and TG/HDL ratio showed a decrease over time that became significant at weeks 24 and 48. Blood glucose levels did not significantly vary during the observation period in patients switching from both bPI and NNRTI-based regimens.Conclusion: Our data suggest an improvement in lipid profile and TG/HDL ratio in pretreated HIV-1-infected patients who switched to DTG/ABC/3TC over 48 weeks, especially in those previously receiving a bPI-based regimen.Keywords: HIV-1 infection, dolutegravir/abacavir/lamivudine, lipid profile

Paola Bagella,1 Giuseppe VL De Socio,2 Elena Ricci,3 Barbara Menzaghi,4 Canio Martinelli,5 Nicola Squillace,6 Paolo Maggi,7 Giancarlo Orofino,8 Leonardo Calza,9 Laura Carenzi,3 Benedetto Maurizio Celesia,10 Giovanni Penco,11 Antonio Di Biagio,12 Laura Valsecchi,3 Francesca Vichi,13 Valeria Colombo,14 Giustino Parruti,15 Chiara Dentone,16 Katia Falasca,17 Paolo Bonfanti,18 Giordano Madeddu1 On behalf of the C.I.S.A.I. Study Group, Italy 1Unit of Infectious Diseases, Department of Medical, Surgical and Experimental Sciences, University of Sassari, Sassari, Italy; 2Infectious Diseases Unit, Department of Medicine, Azienda Ospedaliero-Universitaria di Perugia, Santa Maria Hospital, Perugia, Italy; 3Department of Infectious Diseases, ASST Fatebenefratelli Sacco, Milan, Italy; 4Unit of Infectious Diseases, ASST della Valle Olona, Busto Arsizio, Italy; 5Unit of Infectious and Tropical Diseases, Careggi Hospital Florence, Florence, Italy; 6Infectious Diseases Unit, San Gerardo Hospital, University of Milano-Bicocca, Monza, Italy; 7Infectious Diseases Clinic, University of Bari, Policlinico Consorziale, Bari, Italy; 8Amedeo di Savoia Hospital Unit of Infectious Diseases, Torino, Italy; 9University of Bologna, Department of Infectious Diseases, S. Orsola Malpighi Hospital, Bologna, Italy; 10Unit of Infectious Diseases, Garibaldi Hospital, Catania, Italy; 11Infectious Diseases Unit, Galliera Hospital, Genoa, Italy; 12Unit of Infectious Diseases, IRCCS San Martino Hospital – IST, Genoa, Italy; 13Infectious Diseases Unit, Santa Maria Annunziata Hospital, Florence, Italy; 14Infectious Diseases Unit, University of Milan, DIBIC Luigi Sacco, Milan, Italy; 15Unit of Infectious Diseases, Pescara Hospital, Pescara, Italy; 16Unit of Infectious Diseases, Sanremo Hospital, Sanremo, Italy; 17Infectious Diseases Unit, University of Chieti, Chieti, Italy; 18Unit of Infectious Diseases, A. Manzoni Hospital, Lecco, Italy Abstract: Rilpivirine is associated with a good efficacy and safety profile. However, data from real-life settings are scarce. Methods: We investigated the durability, safety and efficacy of Rilpivirine-based antiretroviral therapy in a prospective, observational, multicenter study. Results: We enrolled 499 HIV-infected patients, 360 (72.1%) males, mean age 43.4 ± 10.5 years, mean CD4 600 ± 327 cell/μL, mean HIV-RNA 3.80 ± 1.15 log10 cp/mL. After a median follow up of 16 months, 81 (16.2%) interruptions were reported, 36 (7.2%) of which for adverse events (16 of grade ≥3), most commonly neurological and gastrointestinal. We observed virological failures in only 8 (1.6%) patients. Naive patients showed a significant reduction in eGFR at week 24, 48 and 72 and in total cholesterol (TC)/HDL ratio at week 48 (p=0.007). In patients switching from PI we found a significant decrease at week 24 and 48 in TC and triglycerides at week 24, 48 and 72. eGFR showed a significant decrease at week 48 and 72. TC/HDL ratio showed a statistically significant decrease at week 24 (p=0.0008) and 72 (p=0.04). A significant increase at week 24 and 48 in AST and ALT values was observed. Patients switching from TDF/FTC/EFV showed a reduction in HDL, total cholesterol and triglycerides at week 24 and 48 and in eGFR at all follow up times. TC/HDL ratio showed a significant decrease at week 48 (p=0.01). CDC stage C and antiretroviral-experience (especially Protease Inhibitors) were associated with RPV discontinuation. Conclusion: In conclusion, our data confirm Rilpivirine efficacy, safety and tolerability with improvement in lipid profile. Although hepatic and renal events rarely caused discontinuation, liver and kidney parameters should be monitored. Keywords: HIV-1, combination antiretroviral therapy, NNRTI, cohort study, naive, experienced patients

Monica Tontodonati,1,2 Giovanni Cenderello,3 Benedetto Maurizio Celesia,4 Michele Trezzi,5 Tamara Ursini,2 Andrea Costantini,6 Domenico Marra,7 Ennio Polilli,8 Corrado Catalani,5 Luca Butini,6 Federica Sozio,8 Elena Mazzotta,9 Antonina Sciacca,8 Giuliano Rizzardini,10 Lamberto Manzoli,11 Alessandro Cozzi-Lepri,12 Giustino Parruti8 1Infectious Disease Unit, Pescara General Hospital, Pescara, 2Clinic of Infectious Diseases, G D'Annunzio University of Chieti-Pescara, Chieti, 3Division of Infectious Disease, Galliera General Hospital, Genoa, 4Infectious Diseases Unit, ARNAS Garibaldi, Catania, 5Infectious Diseases Unit, Pistoia General Hospital, Pistoia, 6Clinical Immunology Unit, Ancona Hospital, Ancona, 7Division of Oncology, Galliera General Hospital, Genoa, 8Infectious Diseases Unit, Pescara General Hospital, Pescara, 9Internal Medicine Department, G D'Annunzio University of Chieti-Pescara, Chieti, 10First Infectious Diseases Department, Luigi Sacco Hospital, Milan, 11Section of Hygiene, Epidemiology, Pharmacology and Legal Medicine, G D'Annunzio University of Chieti-Pescara, Chieti, Italy; 12Research Department of Infection and Population Health, University College London, London, UK Background: As HIV infection turned into a chronic treatable disease, now ranking as one of the most costly in medicine, long-term sustainability of highly active antiretroviral treatment (HAART) expenses became a major issue, especially in countries with universal access to care. Identification of determinants of higher HAART costs may therefore help in controlling costs of care, while keeping high levels of retention in care and viral suppression. Methods: With this aim, we enrolled a large multicentric sample of consecutive unselected human immunodeficiency virus (HIV) patients followed at five sites of care in Italy, and evaluated annual individual HAART costs in relation to a number of sociodemographic, clinical, and laboratory variables. Results: We enrolled 2,044 patients, including 1,902 on HAART. Mean HAART costs were €9,377±€3,501 (range 782–29,852) per year, with remarkable site-based differences, possibly related to the different composition of local assisted populations. Percentages of patients on viral suppression were homogeneously high across all study sites. The factors identified by cross-validation were line of HAART, diagnosis of acquired immune deficiency syndrome, current CD4 T-cell count, and detectable HIV viremia >50 copies/mL. In the final multivariable model, HAART costs were independently directly associated with more advanced HAART line (P

Objectives: The aim of this study was to evaluate the factors that can influence an incomplete viral response (IVR) after acute and early HIV infection (AEHI). Methods: This was a retrospective, observational study including patients with AEHI (Fiebig stages I–V) diagnosed between January 2008 and December 2014 at 20 Italian centres. IVR was defined by: (1) viral blip (51–1000 HIV‐1 RNA copies/mL after achievement of < 50 HIV‐1 RNA copies/mL); (2) virologic failure [> 1000 copies/mL after achievement of < 200 copies/mL, or ≥ 200 copies/mL after 24 weeks on an antiretroviral therapy (ART)]; (3) suboptimal viral response (> 50 copies/mL after 48 weeks on ART or two consecutive HIV‐1 RNA levels with ascending trend during ART). Cox regression analysis was used to calculate the hazard ratios (HRs) and 95% confidence intervals (95% CIs) for IVR. Results: In all, 263 patients were studied, 227 (86%) males, with a median [interquartile range (IQR)] age of 38 (30–46) years. During a median follow‐up of 13.0 (5.7–31.1) months, 38 (14.4%) had IVR. The presence of central nervous system (CNS) symptoms was linked to a higher risk of IVR (HR = 4.70, 95% CI: 1.56–14.17), while a higher CD4/CD8 cell count ratio (HR = 0.13, 95% CI: 0.03–0.51 for each point increase) and first‐line ART with three‐drug regimens recommended by current guidelines (HR = 0.40, 95% CI: 0.18–0.91 compared with other regimens including four or five drugs, older drugs or non‐standard backbones) were protective against IVR. Conclusions: Patients with lower CD4/CD8 ratio and CNS symptoms could be at a higher risk of IVR after AEHI. The use of recommended ART may be relevant for improving short‐term viral efficacy in this group of patients. [ABSTRACT FROM AUTHOR]

Background/Objectives : Out of 39.9 million adults living with HIV in 2022, 20 million were women. Despite bearing a significant burden, women remain underrepresented in clinical trials, including those for antiretroviral treatments (ART). This study evaluates the safety and efficacy of the bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) regimen in a real-life cohort of 99 women with HIV (females with HIV, FWH) over 48 and 96 weeks. Methods : A retrospective cohort study utilized data from the Sardinian HIV Network and Sicilian HIV Cohort (SHiNe-SHiC) research group. The study included FWH, who started B/F/TAF as a treatment switch. The primary objectives were achieving and maintaining an HIV RNA level of <50 copies/mL at 48 and 96 weeks. Secondary objectives included treatment safety, durability, and reasons for discontinuation. Data on demographics, viro-immunological markers, lipid profiles, and treatment interruptions were extracted for analysis. Results : Among the 99 FWH, the median age was 51.9 years, and the median duration of HIV was 15.1 years. At baseline, 80.8% had undetectable HIV-RNA, which increased to 93.8% at 96 weeks. There was a statistically significant increase in CD4 cells/mL (48w p < 0.001, 96w p < 0.001) and CD4/CD8 ratio (48w p < 0.009, 96w p < 0.048), and reductions in total cholesterol (48w p < 0.003, 96w p < 0.006) and LDL (48w p < 0.004, 96w p < 0.009) levels at 48 and 96 weeks. Nine treatment interruptions were noted, with one due to adverse events. The regimen was well-tolerated overall. Conclusions : B/F/TAF demonstrated high efficacy and safety in this real-world cohort of FWH, highlighting the critical need for gender-focused research in HIV treatment. Ensuring equitable access to effective treatment options for women is imperative for the global health community's efforts to eliminate HIV.

Bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF), as a fixed dosed combination, is effective in people living with human immunodeficiency virus (PLWH) previously treated with other therapeutic regimens. The aim of the present retrospective observational real-life study was to analyze virological suppression and immunological, metabolic and safety profile of B/F/TAF. Data were collected from 127 PLHW who switched from any regimen to B/F/TAF. Viral load and virological suppression (viral load <50 copies/ml) were assessed by using real-time PCR methodologies; CD4 and CD8 T cell count as well as CD4/CD8 ratio were determined by cytofluorimetric analyses; other metabolic parameters such as total cholesterol, triglycerides, High- and Low-Density Lipoproteins were assessed by using immunoenzymatic assay. All of the aforementioned parameters were assessed at different timepoints (Baseline, 48 and 96 weeks) for the patients switching to B/F/TAF. Of 127 PLHW [96 (75.6%) male and 31 (24.4%) female, with a mean age of 46.8±10.7 years], 107 PLHW were included in the analysis. The percentage of virologically suppressed PLWH increased from 66.4 to 74.8% at 96 weeks. A statistically significant increase in absolute CD4 (P<0.0001) and CD8 T cell count (P=0.002) was observed. Of importance, there was a significant increase in CD4/CD8 ratio from 0.95 (0.52-1.31) to 1.16 (0.75-1.39) (P=0.003) after 96 weeks. There was a significant decrease in the median values of triglycerides (P<0.0001) and total cholesterol (P<0.0001). Serum creatinine showed a significant increase (P=0.0001). In real life, switching to B/F/T was safe and highly effective both virologically and immunologically. Decrease in cholesterol and triglyceride levels suggested a favorable metabolic profile, which may decrease inflammation, leading to a healthier state and less organ damage., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Spampinato et al.)

Cervical cancer (CC) remains a prevalent malignancy and a significant global public health concern, primarily driven by persistent human papillomavirus (HPV) infections. The infectious nature of HPV underscores the preventability of CC through vaccination and screening programs. In addition to HPV, factors such as age, parity, smoking, hormonal contraceptives, and HIV co-infection elevate the risk of CC. HIV-associated immunodeficiency exacerbates susceptibility to infections and cancers, making CC a defining condition for acquired immune deficiency syndrome (AIDS) and one of the most commonly diagnosed cancers among women living with HIV (WLWH). These women face higher risks of HPV exposure due to sexual behavior and often encounter economic, social, and psychological barriers to screening. HIV and HPV co-infection can potentially accelerate CC carcinogenesis, with WLWH typically being diagnosed with CC earlier than their HIV-negative counterparts. Antiretroviral therapy (ART), which reduces AIDS-related mortality, also lowers the risk of invasive CC. The interaction between HIV and HPV is intricate and bidirectional. This summary reviews current evidence on HPV infection and CC in WLWH, highlighting the connections across pathogenesis, prevention, diagnosis, and treatment.

Background: To clarify whether next-generation sequencing (NGS) can be useful for resistance assessment in virologically suppressed highly treatment-experienced (HTE) individuals with MDR HIV., Methods: Ninety-one participants from the PRESTIGIO Registry were included. NGS was performed on HIV-DNA at 1%, 5% and 20% cut-offs; major drug resistance mutations (DRMs) were evaluated and compared with those detected in historical plasma genotypic resistance testing (h-GRT). APOBEC editing was also characterized., Results: Participants had a complex and long treatment history [median 23 (IQR 21-25) years of ART exposure) and had been virologically suppressed since a median of 3 (IQR 2-5) years. Among all major DRMs detected by HIV-DNA NGS and/or h-GRT, 30% were exclusively found through NGS. The highest detection rate of historical major DRMs was reached with NGS set at 1%, but unusual substitutions and extensive APOBEC hypermutations suggest technical issues and poor clinical relevance in the 1%-5% interval. At NGS set at 5%, 67.2% of historical major DRMs were detected. The number of major DRMs detected exclusively by DNA-NGS as minority variants (frequency 5%-20%) was significantly higher in individuals who later experienced virological rebound compared with those who maintained virological control [median 2 (IQR 1-3) versus 1 (0-2), P = 0.030] and positively correlated with viraemia levels at rebound (rho = 0.474, P = 0.030)., Conclusions: In non-viraemic people with an MDR virus, HIV-1 DNA NGS set at 5% is an acceptable technical cut-off that might help to reveal mutations with a potential clinical relevance. Moreover, the number of minority resistance mutations additionally detected by NGS might be associated with loss of virological control., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)