BACKGROUND:Monitoring HIV-1 subtype circulation and transmitted drug resistance (TDR) remains a key priority, particularly since the rollout of high-sensitivity next-generation sequencing (NGS). METHODS:Routine plasma HIV-1 RNA NGS genotyping data were collected from newly diagnosed individuals in Italy over 2022-24. HIV-1 TDR and genotypic susceptibility were evaluated through HIVdb with NGS set at 10% and 20%. Subtype and transmission clusters (TC) were determined through the maximum likelihood phylogeny based on the GTR + F + R9 model. RESULTS:Seven hundred and forty-two individuals were included, 51.9% harbouring non-B strains [CRF02_AG (18.1%); CRF BF (6.1%); A1/A3/A6 (7.1%); others (20.5%)]. TDR prevalence to any class was 11.7% at Sanger-like NGS-setting (>20%), slightly increased (15.0%) at 10% NGS-setting, and significantly varied across subtypes, with the highest prevalence observed in B subtype. Most antiretrovirals showed full genotypic activity in nearly 99% of individuals, except for efavirenz and rilpivirine (proportion of individuals with full activity <92%). A total of 57 TC were detected: 40 pairs, 17 clusters (>2 sequences). Thirteen TC (22.8%, 8 pairs, 5 clusters) involved individuals harbouring TDR. TDR was detected as minority mutations in five TC. CONCLUSIONS:A high proportion of HIV-1 non-B subtypes circulate in Italy. TDR prevalence is around 12% using NGS at Sanger-like threshold and moderately increases to 15% when NGS is set at 10%. However, the impact of the detected TDR on the susceptibility to currently used antiretrovirals in clinical practice is negligible.

BACKGROUND:Although COVID-19 is no longer a public health emergency, it remains the most prevalent circulating infectious-like-illness in Europe. Whether immunocompromising conditions (ICCs) still carry increased mortality risk during the Omicron era is unclear. METHODS:We conducted a cohort study across EuCARE sites in 8 countries among adults admitted to hospital with COVID-19 between 2020-2023. ICCs and COVID-19 pneumonia at hospitalization were defined using clinical information and ICD-10 codes. Logistic regression and counterfactual mediation analysis was used to compare 28-day in-hospital mortality risk associated with ICCs using COVID-19 pneumonia and vaccination at hospital entry as intermediates. Proportion of the total effect of ICCs mediated and the controlled direct effects (CDEs) were calculated. We also formally tested for interaction between SARS CoV-2 variants and ICCs for mortality risk. FINDINGS:42,488 individuals were included, of which 1,675 (3.9%) had an ICC. 55% were male, median (IQR) age was 67 (52, 79) years. Overall, 4,344 (10.2%) individuals died in hospital. ICCs were associated with increased mortality, OR = 1.49 (1.25, 1.79) with no evidence for an attenuation during the Omicron phase (p-interaction=0.60). Mediation analyses showed that the total effect of ICCs was mediated by vaccination but only weakly by pneumonia. With Omicron, the excess mortality associated with ICC was higher under the scenario that everyone in the cohort was to develop COVID-19 pneumonia [CDE =1.22 (0.09, 1.65)]. INTERPRETATION:ICC remains a significant risk factor for in-hospital death, even during the Omicron era, particularly if the infection led to the development of pneumonia.

OBJECTIVES:To estimate the prevalence of NRTI and integrase strand transfer inhibitor (INSTI), drug resistance mutations (DRMs) in antiretroviral therapy (ART)-experienced people with HIV-1 (PWH) switched to bictegravir/emtricitabine/tenofovir alafenamide fumarate (B/F/TAF) and among those with HIV-RNA ≤50 copies/mL to assess the association with risk of viral rebound (VR). METHODS:Data from the ARCA cohort were used to estimate resistance prevalence assuming a binomial distribution; 95% confidence intervals (CIs) were reported. Standard survival analysis with time-fixed covariate measured at B/F/TAF initiation was used to evaluate the association between detected resistance and risk of confirmed VR >50 copies/mL (2 consecutive values). FINDINGS:We included 1414 PWH (973-69%-in the VR analysis). Overall, 27% were female, median age was 53 years (IQR 44-59). Major NRTI-DRMs were detected in 25% (95% CI: 22.5, 27.1). Major INSTI-DRMs affecting bictegravir were rare (0.6%, 95% CI: 0.2, 1.4). The overall rate of VR by 36 months was 5.3% (95% CI: 3.7-6.9%), confirming that viral rebound on B/F/TAF is a rare event. After controlling for confounding, NRTI resistance was not associated with VR, whereas a history of INSTI virological failure (VF) and major INSTI-DRMs were associated with VR (aRH 2.68, 95% CI: 1.40, 5.12; and aRH 4.21, 95% CI: 1.18, 15.02, respectively). CONCLUSIONS:In our cohort of PWH who were switched to B/F/TAF, NRTI-DRMs were common, but B/F/TAF remained effective in maintaining viral suppression despite their presence. However, previous failure to INSTI-based regimens and major INSTI-DRMs were risk factors for VR.

Background:HIV-1 sub-subtype A6 is predominant in Eastern Europe and was associated with increased risk of treatment failure with the long-acting cabotegravir plus rilpivirine regimen. In this study, we aimed to evaluate the in vitro susceptibility and the genetic barrier to resistance to INSTI in recombinant viruses harboring clinically derived A6 integrase coding regions. Methods:We generated 23 NL4-3 strain-based recombinant viruses harboring clinically derived integrase coding region. We measured their susceptibility to second-generation INSTIs dolutegravir, bictegravir, and cabotegravir in a TZM-bl cell-based phenotypic assay. The genetic barrier to resistance was evaluated by exposing MT-2 cell cultures infected with 4 A6 integrase recombinant viruses, as well as the NL4-3 and HXB2 subtype B reference strains. Results:All 23 recombinant viruses generated with clinically derived A6 integrase displayed full susceptibility to dolutegravir, bictegravir, and cabotegravir, showing median (interquartile range) fold-change values of 1.2 (0.9-1.5), 1.1 (0.7-1.5), and 0.9 (0.6-1.1), respectively. Of 4 A6 viruses assessed for their genetic barrier to resistance in vitro, only 1 showed emerging integrase mutations E138K or Q148R at subinhibitory concentrations of dolutegravir or cabotegravir, respectively. Conclusions:These data suggest that sub-subtype A6 integrase has full susceptibility and largely maintains a high genetic barrier to resistance to second-generation integrase strand transfer inhibitors.

OBJECTIVE:The objective of the study was to establish whether HIV-1 sub-subtype A6 (HIV-1A6) is a risk factor for virological failure in people with HIV (PWH) treated with the high genetic barrier integrase strand transfer inhibitors (INSTIs) dolutegravir (DTG) or bictegravir (BIC). METHODS:The virological outcome of first-line DTG or BIC-containing antiretroviral therapy (ART) was assessed in 261 people with HIV-1A6 (PWH-1A6) and 1042 people with HIV-1B (PWH-1B) starting treatment between January 2014 and May 2025 with follow-up for at least one year in the EuResist Integrated Database. RESULTS:Most PWH-1A6 were recent migrants from Ukraine. The event rates per 100 person-years follow-up were higher in PWH-1A6 for low-level viremia (LLV, 2.71 vs. 1.71, P = 0.049) and virological failure with more than 1000 HIV RNA copies/mL (4.12 vs. 2.03, P < 0.001) than in PWH-1B. At the end of follow-up, 226/261 (86.6%) PWH-1A6 and 936/1042 (89.8%) PWH-1B had viral load below 50 copies/ml (P = 0.132). INSTI DRMs were observed in 5/219 (2.3%) available integrase sequences of PWH-1A6, including two cases detected at virological failure with more than 1000 HIV RNA copies/ml and two cases in virologically suppressed PWH-1A6. In total, 12/261 PWH-1A6 discontinued DTG- or BIC-containing ART, including three individuals who were not virologically suppressed at discontinuation. CONCLUSION:In PWH treated with DTG or BIC-containing first-line ART, LLV and virological failure with more than 1000 HIV RNA copies/ml were observed more frequently in PWH-1A6 than in PWH-1B. However, such events rarely resulted in INSTI resistance or discontinuation of INSTI-containing ART.

INTRODUCTION:Data on soluble gp120 (sgp120) and anti-HIV antibodies are lacking in people with HIV (PWH) and multidrug resistance, characterized by high inflammation and disease burden. We aimed to investigate the relationship between immuno-virological features and sgp120, anti-cluster A, anti-p24, and anti-CD4 binding site (anti-CD4bs) antibodies in 4-class drug-resistant (4DR) individuals. METHODS:Cross-sectional study on PWH with resistance to nucleoside and non-nucleoside reverse transcriptase, protease, and integrase inhibitors. Sgp120, anti-cluster A, anti-p24, and anti-CD4bs antibodies were measured using enzyme-linked immunosorbent assays. K-means clustering based on normalized anti-cluster A and anti-p24 levels identified antibody profiles. Associations with clinical variables were assessed using descriptive statistics and multinomial logistic regression. RESULTS:Overall, 80 4DR-PWH evaluated. Sgp120 and anti-CD4bs antibodies were detected in 12.5 % and 8.8 %, respectively, and not significantly associated with immuno-virological characteristics. Based on anti-cluster A and anti-p24 levels, four PWH clusters were identified. Participants with low anti-p24 and high anti-cluster A antibodies showed more frequent detectable viremia (p = 0.018), lower CD4+/CD8+ (p = 0.044), and shorter ART duration (p = 0.025). CD4+ nadir (p = 0.036) followed a similar trend, except with high anti-p24 levels. Recent 4DR onset (p = 0.029) was linked to increasing anti-cluster A antibodies. At multivariable analysis, detectable viremia (p = 0.035) and ART duration (p = 0.022) remained significantly associated with cluster assignment. CONCLUSIONS:Among 4DR-PWH, a specific antibody signature characterized by high anti-cluster A and low anti-p24 levels was associated with unsuppressed viremia and, potentially, immunological impairment. These findings provide preliminary insights into the interplay between anti-HIV humoral responses and immuno-virological features in this fragile population.

Motivation:The emergence of multidrug class resistance (MDR) in Human Immunodeficiency Virus (HIV) is a rare but significant challenge in antiretroviral therapy (ART). MDR, which may arise from prolonged drug exposure, treatment failures, or transmission of resistant strains, accelerates disease progression and poses particular challenges in resource-limited settings with restricted access to resistance testing and advanced therapies. Early prediction of future MDR development is important to inform therapeutic decisions and mitigate its occurrence. Results:In this study, we employ various machine learning classifiers to predict future resistance to all four major antiretroviral drug classes using features extracted from clinical HIV sequence data. We systematically explore several variations of the problem that differ in the pre-existing resistance level and the temporal gap between sample collection and observed MDR occurrence. Our models show the ability to predict multidrug class resistance even in the most challenging variations, albeit at a reduced accuracy. Feature importance analysis reveals that our models primarily utilize known drug resistance mutations for easier classification tasks, but rely on new mutations for the difficult task of distinguishing four class drug resistance from three class drug resistance. Availability and implementation:All analysis was performed using the Euresist Integrated DataBase (EIDB). Researchers wishing to reproduce, validate or extend these findings can request access to the latest EIDB release via the Euresist Network.

BACKGROUND:A dolutegravir plus darunavir/cobicistat regimen has been used as a simplified option in heavily treatment-experienced people with HIV (PWH). We report on long-term results of this regimen as assessed by treatment discontinuation (TD) for any reason. METHODS:This was a retrospective, observational, multicentre study. PWH started on dolutegravir plus darunavir/cobicistat from 1 December 2015 to 31 December 2022 were included. The primary endpoint was the rate of TD for any reason. Survival analysis with the Kaplan-Meier estimator was used to assess the probability of TD over time. Multiple Cox regression was used to estimate the probability of TD at 1 year following regimen initiation. RESULTS:Three hundred and twenty-seven subjects were included. At baseline, 63.6% of individuals had HIV RNA of <50 copies/mL. Primary resistance-associated mutations for NRTIs, NNRTIs, PIs and integrase inhibitors were documented in 88.0%, 70.0%, 24.5% and 6.6%, respectively. Median follow-up was 4 years (IQR 3.3-6.9). Probability of TD was 8.3%, 13.0%, 18.0%, 22.0%, 25.0%, 30.0% and 35.0% after 1, 2, 3, 4, 5, 6 and 7 years of treatment, respectively. TD occurred in 83 subjects, largely due to death (n = 20), simplification (n = 13), toxicity (n = 11), intolerance (n = 9), drug interactions (n = 10) and virological failure (n = 7). At Cox regression analysis, factors associated with a higher probability of TD over time were baseline HIV RNA of >50 copies/mL (HR = 2.14, 95% CI 1.20-3.81; P = 0.01) and the presence of PI or integrase strand transfer inhibitor resistance mutations (HR = 5.48, 95% CI 2.42-12.4; P < 0.001). CONCLUSIONS:Dolutegravir plus darunavir/cobicistat is a durable combination in heavily treatment-experienced PWH. Those who were viraemic at the time of switch were more likely to discontinue, although most reasons for TD were other than virological failure.

We evaluated the HIV-1 reservoir in people with four-class drug-resistant (4DR) virus compared with those without drug resistance. Using the intact proviral DNA assay, we found that both groups had similar levels of intact HIV-1 DNA, suggesting that multidrug-resistant HIV does not significantly alter the size of the reservoir. These findings are a first step in studying the HIV-1 reservoir in the population with multidrug-resistant virus, and further studies are needed to confirm our results.

The study evaluated the effects of violet-blue light (VBL) on cell viability and replication, carbonylation of three structural proteins (S, E, and N) and one non-structural protein (NSP13), and direct damage to the RNA of SARS-CoV-2. The virus was exposed to increasing doses of VBL along with influenza A and B viruses to compare their susceptibility. At the highest dose (21.6 J/cm2), SARS-CoV-2 was significantly more susceptible to VBL than the influenza viruses, with a reduction in viral titer of 2.33 log10. Viral RNA did not show significant changes after exposure to VBL, as demonstrated by next-generation sequencing and real-time PCR quantification, suggesting that the inactivation process does not involve direct nucleic acid damage. To exclude the role of the culture suspension in the inactivation process, virus viability experiments were performed using different dilutions of Dulbecco's modified Eagle's medium (DMEM) in phosphate-buffered saline (PBS). The results indicated that the suspension medium played a secondary role in virus inactivation, as viability did not increase with increasing DMEM dilution. Subsequent tests with three different antioxidants (NAC, AsA, and SOD) at different concentrations prevented viral inactivation, from 99.99% to 85.43% (with SOD 0.003 mM). Carbonylation of S and E proteins was more pronounced when viruses were suspended in DMEM rather than PBS, although the tests demonstrated that the intrinsic properties of the viral membrane were a crucial element to consider in relation to its susceptibility to VBL.IMPORTANCELight-based disinfection methods are often used in combination with other cleaning methods due to their non-invasive nature, versatility, and environmental benefits. VBL is an effective approach as it induces the production of reactive oxygen species that reduce microbial viability. In this study, lipid peroxidation was identified as an important factor affecting the structural integrity and function of the viral envelope, reducing its ability to interact with host cells and consequently its ability to be infectious. The lipid envelope of SARS-CoV-2, composed mainly of glycerophospholipids and lacking cholesterol and sphingolipids, appears to be the critical factor in its susceptibility, distinguishing it from influenza viruses, which have a lipid profile richer in components that protect against oxidative stress.

INTRODUCTION:A reliable quantification of hepatitis D virus (HDV) RNA is of paramount importance for monitoring patients under antiviral therapy. This quality control study compares the diagnostic performances of quantitative HDV-RNA assays used in clinical practice. METHODS:Two HDV-RNA sample panels were quantified in 30 centers by RoboGene (N = 9 laboratories), EurobioPlex (N = 7), RealStar (N = 4), AltoStar (N = 1), Bosphore (N = 3), Bosphore-on-InGenius (N = 1), Dia.Pro (N = 2), Nuclear-Laser-Medicine (N = 1) and 3 in-house assays. Panel A and B comprised 8 serial dilutions of WHO/HDV standard (range: 0.5-5.0 log10 IU/ml) and 20 clinical samples (range: 0.5-6.0 log10 IU/ml), respectively. The following parameters were determined: sensitivity by 95 % LOD (limit of detection), precision by intra- and inter-run CV (coefficient of variation), accuracy by the differences between expected-observed HDV-RNA, linearity by linear regression analysis. RESULTS:95 % LOD varied across assays and centers underlining heterogeneous sensitivities: AltoStar had the lowest 95 % LOD (3 IU/ml) followed by RealStar (10 [min-max: 3-316] IU/ml), Bosphore-on-InGenius (10 IU/ml), RoboGene (31 [3-316] IU/ml), Nuclear-Laser-Medicine (31 IU/ml) and EuroBioplex (100 [100-316] IU/ml). Moreover, 6 assays (RoboGene, EurobioPlex, RealStar, AltoStar, Nuclear-Laser-Medicine and In-house) showed <0.5 log10 IU/ml differences between expected and observed HDV-RNA for all dilutions while other assays had >1 log10 IU/ml underestimations. RealStar, Bosphore-on-InGenius and EurobioPlex had the highest precision (mean intra-run CV < 20 %). Inter-run CV was higher for all assays, with CVs < 25 % for RealStar, AltoStar, Nuclear-Laser-Medicine and EurobioPlex. Seven assays (RoboGene/AltoStar/RealStar/EurobioPlex/Nuclear-Laser-Medicine/In-house) showed a good linearity (R2 > 0.90), but for HDV-RNA < 1000 IU/ml only Bosphore-on-InGenius, AltoStar, RealStar and Robogene showed a R2 > 0.85. CONCLUSIONS:This study underlines heterogeneous sensitivities (inter- and intraassays), that could hamper proper HDV-RNA quantification, particularly at low viral loads. This raises the need to improve the diagnostic performance of most assays for properly identifying virological response to anti-HDV drugs.

We characterized sensitivity to teropavimab (TAB) and zinlirvimab (ZAB) in people living with four-class drug-resistant HIV (4DR-PWH). This was a multicenter, observational study using plasma or peripheral blood mononuclear cells collected from 50 4DR-PWH (25 with HIV-1 RNA > 1,000 copies/mL matched by age, sex, nadir CD4+, and years on ART to 25 virologically suppressed [HIV-1 RNA < 50 copies/mL]) enrolled in the PRESTIGIO Registry (NCT04098315) with a documented 4DR (NRTIs, NNRTIs, PIs, and INSTIs). Phenotypic sensitivity to bNAbs was determined using the PhenoSense monoclonal antibody assay (Monogram), with susceptibility defined as IC90 ≤ 2 µg/mL. The HIV-1 envelope was genotyped by next-generation sequencing, and sequences were analyzed for the presence of multi-position HIV-1 envelope amino acid signatures associated with in vitro phenotypic susceptibility to TAB and ZAB. Of 46/50 (92%) participants with PhenoSense mAb assay results, 35 (76%) were phenotypically sensitive to TAB, 23 (50%) to ZAB, and 19 (41%) to both bNAbs; seven (15%) were phenotypically resistant to both bNAbs. The proportion of individuals with sensitivity to both bNAbs was similar in participants with viremia (41%) and those with virologic suppression (42%; P = 0.99). We observed marginal correlations between TAB 90% inhibitory concentration (IC90) values and years since HIV diagnosis at the time of sample collection (Spearman r = 0.29, P = 0.05) as well as between ZAB IC90 values and CD8+ cell count (Spearman r = -0.32, P = 0.05). A significant number of the 4DR-PWH analyzed were found to have virus susceptible to TAB and ZAB. These data provide proof-of-concept that selected multidrug-resistant PWH may be candidates for future trials investigating bNAbs-containing regimens to achieve or maintain virologic suppression.IMPORTANCEMultidrug-resistant HIV presents significant challenges for treatment, often leaving individuals with a limited range of therapeutic alternatives. This study provides crucial insights into the efficacy of two promising broadly neutralizing antibodies, teropavimab (TAB) and zinlirvimab (ZAB), in individuals living with HIV who have developed resistance to multiple drug classes. The findings indicate that a significant proportion of the population remains susceptible to these novel treatments, irrespective of their viral suppression status. These results offer a promising basis for developing new therapeutic strategies to improve outcomes for individuals with multidrug-resistant HIV who have a history of extensive treatment, paving the way for future clinical trials aimed at achieving long-term viral suppression with novel drug regimens.

ObjectiveTo explore the durability of multi-drug antiretroviral regimens in treatment-experienced PWH.DesignThis retrospective observational study including PWH who started mega-ART regimens between 1 January 2009 and 31 December 2019, selected from the ARCA cohort.MethodsTime-dependent events were analysed by Kaplan-Meier methods, while Cox regression models were used to define the predictors of mega-ART discontinuation.ResultsA total of 1,514 ART-experienced PWH were included. Over a median follow-up of 47 weeks (IQR 15-127), 1,299 (83%) mega-ART were interrupted, with an incidence of 85.62 per 100 person-years of follow-up. In the multivariable analysis, predictors of higher risk of mega-ART discontinuation were a higher number of antiretroviral drugs included in baseline regimens (aHR 1.206, CI 95% 1.016-1.431, p = .032) and a higher baseline HIV RNA log10 (aHR 1.113, CI 95% 1.048-1.181, p < .001); otherwise, shorter duration of previous ART was associated with a lower risk of discontinuation (aHR 0.982, CI 95% 0.965-0.999, p = .037). When mega-ART was stopped, 299 PWH (23%) had HIV RNA levels above 50 copies/ml, 16/299 (1%) had HIV RNA levels >50 copies/ml but less than 200 copies/ml, 792 PWH (61%) had HIV RNA levels below 50 copies/ml, and 208 PWH (16%) had an undetermined HIV RNA load.ConclusionsMega-ART was characterized by limited durability and poor virological success.

In the last few years, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been the cause of a worldwide pandemic, highlighting the need for novel antiviral agents. The main protease (Mpro) of SARS-CoV-2 was immediately identified as a crucial enzyme for viral replication and has been validated as a drug target. Here, we present the design and synthesis of peptidomimetic Mpro covalent inhibitors characterized by quinoline-based P3 moieties. Structure-activity relationships (SARs) were also investigated at P1 and P2, as well as for different warheads. The binding modes of the designed inhibitors were assessed using X-ray crystallographic and molecular docking studies. The identified Mpro inhibitors were tested for their antiviral activities in cell-based assays, and the results were encouraging. The SAR studies presented here can contribute to the future design of improved inhibitors by addressing some of the current or prospective issues regarding Mpro inhibitors currently used in therapy.