Cohort profile: PRESTIGIO, an Italian prospective registry-based cohort of people with HIV-1 resistant to reverse transcriptase, protease and integrase inhibitors.

PURPOSE: The PRESTIGIO Registry was established in 2017 to collect clinical, virological and immunological monitoring data from people living with HIV (PLWH) with documented four-class drug resistance (4DR). Key research purposes include the evaluation of residual susceptibility to specific antiretrovirals and the validation of treatment and monitoring strategies in this population. PARTICIPANTS: The PRESTIGIO Registry collects annual plasma and peripheral blood mononuclear cell samples and demographic, clinical, virological, treatment and laboratory data from PLWH followed at 39 Italian clinical centres and characterised by intermediate-to-high genotypic resistance to ≥1 nucleoside reverse transcriptase inhibitors, ≥1 non-nucleoside reverse transcriptase inhibitors, ≥1 protease inhibitors, plus either intermediate-to-high genotypic resistance to ≥1 integrase strand transfer inhibitors (INSTIs) or history of virological failure to an INSTI-containing regimen. To date, 229 people have been recorded in the cohort. Most of the data are collected from the date of the first evidence of 4DR (baseline), with some prebaseline information obtained retrospectively. Samples are collected from the date of enrollment in the registry. FINDINGS TO DATE: The open-ended cohort has been used to assess (1) prognosis in terms of survival or development of AIDS-related or non-AIDS-related clinical events; (2) long-term efficacy and safety of different antiretroviral regimens and (3) virological and immunological factors predictive of clinical outcome and treatment efficacy, especially through analysis of plasma and cell samples. FUTURE PLANS: The registry can provide new knowledge on how to implement an integrated approach to study PLWH with documented resistance to the four main antiretroviral classes, a population with a limited number of individuals characterised by a high degree of frailty and complexity in therapeutic management. Given the scheduled annual updates of PLWH data, the researchers who collaborate in the registry can send study proposals at any time to the steering committee of the registry, which evaluates every 3 months whether the research studies can be conducted on data and biosamples from the registry and whether they are aimed at a better understanding of a specific health condition, the emergence of comorbidities or the effect of potential treatments or experimental drugs that may have an impact on disease progression and quality of life. Finally, the research studies should aim to be inclusive, innovative and in touch with the communities and society as a whole. TRIAL REGISTRATION NUMBER: NCT04098315.

A Novel UHPLC-MS/MS Method for the Quantification of Seven Opioids in Different Human Tissues.

BACKGROUND: Opioids are considered the cornerstone of pain management: they show good efficacy as a first-line therapy for moderate to severe cancer pain. Since pharmacokinetic/pharmacodynamic information about the tissue-specific effect and toxicity of opioids is still scarce, their quantification in post-mortem autoptic specimens could give interesting insights. METHODS: We describe an ultra-high-performance liquid chromatography coupled with tandem mass spectrometry method for the simultaneous quantification of methadone, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone and fentanyl in several tissues: liver, brain, kidney, abdominal adipose tissue, lung and blood plasma. The presented method has been applied on 28 autoptic samples from different organs obtained from four deceased PLWH who used opioids for palliative care during terminal disease. RESULTS: Sample preparation was based on tissue weighing, disruption, sonication with drug extraction medium and a protein precipitation protocol. The extracts were then dried, reconstituted and injected onto the LX50 QSight 220 (Perkin Elmer, Milan, Italy) system. Separation was obtained by a 7 min gradient run at 40 °C with a Kinetex Biphenyl 2.6 µm, 2.1 × 100 mm. Concerning the analyzed samples, higher opioids concentrations were observed in tissues than in plasma. Particularly, O-MOR and O-COD showed higher concentrations in kidney and liver than other tissues (>15-20 times greater) and blood plasma (>100 times greater). CONCLUSIONS: Results in terms of linearity, accuracy, precision, recovery and matrix effect fitted the recommendations of FDA and EMA guidelines, and the sensitivity was high enough to allow successful application on human autoptic specimens from an ethically approved clinical study, confirming its eligibility for post-mortem pharmacological/toxicological studies.

COVID-19: A Possible Contribution of the MAPK Pathway.

BACKGROUND: COVID-19 is characterized by an uncontrolled inflammatory response with high pro-inflammatory cytokine production through the activation of intracellular pathways, such as mitogen-activated protein kinase (MAPK). Viruses are able to exploit the MAPK pathway to their advantage; this pathway relevance to severe COVID-19 is poorly described. The aim of this study was to quantify biomarkers involved in the MAPK pathway and to clarify its possible role in affecting some COVID-19-related clinical features. METHODS: H-RAS, C-RAF, MAPK1, MAPK2, and ERK were quantified through ELISA, and genetic polymorphisms were evaluated through real-time PCR. RESULTS: We prospectively recruited 201 individuals (158 positive and 43 negative for SARS-CoV-2): 35 were male, and their median age was 65 years. MAPK-related biomarker levels were increased in SARS-CoV-2-positive participants (n = 89) compared to negative ones (n = 29). Dyspnea was reported by 48%; this symptom was associated with PBMC C-RAF levels in positive participants (p = 0.022) and type of ventilation (p = 0.031). The highest degree of ventilation was used by 8% for invasive ventilation and 41% for continuous positive airway pressure (CPAP). CONCLUSIONS: This is the first study that showed a possible contribution of MAPK-related biomarkers in affecting COVID-19 clinical features, and this may be relevant for identifying COVID-19 positive participants at risk of serious complications.

COVID-19: Focusing on the Link between Inflammation, Vitamin D, MAPK Pathway and Oxidative Stress Genetics.

An uncontrolled inflammatory response during SARS-CoV-2 infection has been highlighted in several studies. This seems to be due to pro-inflammatory cytokines whose production could be regulated by vitamin D, ROS production or mitogen-activated protein kinase (MAPK). Several genetic studies are present in the literature concerning genetic influences on COVID-19 characteristics, but there are few data on oxidative stress, vitamin D, MAPK and inflammation-related factors, considering gender and age. Therefore, the aim of this study was to evaluate the role of single nucleotide polymorphisms in these pathways, clarifying their impact in affecting COVID-19-related clinical features. Genetic polymorphisms were evaluated through real-time PCR. We prospectively enrolled 160 individuals: 139 patients were positive for SARS-CoV-2 detection. We detected different genetic variants able to affect the symptoms and oxygenation. Furthermore, two sub-analyses were performed considering gender and age, showing a different impact of polymorphisms according to these characteristics. This is the first study highlighting a possible contribution of genetic variants of these pathways in affecting COVID-19 clinical features. This may be relevant in order to clarify the COVID-19 etiopathogenesis and to understand the possible genetic contribution for further SARS infections.

Reduced probability of improving viro-immunological state in subjects with vertical transmission of HIV reaching adult age: A multicenter retrospective cohort study.

INTRODUCTION: Young adults with vertical transmission (VT) of human immunodeficiency virus (HIV) represent a fragile population. This study evaluates factors associated with viro-immunological outcome of these patients. METHODS: We performed a multicenter study including HIV-infected subjects with VT ≥ 18 years old from six Italian clinics. Subjects were observed from birth to death, lost to follow-up, or last visit until December 31, 2019. Condition of "optimal viro-immunological status" (OS) was defined as the simultaneous presence of HIV ribonucleic acid (RNA) < 50 copies/mL, CD4+ > 500 cells/mm3 , and CD4+/CD8+ ratio ≥ 1. RESULTS: A total of 126 subjects were enrolled. At 18 years of age, 52/126 (44.4%) had HIV-RNA > 50 copies/mL, 47/126 (38.2%) had CD4+ < 500/mm3 , and 78/126 (67.2%) had CD4+/CD8+ < 1; 28 subjects (23.7%) presented in the condition of OS. Having a CD4+/CD8+ ratio ≥ 1 at 18 years of age was related with an increased probability of shift from suboptimal viro-immunological status (SOS) to OS (HR: 7.7, 95% confidence interval [CI]: 4.23-14.04), and a reduced risk of shift from the OS to the SOS (HR: 0.49, 95% CI: 0.26-0.92). Acquired immunodeficiency syndrome (AIDS) diagnosis significantly reduced the probability of shift from a viro-immunological SOS to OS (HR: 0.09, 95% CI: 0.03-0.30). Subjects who had not achieved an OS at 18 years of age had an increased risk of discontinuation of combination antiretroviral therapy (cART, p = .019). CONCLUSIONS: Only a small proportion of subjects with VT of HIV reached the adult age with "OS". Transition to the adult care with a compromised viro-immunological condition represents a negative driver for future optimal infection control, with a higher risk of discontinuation of cART and a reduced probability to improve the immunological status later in the years.

Residual phenotypic susceptibility to doravirine in multidrug-resistant HIV-1 from subjects enrolled in the PRESTIGIO Registry.

OBJECTIVES: Doravirine shows a rather distinct resistance profile within the nonnucleoside reverse transcriptase inhibitor (NNRTI) class. This study aimed to evaluate the phenotypic susceptibility to doravirine, rilpivirine and etravirine in a panel of multidrug-resistant (MDR) HIV-1 isolates collected from people living with HIV (PLWH) enrolled in the PRESTIGIO Registry. METHODS: Recombinant viruses expressing PLWH-derived protease, reverse transcriptase coding regions were generated from plasma samples at virological failure with documented resistance to protease inhibitors, nucleoside reverse transcriptase inhibitors, NNRTIs and integrase strand transfer inhibitors. In vitro susceptibility was assessed through a phenotypic assay measuring fold-change values with respect to the reference NL4-3 virus. Genotypic susceptibility was computed by the Stanford HIVdb algorithm 8.9-1. RESULTS: Plasma samples were collected from 22 PLWH: 20 (91%) were male, median age 55 years (IQR 50-58), time since HIV-1 diagnosis 27 years (23-31) and time on antiretroviral treatment 23 years (22-26). Median doravirine, etravirine and rilpivirine fold-change values were 9.8 (2.9-40.4), 42.9 (3.1-100.0) and 100.0 (17.9-100.0), respectively. According to the fold-change cut-offs, full susceptibility was observed in five (23%), four (18%) and one (5%) cases with doravirine, etravirine and rilpivirine, respectively. Irrespective of the presence of specific doravirine mutations, higher numbers of NNRTI mutations correlated with higher fold-change values for doravirine. By comparing the distribution of fold-change values with the Stanford HIVdb predicted susceptibility, a significant correlation was detected for doravirine and rilpivirine but not etravirine. CONCLUSION: Despite extensive cross-resistance among NNRTIs, doravirine can be a valid option in a proportion of PLWH with MDR HIV-1. Doravirine activity appeared to be inferred with fair accuracy by the HIVdb algorithm.

Inflammation and intracellular exposure of dolutegravir, darunavir, tenofovir and emtricitabine in people living with HIV.

AIMS: Antiretroviral (ARV) therapy reduces inflammation and immune activation in people with HIV, but not down to the levels observed in people without HIV. Limited drug penetration within tissues has been argued as a potential mechanism of persistent inflammation. Data on the inflammation role on ARV plasma/intracellular (IC) pharmacokinetics (PK) through to expression of cytochrome P450 3A/membrane transporters are limited. The aim of this study was to investigate the correlation between inflammation markers (IM) and plasma/IC PK of ARV regimen in HIV-positive patients. METHODS: We included ART-experienced patients switching to three different ARV regimens. Plasma and IC ARV drug concentration means at the end of dosing interval (T0 ), IM on samples concomitantly with ARV PK determination: sCD14, CRP, IL-6 and LPS were analysed. RESULTS: Plasma and IC drug concentrations were measured in 60 samples. No significative differences between CRP, sCD14, IL-6 and LPS values in the three arms were observed. A significant inverse correlation between tenofovir plasma concentration and sCD14 (rho = -0.79, P < .001), and between DRV IC/plasma ratio and Log10 IL-6 concentrations (rho = -0.36, P = .040), and a borderline statistically significant positive trend between DRV plasma concentration and sCD14 (rho = 0.31, P = .070) were suggested. Furthermore, a borderline statistically significant inverse trend between DTG IC concentrations and sCD14 (rho = -0.34, P = .090) was observed in 24 patients on DTG-based triple therapy. CONCLUSIONS: Our preliminary data support the hypothesis of lower DRV and DTG IC concentrations and lower TFV plasma exposure in patients with higher plasma IM suggesting an interplay between HIV drug penetration and persistent inflammation in cART-treated HIV-positive patients.

Durability of Integrase STrand Inhibitor (InSTI)-based regimen in geriatric people living with HIV in the GEPPO cohort.

OBJECTIVE: To investigate the durability of the first integrase inhibitor-based regimen in a HIV geriatric multicentric prospective cohort and to explore the reasons of regimen discontinuation. DESIGN: This is an analysis conducted on the Geriatric Patients Living with HIV/AIDS (GEPPO) cohort, an Italian prospective observational multicentre cohort of people living with HIV with 65 years of age or more. METHODS: The analysis was performed using R (version 4.0.2). The tests performed were two sided assuming a 5% significance level (Kruskal-Wallis test, Chi-squared test, log-rank test and a Cox Proportional Hazard model). The proportion of participants discontinuing the three regimens was displayed using cumulative curves. RESULTS: Among 1531 patients enrolled between 2017 and 2019 in the GEPPO cohort, we included 822 participants in this analysis. At baseline, median age was 69.8, the immunovirological profile good, multimorbidity was present in 42.3% of participants, while 27.4% were on polypharmacy. Overall, 483, 243 and 96 participants received DTG, RAL and EVG/c respectively as first InSTI. At the end of the follow up 6.4%, 21.1% and 22.9% participants discontinued DTG, RAL and EVG/c respectively. Using a log-rank test, EVG showed a significantly lower durability than DTG (p<0.001) or RAL (p 0.05) or both, DTG and RAL (p<0.001). Among participants who discontinued their regimen we found 0 virological failure and 56.7% simplification/deprescription. CONCLUSIONS: The three integrase inhibitors considered showed a good durability and no virological failures in geriatric patients such as those enrolled in the GEPPO cohort when used in a two or three drug regimen.

Development and Validation of an Up-to-Date Highly Sensitive UHPLC-MS/MS Method for the Simultaneous Quantification of Current Anti-HIV Nucleoside Analogues in Human Plasma.

Therapeutic options to treat HIV infection have widened in the past years, improving both effectiveness and tolerability, but nucleoside reverse transcriptase inhibitors (NRTIs) are still considered the standard backbone of the combination regimens. Therapeutic drug monitoring (TDM) can be useful for these drugs, due to concentration-effect relationship, with risk of ineffectiveness, toxicity or adherence concerns: in this scenario, robust and multiplexed methods are needed for an effective TDM activity. In this work, the first validated ultra-high spectrometry liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method is described for the high-sensitive simultaneous quantification of all the currently used NRTIs in human plasma, including tenofovir alafenamide (TAF), following FDA and EMA guidelines. The automated sample preparation consisted in the addition of an internal standard (IS) working solution, containing stable-isotope-linked drugs, protein precipitation and drying. Dry extracts were reconstituted with water, then, these underwent reversed phase chromatographic separation: compounds were detected through electrospray ionization and multiple reaction monitoring. Accuracy, precision, recovery and IS-normalized matrix effect fulfilled guidelines' requirements. The application of this method on samples from people living with HIV (PLWH) showed satisfactory performance, being capable of quantifying the very low concentrations of tenofovir (TFV) in patients treated with TAF.

Medication burden and clustering in people living with HIV undergoing therapeutic drug monitoring.

AIM: People living with HIV (PLWH) have a high burden of comorbidities and concomitant medication use. The aim of this study was to analyse the prevalence, predictors and patterns of polypharmacy (PP) in a large therapeutic drug monitoring (TDM) registry. METHODS: We searched our TDM registry and categorized co-medications into 26 drug classes. We included patients with at least one medication recorded: PP and severe polypharmacy (sPP) were defined as the concomitant use of ≥5 or ≥10 nonantiretroviral/nonantitubercular drugs. Multivariable binary logistic analysis were conducted for identifying PP/sPP predictors. A hierarchical average-linkage cluster analysis was performed among drug classes. RESULTS: We included 2432 participants (1158 PLWH) aged 49.6 years (± 14.4) in the 2016-2020 period. A higher number of concomitant medications (4 vs 3.1, P < .001) and a higher prevalence of PP (26.1% vs 21.8%, P = .015) were recorded in controls. At multivariable binary logistic analysis older age, female gender and HIV-positive serostatus (P = .015) were independent predictors of PP; older age and year of inclusion were independent predictors of sPP. Cluster analysis showed that patients receiving oral drug for type 2 diabetes have a high probability of receiving several other drugs; a cluster of co-medications was observed with opioids, diuretics and central nervous system-affecting drugs. CONCLUSION: We observed a moderately high prevalence of polypharmacy in middle-aged PLWH: advanced age and female gender were associated with the greatest prevalence. The observation of co-medication clusters suggests groups of comorbidities but also identifies groups of patients at risk of similar drug-to-drug interactions.

Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV.

Recently, anti-HIV treatment has achieved high efficacy and tolerability. Nevertheless, few data are available about the intracellular penetration of antiretrovirals, partly due to the technical challenges related to intracellular quantification. This work aimed to validate an ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the simultaneous quantification of maraviroc, nevirapine, rilpivirine, dolutegravir, raltegravir, cobicistat, darunavir, ritonavir, atazanavir, efavirenz, elvitegravir, and etravirine within peripheral blood mononuclear cells (PBMCs) and apply it to samples from patients. PBMCs were isolated by density gradient on cell preparation tubes (CPT). Samples were prepared by addition of internal standards (IS), sonication, centrifugation, and drying. Reconstituted extracts underwent chromatographic separation by reversed phase UHPLC and detection was performed by electrospray ionization and multiple reaction monitoring. Method validation followed FDA and EMA guidelines, showing acceptable accuracy, precision, recovery and IS-normalized matrix effect. The application to 56 samples from patients undergoing antiretroviral treatment provided description of intracellular penetration, showing method eligibility for future studies.

Antiretroviral drug concentrations in brain tissue of adult decedents.

OBJECTIVE: Determine concentrations of antiretroviral therapy (ART) drugs in the human brain. DESIGN: Cohort study of persons with HIV who consented to antemortem assessment and postmortem autopsy. METHODS: Eleven persons with HIV who were taking ART at the time of death and had detectable concentrations of at least one ART drug in intracardiac aspirate at autopsy were evaluated. Autopsies were performed within 24 h of death and brain tissue was stored at -80 °C. Concentrations of 11 ART drugs were measured in three brain regions (globus pallidus, cortical gray matter, white matter) by HPLC tandem mass spectrometry with a lower limit of quantification of 25 ng/ml. RESULTS: Participants were mostly men (82%) with a mean age of 40.4 years. Drug concentrations in brain tissue were highly variable and exceeded published concentrations in cerebrospinal fluid for several drugs, including for tenofovir, efavirenz, and lopinavir. Drug concentrations correlated most strongly between cortical gray matter and globus pallidus (rho = 0.70) but less well between globus pallidus and white matter (rho = 0.43). Combining all drugs and brain regions (n = 89), higher drug concentrations in brain were associated with longer estimated duration of HIV infection (P = 0.015), lower HIV RNA in plasma (P = 0.0001), lower nadir CD4 T-cell count (P = 0.053), and worse neurocognitive performance (P = 0.017). CONCLUSION: This is the first analysis of ART drug concentrations in human brain tissue. Concentrations of several drugs in this analysis were similar to published concentrations in cerebrospinal fluid but others exceeded published concentrations. The association between higher drug concentrations in the brain and worse neurocognitive performance may indicate ART neurotoxicity.

Enhanced Immunological Recovery With Early Start of Antiretroviral Therapy During Acute or Early HIV Infection-Results of Italian Network of ACuTe HIV InfectiON (INACTION) Retrospective Study.

BACKGROUND: Viral load peak and immune activation occur shortly after exposure during acute or early HIV infection (AEHI). We aimed to define the benefit of early start of antiretroviral treatment (ART) during AEHI in terms of immunological recovery, virological suppression, and treatment discontinuation. SETTING: Patients diagnosed with AEHI (Fiebig stages I-V) during 2008-2014 from an analysis of 20 Italian centers. METHODS: This was an observational, retrospective, and multicenter study. We investigated the effect of early ART (defined as initiation within 3 months from AEHI diagnosis) on time to virological suppression, optimal immunological recovery (defined as CD4 count ≥500/µL, CD4 ≥30%, and CD4/CD8 ≥1), and first-line ART regimen discontinuation by Cox regression analysis. RESULTS: There were 321 patients with AEHI included in the study (82.9% in Fiebig stage III-V). At diagnosis, the median viral load was 5.67 log10 copies/mL and the median CD4 count was 456 cells/µL. Overall, 70.6% of patients started early ART (median time from HIV diagnosis to ART initiation 12 days, IQR 6-27). Higher baseline viral load and AEHI diagnosis during 2012-2014 were independently associated with early ART. HBV co-infection, baseline CD4/CD8 ≥1, lower baseline HIV-RNA, and AEHI diagnosis in recent years (2012-2014) were independently associated with a shorter time to virological suppression. Early ART emerged as an independent predictor of optimal immunological recovery after adjustment for baseline CD4 (absolute and percentage count) and CD4/CD8 ratio. The only independent predictor of first-line ART discontinuation was an initial ART regimen including > 3 drugs. CONCLUSIONS: In a large cohort of well-characterized patients with AEHI, we confirmed the beneficial role of early ART on CD4+ T-cell recovery and on rates of CD4/CD8 ratio normalization. Moreover, we recognized baseline CD4/CD8 ratio as an independent factor influencing time to virological response in the setting of AEHI, thus giving new insights into research of immunological markers associated with virological control.

96-week results of a dual therapy with darunavir/ritonavir plus rilpivirine once a day vs triple therapy in patients with suppressed viraemia: virological success and non-HIV related morbidity evaluation.

Antiretroviral therapies have been tested with the goal of maintaining virological suppression with a particular attention in limiting drug-related toxicity. With this aim we designed the DUAL study: a randomized, open-label, multicenter, 96 weeks-long pilot exploratory study in virologically suppressed HIV-1+ patients with the aim of evaluating the immunovirological success and the impact on non-HIV related morbidity of switching to a dual therapy with darunavir-ritonavir (DRV/r) and rilpivirine (RPV). We recruited patients who received a PI/r-containing HAART for ≥6 months, HIV-RNA < 50 cp/mL for ≥3 months, eGFR > 60 mL/min/1,73m2, without DRV or RPV RAMs. We randomized patients in arm A: RPV + DRV/r QD or arm B: ongoing triple therapy. The primary endpoint has been defined as the percentage of patients with HIV-RNA < 50 cp/mL at week 48 (ITT). VACS index, Framingham CVD risk (FRS) and urinary RBP (uRBP) were calculated. We used Chi-square or Fisher statistics for categorical variables and Mann-Whitney U for continuous ones. Forty-one patients were enrolled (22 in arm A, 14 in arm B, plus 5 screening failures): 30 patients reached 96 weeks: 100% had HIV-RNA < 50 cp/mL in arm A versus 91.7% in arm B. Similar changes were observed in median CD4/mL between baseline and week 96 (+59 versus - 31, p: n.s.). Thirty-one in arm A and 23 in arm B adverse events took place, whereas only 1 was serious (arm A: turbinate hypertrophy, unrelated to HAART). Among the 6 discontinuations (3 in A, 3 in B), only 1 was related to adverse event (arm A: G3 depression, insomnia, weakness). VACS index, median FRS and median uRBP values did not vary from baseline to week 96. At 96-weeks all patients switched to a QD 2-drug regimen based on DRV/r + RPV maintained HIV-RNA suppression, but a single patient who showed a virological failure at week 4. CD4 counts increased overtime without significant differences between the two arms. The novel dual regimen was well tolerated with the same amount of discontinuation as the control arm. VACS index, FRS and uRBP did not differ between arms at week 96.

Correlates of HIV RNA concentrations in cerebrospinal fluid during antiretroviral therapy: a longitudinal cohort study.

BACKGROUND: Few large projects have evaluated the factors that influence the HIV RNA concentrations (viral load) in cerebrospinal fluid (CSF) during antiretroviral therapy (ART) over time. We aimed to determine the correlates of HIV RNA in CSF in a large cohort. METHODS: We analysed longitudinal data from adults living with HIV in the US CHARTER cohort. Participants in the CHARTER study were recruited from six US academic medical centres-in Baltimore (MD), Galveston (TX), New York (NY), St Louis (MO), San Diego (C92A), and Seattle (WA). Participants in this study had been assessed at least three times between Sept 4, 2003, and Sept 14, 2010, and were taking ART and underwent venous and lumbar puncture with measurement of HIV RNA concentration at all assessments. The lower limit of quantification of the HIV RNA assays was 50 copies per mL. Data were analysed with longitudinal mixed effects logistic regression to identify correlates of HIV RNA concentration (as a binary [detectable or not] and as a continuous variable) in CSF over time. We tested demographic characteristics, plasma HIV RNA, nadir and current CD4 cell count in blood, current CD8 cell count in blood, estimated duration of HIV infection, AIDS diagnosis, duration of ART, adherence to ART, ART characteristics, and CSF characteristics as potential correlates. FINDINGS: At the time of analysis, 2207 assessments from 401 participants met the criteria for inclusion in this study. Mean duration of observation was 33·7 months (range 12-84). HIV RNA concentrations in 710 (32·2%) plasma specimens and in 255 (11·6%) CSF specimens were greater than the lower limit of quantification. The best multivariate model of HIV RNA concentration in CSF greater than the lower limit of quantification over time included increased plasma HIV RNA concentration (odds ratio 18·0 per 1 log10 copy per mL, 95% CI 11·3 to 28·8; p<0·0001), increased CSF leucocyte count (2·01 per 5 cells per µL, 1·61 to 2·39; p<0·0001), decreased CD4 cell count (0·53 per 5 square-root cells per µL, 0·35 to 0·79; p=0·0025), decreased CNS penetration-effectiveness value (0·71 per unit, 0·56 to 0·92; p=0·0078), increased CD8 cell count (1·51 per 5 square-root cells, 1·11 to 2·06; p=0·0089), and protease inhibitor use (3·26, 1·04 to 10·23; p=0·039; model R2=0·22, p<0·0001). Analyses of continuous HIV RNA concentration in CSF that accounted for censoring below the lower limit of quantification had similar findings, although increased HIV RNA concentrations in CSF were also associated with black ethnicity (change in log10 HIV RNA concentration in CSF 0·205, 0·0367 to 0·3733; p=0·017), increased total protein in CSF (0·0025, -0·0002 to 0·0052; p=0·069), and the presence of addictive-drug metabolites in urine (0·103, -0·013 to 0·219; p=0·081). INTERPRETATION: The identified correlates of HIV RNA concentration in CSF during ART could strengthen clinical prediction of risk for failure to achieve or maintain HIV RNA suppression in CSF. Because most participants in this analysis were ART-experienced and were taking a three-drug regimen that did not include an integrase inhibitor, future research should focus on participants who are taking their first ART regimens or regimens that include integrase inhibitors or two drugs. FUNDING: The work was supported by the National Institute of Mental Health and the National Institute of Neurological Disorders and Stroke.

Symptomatic cerebrospinal fluid HIV-1 escape with no resistance-associated mutations following low-level plasma viremia.

The majority of neurologically symptomatic cerebrospinal fluid HIV-1 escape cases are connected with resistance-associated mutations and potentially explained by low cerebrospinal fluid antiretroviral concentrations. However, there are still significant knowledge gaps regarding the physiopathology and long-term management of neurosymptomatic viral escape. We report a case of Parkinson-like syndrome following cerebrospinal fluid HIV-1 escape in a 40-year-old female patient with an history of persistent low-level plasma viremia under treatment. No resistance-associated mutations, high viral diversity (env deep sequencing), adequate pharmacokinetics, atypical CD3-CD14-CD4+CD5-CD2-/+CD7-/+ lymphocytes, low-level Epstein-Barr virus replication, and white matter autoimmune reactivity were observed in the cerebrospinal fluid. Antiretroviral regimen modification led to rapid clinical and radiological improvements. This case may increase the current uncertain knowledge on the origin of cerebrospinal fluid HIV-1 and illustrates the consequences of uncontrolled compartmental viral replication; it also highlights the relevance and persistence of immune activation and the possibility of various detrimental mechanisms underlying neurosymptomatic viral escape.

Long-term efficacy of dolutegravir in treatment-experienced subjects failing therapy with HIV-1 integrase strand inhibitor-resistant virus.

OBJECTIVES: This study evaluated the virological efficacy of dolutegravir 50 mg twice daily in 190 HIV-1 failing antiretroviral-experienced patients with previous exposure to first-generation integrase strand transfer inhibitor (INSTI) over a 5 year follow-up using data from clinical practice. PATIENTS AND METHODS: This analysis included HIV-1-infected patients who were ≥18 years of age, treatment experienced, had HIV-1 RNA >50 copies/mL, with INSTI-resistant virus, who started dolutegravir 50 mg twice daily plus optimized background therapy (OBT), recorded in the national prospective database PRESTIGIO (www.progettoprestigio.it). Follow-up accrued from the start of dolutegravir 50 mg twice daily + OBT until virological failure (VF) or dolutegravir discontinuation for any reason or the last treatment visit on dolutegravir 50 mg twice daily treatment. VF was defined by the lack of achievement of HIV-1 RNA <50 copies/mL by 6 months and thereafter, or the occurrence of two consecutive HIV-1 RNA ≥50 copies/mL after achievement of undetectable viral load. RESULTS: The estimated VF probabilities were 17% (95% CI = 12%-24%), 28% (95% CI = 21%-37%), 33% (95% CI = 25%-43%), 39% (95% CI = 29%-51%) and 52% (95% CI = 39%-67%) at 12, 24, 36, 48 and 60 months since baseline, respectively. A higher risk of VF was independently associated with baseline viral load >100000 copies/mL (adjusted HR = 4.73, 95% CI = 1.33-16.78, P = 0.016) and with ≥1 INSTI mutations plus Q148H/K/R/N and the G140S/A/C as compared with other subjects (adjusted HR = 4.18, 95% CI = 1.32-13.23, P = 0.015). CONCLUSIONS: Our data showed a favourable long-term efficacy of dolutegravir 50 mg twice daily in association with OBT in treatment-experienced failing subjects, with INSTI-resistant virus, in the real world. A close monitoring of adherence is crucial for maintenance of virological response in this fragile subgroup of subjects.

Cerebrospinal fluid viral load and neopterin in HIV-positive patients with undetectable viraemia.

BACKGROUND: Cerebrospinal fluid (CSF) HIV RNA is commonly used as a marker of compartmental antiviral activity in HIV-positive patients. Undetectable CSF HIV RNA levels have been associated with low CSF neopterin levels and better neurocognitive performances. The aim of this study was to analyse the prevalence and predictors of non-detectable CSF HIV RNA using a commercial assay. METHODS: In adult HIV-positive HAART-treated patients with confirmed plasma HIV RNA <50 copies/ml, CSF HIV RNA (with Roche Amplicor Assay) and neopterin were measured. RESULTS: 112 adult patients were included. Plasma and CSF HIV RNA were non-detectable (target not detected [TND]) in 29 (25.9%) and 36 (32.1%) patients, respectively. CSF TND was observed more frequently in patients with plasma TND (P=0.005, OR=3.87). CSF neopterin levels were associated with age (rho =0.333, P=0.002) and current (rho= -0.272, P=0.015) and nadir (rho =-0.240, P=0.038) CD4+ T-lymphocytes; the lowest CSF neopterin concentration was observed in patients with CSF TND versus other viral load strata (0.62 mg/dl versus 0.78 mg/dl; P=0.048). CONCLUSIONS: Efficaciously treated HIV-positive patients with detectable plasma HIV RNA might imperfectly control CSF viral replication. Prospective studies addressing the management and neurocognitive consequences of CSF low-level viraemia are warranted.