Use of Drug-level Testing and Single-genome Sequencing to Unravel a Case of Human Immunodeficiency Virus Seroconversion on Pre-exposure Prophylaxis.

Cases of seroconversion on pre-exposure prophylaxis (PrEP) should be carefully investigated, given their public health implications and rarity. We report a case of transmitted drug resistance causing seroconversion on PrEP in spite of high adherence, confirmed with dried blood spot and segmental hair drug-level testing and single-genome sequencing.

Antiretroviral Concentrations in Hair Strongly Predict Virologic Response in a Large Human Immunodeficiency Virus Treatment-naive Clinical Trial.

Concentrations of antiretrovirals in hair are associated with virologic outcomes in cohorts of human immunodeficiency virus (HIV)-positive individuals but have never been examined in a clinical trial. We show for the first time the predictive utility of hair antiretroviral concentrations in a large HIV treatment-naive trial (AIDS Clinical Trials Group protocol A5257).

Cumulative Antiretroviral Exposure Measured in Hair Is Not Associated With Measures of HIV Persistence or Inflammation Among Individuals on Suppressive ART.

Data on the relationship of antiretroviral exposure to measures of human immunodeficiency virus (HIV) persistence are limited. To address this gap, multiple viral, immunologic, and pharmacologic measures were analyzed from individuals with sustained virologic suppression on therapy (median 7 years) in the AIDS Clinical Trials Group A5321 cohort. Among 110 participants on tenofovir-(TFV)-disoproxil-fumarate (TDF)/emtricitabine (FTC)-containing regimens, we found no significant correlation between hair concentrations of individual antiretrovirals (ARVs) in the regimen and measures of HIV persistence (plasma HIV-1 RNA by single copy assay, cell-associated-DNA, cell-associated RNA) or soluble markers of inflammation. These findings suggest that higher systemic ARV exposure may not impact HIV persistence or inflammation.

Development and validation of an assay to analyze atazanavir in human hair via liquid chromatography/tandem mass spectrometry.

RATIONALE: Assays to quantify antiretrovirals in hair samples are increasingly used to monitor adherence and exposure in both HIV prevention and treatment studies. Atazanavir (ATV) is a protease inhibitor used in combination antiretroviral therapy (ART). We developed and validated a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based method to quantify ATV in human hair, per the NIH Division of AIDS Clinical Pharmacology Quality Assurance (CPQA) program and the FDA bioanalytical method validation guidelines. METHODS: ATV was extracted from hair using optimized methods and the extracts were injected onto a BDS C-18 column (5 µm, 4.6 × 100 mm), followed by isocratic elution via a mobile phase composed of 55% acetonitrile, 45% water, 0.15% acetic acid, and 4 mM ammonium acetate, at a flow rate of 0.8 mL/min prior to analysis by MS/MS. Levels were quantified using positive electrospray ionization by multiple reaction monitoring (MRM) for the transitions MH+ m/z 705.3 to m/z 168.0 and MH+ m/z 710.2 to m/z 168.0 for ATV and ATV-d5 (internal standard), respectively. RESULTS: Our assay demonstrated a linear standard curve (r = 0.99) over the concentration range of 0.0500 ng ATV/mg hair to 20.0 ng/mg hair. The inter- and intraday accuracy of ATV quality control (QC) samples was -1.33 to 4.00% and precision (% coefficient of variation (%CV)) was 1.75 to 6.31%. The %CV for ATV levels in hair samples from highly adherent patients (incurred samples) was less than 10%. No significant endogenous peaks or crosstalk were observed in the specificity test with other HIV drugs. The overall extraction efficiency of ATV from incurred hair samples was greater than 95%. CONCLUSIONS: This highly sensitive, highly specific and validated assay can be considered for therapeutic drug monitoring for HIV-infected patients on ATV-based ART.

Tenofovir and emtricitabine concentrations in hair are comparable between individuals on tenofovir disoproxil fumarate versus tenofovir alafenamide-based ART.

Tenofovir disoproxil fumarate (TDF) in combination with emtricitabine (FTC) is the backbone for both human immunodeficiency virus (HIV) treatment and pre-exposure prophylaxis (PrEP) worldwide. Tenofovir alafenamide (TAF) with FTC is increasingly used in HIV treatment and was recently approved for PrEP among men-who-have-sex-with-men. TDF and TAF are both metabolized into tenofovir (TFV). Antiretrovirals in plasma are taken up into hair over time, with hair levels providing a long-term measure of adherence. Here, we report a simple, robust, highly sensitive, and validated high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS)-based analytical method for analyzing TFV and FTC from individuals on either TDF/FTC or TAF/FTC in small hair samples. TFV/FTC are extracted from ~5 mg hair and separated on a column using a gradient elution. The lower quantification limits are 0.00200 (TFV) and 0.0200 (FTC) ng/mg hair; the assay is linear up to 0.400 (TFV) and 4.00 (FTC) ng/mg hair. The intra-day and inter-day coefficients of variance (CVs) are 5.39-12.6% and 6.40-13.5% for TFV and 0.571-2.45% and 2.45-5.16% for FTC. TFV concentrations from participants on TDF/FTC-based regimens with undetectable plasma HIV RNA were 0.0525 ± 0.0295 ng/mg, whereas those from individuals on TAF/FTC-based regimens were 0.0426 ± 0.0246 ng/mg. Despite the dose of TFV in TDF being 10 times that of TAF, hair concentrations of TFV were not significantly different for those on TDF versus TAF regimens. Pharmacological enhancers (ritonavir and cobicistat) did not boost TFV concentrations in hair. In summary, we developed and validated a sensitive analytical method to analyze TFV and FTC in hair and found that hair concentrations of TFV were essentially equivalent among those on TDF and TAF.

Type 2 diabetes mellitus duration and obesity alter the efficacy of autologously transplanted bone marrow-derived mesenchymal stem/stromal cells.

Human bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) represent promising stem cell therapy for the treatment of type 2 diabetes mellitus (T2DM), but the results of autologous BM-MSC administration in T2DM patients are contradictory. The purpose of this study was to test the hypothesis that autologous BM-MSC administration in T2DM patient is safe and that the efficacy of the treatment is dependant on the quality of the autologous BM-MSC population and administration routes. T2DM patients were enrolled, randomly assigned (1:1) by a computer-based system into the intravenous and dorsal pancreatic arterial groups. The safety was assessed in all the treated patients, and the efficacy was evaluated based on the absolute changes in the hemoglobin A1c, fasting blood glucose, and C-peptide levels throughout the 12-month follow-up. Our data indicated that autologous BM-MSC administration was well tolerated in 30 T2DM patients. Short-term therapeutic effects were observed in patients with T2DM duration of <10 years and a body mass index <23, which is in line with the phenotypic analysis of the autologous BM-MSC population. T2DM duration directly altered the proliferation rate of BM-MSCs, abrogated the glycolysis and mitochondria respiration of BM-MSCs, and induced the accumulation of mitochondria DNA mutation. Our data suggest that autologous administration of BM-MSCs in the treatment of T2DM should be performed in patients with T2DM duration <10 years and no obesity. Prior to further confirming the effects of T2DM on BM-MSC biology, future work with a larger cohort focusing on patients with different T2DM history is needed to understand the mechanism underlying our observation.

Measuring Adherence to Antiretroviral Therapy via Hair Concentrations in India.

BACKGROUND: Objective adherence measures are of increasing interest in antiretroviral treatment (ART) monitoring. Hair ART levels predict virologic suppression, and hair is easy to collect and store. No previous study has examined hair levels in an India-based cohort or laboratory. METHODS: Small hair samples were collected from HIV-positive participants on either efavirenz (EFV)-based or nevirapine (NVP)-based ART in a South India-based study. Hair samples were split and analyzed for EFV or NVP in the University of California, San Francisco -based Hair Analytical Laboratory and the analytic laboratory of the Division of Nutrition at St. John's Research Institute, Bangalore, India, using liquid chromatography/tandem mass spectrometry. Agreement (using Bland-Altman methods) and rank correlation between the 2 laboratories' hair levels were calculated. Rank correlation between self-reported adherence (SRA) over the previous month using a visual analog scale and hair ART levels was calculated. RESULTS: Among 75 participants (38 on NVP; 37 on EFV), the correlation between NVP levels generated by the 2 laboratories was 0.66 (P < 0.0001) and between EFV levels was 0.87 (P < 0.0001). Measurements from St. John's Research Institute were usually within 20% of those from the University of California, San Francisco Hair Analytical Laboratory. SRA was essentially uncorrelated with hair antiretroviral levels for either drug (all correlations < 0.04). Hair levels showed variability in adherence although SRA was >85% in all participants. CONCLUSIONS: Hair ART levels measured by both an India-based laboratory and the standard U.S.-based laboratory showed generally high agreement and correlation, demonstrating local capacity. As in many other cohorts, hair ART levels and SRA were not well-correlated, likely indicating limitations in self-report and the need for objective adherence monitoring in resource-limited settings.

Tenofovir concentrations in hair strongly predict virologic suppression in breastfeeding women.

BACKGROUND: Antiretroviral treatment (ART) adherence is often suboptimal in the perinatal period. We measured hair tenofovir (TFV) concentrations as a metric of adherence in postpartum women to understand patterns and predictors of adherence throughout this critical period. In addition, we examined the association between hair TFV concentrations and virologic outcomes. METHODS: Between 12/2012 and 09/2016, hair samples were collected longitudinally from delivery through breastfeeding from women on ART in the Promoting Maternal and Infant Survival Everywhere study (NCT01061151) in sub-Saharan Africa. Hair TFV levels were measured using validated methods. Using generalized estimating equations, we estimated the association between hair TFV levels and virologic suppression (<400 copies/ml) over time and assessed predictors of hair TFV levels. RESULTS: Hair TFV levels were measured at 370 visits in 71 women from delivery through a median of 14 months (interquartile range 12-15) of breastfeeding. Levels ranged from below detection (0.002) to 1.067 ng/mg (geometric mean: 0.047). After at least 90 days on ART, 69 women had at least one viral load measured (median 5 measures, range 1-9); 18 (26%) experienced viremia at least once. Each doubling of TFV level more than doubled odds of concurrent virologic suppression [odds ratio 2.35, 95% confidence interval (CI): 1.44-3.84, P = 0.0006] and was associated with 1.43 times the odds of future suppression (95% CI: 0.75-2.73, P = 0.28). Relative to the first 3 months after delivery, hair levels were highest in months 6-12 (1.42-fold higher, 95% CI: 1.09-1.85, P = 0.01). CONCLUSION: Hair TFV levels strongly predicted concurrent virologic suppression among breastfeeding women. Objective adherence metrics can supplement virologic monitoring to optimize treatment outcomes in this important transition period.

Brief Report: Lopinavir Hair Concentrations Are the Strongest Predictor of Viremia in HIV-Infected Asian Children and Adolescents on Second-Line Antiretroviral Therapy.

BACKGROUND: Children/adolescents display suboptimal antiretroviral therapy (ART) adherence and outcomes versus adults. Hair ART concentrations are objective adherence measures that predict viremia in adults but longitudinal data on hair levels in pediatric populations is limited. We assessed the predictive utility of hair lopinavir (LPV) levels on viremia among youth on second-line ART. METHODS: We examined predictors of viremia (HIV-1 RNA >400 and >1000 copies/mL) at least 24 weeks after switch to LPV-based second-line ART in a cohort of HIV-infected Asian children followed between 2011 and 2014. Small hair samples, HIV-1 RNA, and self-reported adherence were collected biannually. Hair concentrations of LPV were measured through liquid chromatography/tandem mass spectrometry using validated methods. Time-to-first viremia was examined using discrete-time Cox models. RESULTS: Overall, 244 children met the inclusion criteria for the present analysis. Approximately half (55%) were boys and the median age 10 years [interquartile range (IQR) 7-13]; 40% were older than 11 years. At switch to second-line ART, median CD4 count was 300 (IQR 146-547) cells/mm and median HIV-RNA level was 5.0 (IQR 4.3-5.6) log10/mL. Median time of study follow-up was 48 weeks and a median of 3 (range 1-5) hair samples were collected from each participant. Adjusting for age, sex, country, self-reported adherence, CD4, and HIV-RNA, higher LPV hair concentrations were the strongest predictor of lower odds of viremia (HIV-RNA >400 copies/mL adjusted odds ratio = 0.41 per doubling in hair concentration, 95% confidence interval: 0.29 to 0.58, P < 0.001; HIV-RNA >1000 copies/mL, adjusted odds ratio = 0.54, 95% confidence interval: 0.45 to 0.65, P < 0.001). CONCLUSIONS: Hair concentrations predict viremia among children with HIV on second-line ART and could guide clinical decisions for this population.