•Testing of blood for chemistry analytes is usually performed using serum or plasma.•Clinical chemistry analyte concordance was evaluated across different tube types.•Total protein and potassium ...results were not equal in plasma- vs. serum-separator tubes (PST; SST).•Storage stability was shortened for several analytes when collected in PSTs vs. SSTs.•Rapid serum-separator tubes had the least interference from hemolysis and lipemia.
Routine chemistry testing is typically performed using serum or plasma to assess a patient’s clinical status. At our institution, serum is the specimen type used. To reduce processing times, evaluation of plasma-based and rapid serum gel separator tubes was performed.
We compared the results of routine chemistry analytes collected in serum gel separator tubes (SST), plasma gel separator tubes (PST), rapid serum gel separator tubes (RST), and plasma tubes without gel separators (DGT). Result concordance was assessed at baseline (immediate testing after processing) and up to one week of refrigerated storage. Other parameters assessed were the susceptibility to hemolysis and lipemia interference, and changes in results after re-centrifugation. Percent changes were compared against the SST and evaluated according to established bias thresholds.
Total protein and potassium results at baseline in plasma-based tubes had percent changes from the SST that exceeded acceptability thresholds. Stability was significantly shortened for glucose, potassium, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) when collected in the PST as compared to the SST. The RST was the least susceptible to hemolysis and lipemia interferents. Re-centrifugation affected the serum-based analysis of potassium.
Plasma may reduce processing time at the expense of shortened sample stability and may require specimen source-specific reference intervals for potassium and total protein. The RST provides an alternate option to reduce processing time, while maintaining storage stability.
•Specimen integrity is critical for the generation of reliable and actionable clinical results.•Hemolysis, icterus, and lipemia, are common interferents in clinical chemistry assays.•Bio-Rad serum ...indices QC materials serve as an effective way of monitoring specimen integrity.
Evaluation of specimen suitability for downstream analytical testing and identification of potential interferents in the clinical laboratory is critical for the generation of actionable clinical results. Within the clinical laboratory, hemolysis, icterus, and lipemia are commonly assessed spectrophotometrically. While clinical laboratories rely on analyte-specific quality control (QC) materials to monitor test or instrument performance, QC materials evaluating specimen integrity checks are infrequently implemented.
Using commercially available specimen integrity materials, we evaluated the Bio-Rad Liquichek™ Serum Indices product on Roche cobas® c701 analyzers at a large academic medical center. Target arbitrary values for the hemolysis, icterus, and lipemia QC materials were 200, 20, and 500, respectively. Means, standard deviations (SD), and coefficients of variation (%CV) were established for hemolysis, icterus, lipemia, and non-interfered QCs, and performance was monitored over a 60-day period.
Across four c701 instruments, all QC materials performed well, with %CVs ≤ 1.76%, 4.51%, and 3.46% for hemolysis, icterus, and lipemia QC, respectively.
The Bio-Rad Liquichek Serum Indices product can serve as an effective means of monitoring specimen integrity checks in a manner congruous with existing QC programs.
Dried blood spots (DBS) have been utilized as a blood plasma alternative for therapeutic drug monitoring and pharmacologic analysis. There are analytical and physiochemical considerations in bridging ...drug concentrations from plasma to DBS. Recently, the long-acting antiretroviral cabotegravir (CAB) has been approved for HIV prevention, and a co-packaged regimen of long-acting CAB and rilpivirine (RPV) has been approved for HIV treatment. Measurement of these drugs in blood collected as DBS may offer increased capacity and flexibility in translational applications.
Whole blood was spiked with CAB and RPV and spotted on DBS cards. Following extraction and addition of isotopically labeled internal standards, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The method was validated according to regulatory recommendations, and the assay was evaluated in remnant samples from an HIV prevention trial in which paired DBS and plasma samples were collected.
DBS CAB and RPV concentrations were linear from 25 to 20,000 ng/mL and 2–2500 ng/mL, respectively. Precision, accuracy, and matrix effect results were acceptable. DBS RPV demonstrated stability under all tested conditions; DBS CAB showed mean biases of − 23.5% when stored at room temperature for 36 days, and − 18.0% at 40 °C and 100% humidity for two days. DBS measurements for CAB and RPV were an average 54.0% and 14.1% lower, respectively, as compared to paired plasma samples. Derived conversion factors of 1.79 and 1.16 were applied to DBS CAB and RPV measurements, respectively, to estimate plasma concentrations. Estimated plasma CAB and RPV concentrations showed mean biases of 2.2% and 0.6%, respectively. In a CAB clinical trial, application of the conversion factor resulted in agreement between estimated plasma CAB concentrations from DBS and plasma CAB concentrations (y = 1.08x - 79.2, r = 0.932; mean bias of −3.2%; 95% CI: −48.2% to 41.9%).
We developed and validated a novel LC-MS/MS assay for the quantification of CAB and RPV from DBS, and identified conversion factors to estimate plasma concentrations from spotted blood.
•Development and validation of an LC-MS/MS method for CAB and RPV quantification from DBS.•Determination of correction factors to estimate plasma CAB and RPV concentrations from DBS.•Pre-analytical considerations for accurate measurements of CAB and RPV from DBS.
Global efforts aimed at preventing human immunodeficiency virus type one (HIV-1) infection in vulnerable populations appear to be stalling, limiting our ability to control the epidemic. Long-acting, ...controlled drug administration from subdermal implants holds significant potential by reducing the compliance burden associated with frequent dosing. We, and others, are exploring the development of complementary subdermal implant technologies delivering the potent prodrug, tenofovir alafenamide (TAF). The current report addresses knowledge gaps in the preclinical pharmacology of long-acting, subdermal TAF delivery using several mouse models. Systemic drug disposition during TAF implant dosing was explained by a multi-compartment pharmacokinetic (PK) model. Imaging mass spectrometry was employed to characterize the spatial distribution of TAF and its principal five metabolites in local tissues surrounding the implant. Humanized mouse studies determined the effective TAF dose for preventing vaginal and rectal HIV-1 acquisition. Our results represent an important step in the development of a safe and effective TAF implant for HIV-1 prevention.
Long-acting cabotegravir is approved for pre-exposure prophylaxis and combination HIV treatment, both initiated with optional short-term oral lead-in (OLI). We evaluated the impact of OLI on ...long-acting cabotegravir pharmacokinetics. Cabotegravir plasma concentrations were compared between HIV-positive participants initiating injections with (
= 278) or without (
= 110) OLI in phase III treatment study FLAIR and in HIV-negative participants using OLI (
= 263) in pivotal pre-exposure prophylaxis studies HPTN 083 and HPTN 084. Cabotegravir pharmacokinetic profiles were simulated in three populations (assigned-male-at-birth, 50%-assigned-female-at-birth, and assigned-female-at-birth) under three scenarios: first injection given (A) 1 or (B) 3 days after final OLI dose (OLI-injection gap) or (C) without OLI. The PK objective was 80% of participants achieving 4×
protein-adjusted 90% maximal inhibitory concentration (PA-IC
) and 50% achieving 8× PA-IC
. Observed trough concentrations (
) were similar with and without OLI (
> 0.3). With a 3-day OLI-injection gap, simulated pre-injection
remained above PK objective. Approximately 1-2 weeks after the first injection, simulated PK profiles became nearly identical among all scenarios. Without OLI, it was predicted that 80% of participants achieve 4× PA-IC
in 1.2, 1.8, and 2.8 days after the first injection in each population, respectively, and 50% achieve 8× PA-IC
in 1.4, 2.1, and 3.8 days, respectively. Observed long-acting cabotegravir exposure was similar with or without OLI, supporting optional OLI use. Cabotegravir exposure was predicted to remain above PK objective for OLI-injection gaps of ≤3 days and rapidly achieve PK objective after first injection without OLI. Findings are consistent between assigned-male-at-birth and assigned-female-at-birth populations.This study is registered with ClinicalTrials.gov as NCT02720094.
Abstract
Background
HIV Prevention Trials Network 084 demonstrated that long-acting injectable cabotegravir (CAB) was superior to daily oral tenofovir (TFV) disoproxil fumarate (TDF)/emtricitabine ...(FTC) for preventing human immunodeficiency virus (HIV) infection in sub-Saharan African women. This report describes HIV infections that occurred in the trial before unblinding.
Methods
Testing was performed using HIV diagnostic assays, viral load testing, a single-copy RNA assay, and HIV genotyping. Plasma CAB, plasma TFV, and intraerythrocytic TFV-diphosphate concentrations were determined by liquid chromatography–tandem mass spectrometry.
Results
Forty HIV infections were identified (CAB arm, 1 baseline infection, 3 incident infections; TDF/FTC arm, 36 incident infections). The incident infections in the CAB arm included 2 with no recent drug exposure and no CAB injections and 1 with delayed injections; in 35 of 36 cases in the TDF/FTC arm, drug concentrations indicated low or no adherence. None of the cases had CAB resistance. Nine women in the TDF/FTC arm had nonnucleoside reverse-transcriptase inhibitor resistance; 1 had the nucleoside reverse-transcriptase inhibitor resistance mutation, M184V.
Conclusions
Almost all incident HIV infections occurred in the setting of unquantifiable or low drug concentrations. CAB resistance was not detected. Transmitted nonnucleoside reverse-transcriptase inhibitor resistance was common; 1 woman may have acquired nucleoside reverse-transcriptase inhibitor resistance from study drug exposure.
Human immunodeficiency virus (HIV) infections were characterized in women receiving long-acting cabotegravir or oral tenofovir disoproxil fumarate/emtricitabine for HIV prevention. Almost all incident HIV infections occurred in the setting of unquantifiable or low drug concentrations. Cabotegravir resistance was not detected.
Oral preexposure prophylaxis (PrEP) trials report disparate efficacy attributed to variable adherence. HPTN 066 was conducted to establish objective, quantitative benchmarks for discrete, regular ...levels of adherence using directly observed dosing of tenofovir (TFV) disoproxil fumarate (TDF)/emtricitabine (FTC). Healthy, HIV-uninfected men and women were randomized to one of four oral regimens of fixed-dose TDF 300 mg/FTC 200 mg tablet for 5 weeks with all doses observed: one tablet weekly (one/week), one tablet twice weekly (two/week), two tablets twice weekly (four/week), or one tablet daily (seven/week). Trough serum TFV and FTC, peripheral blood mononuclear cell (PBMC), and CD4(+) TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) concentrations were determined throughout dosing and 2 weeks after the last dose. Rectosigmoidal, semen, and cervicovaginal samples were collected for drug assessment at end of dosing and 2 weeks later in a subset of participants. The 49 enrolled participants tolerated the regimens well. All regimens achieved steady-state concentrations by the second dose for serum TFV/FTC and by 7 days for PBMC TFV-DP/FTC-TP. Steady-state median TFV-DP predose concentrations demonstrated dose proportionality: one/week 1.6 fmol/10(6) PBMCs, two/week 9.1, four/week 18.8, seven/week, 36.3. Further, TFV-DP was consistently quantifiable 2 weeks after the last dose for the ≥4/week regimens. Adherence benchmarks were identified using receiver operating characteristic curves, which had areas under the curve ≥0.93 for all analytes in serum and PBMCs. Intersubject and intrasubject coefficients of variation (%CV) ranged from 33% to 63% and 14% to 34%, respectively, for all analytes in serum and PBMCs. Steady-state PBMC TFV-DP was established earlier and at lower concentrations than predicted and was the only analyte demonstrating predose concentration dose proportionality. Steady-state daily dosing serum TFV and PBMC TFV-DP was consistent with highly effective PrEP clinical trials. HPTN 066 provides adherence benchmarks for oral TFV/FTC regimens to assist interpreting study outcomes.
Peritoneal dialysis (PD) is an effective renal replacement modality in people with HIV (PWH) with end-stage kidney disease (ESKD), particularly those with residual kidney function. Data on ...pharmacokinetics (PK) of antiretrovirals in patients on peritoneal dialysis are limited.
A single-participant study was performed on a 49-year-old gentleman with ESKD on PD and controlled HIV on once daily dolutegravir (DTG) 50 mg + tenofovir alafenamide (TAF) 25 mg / emtricitabine (FTC) 200 mg. He underwent serial blood plasma, peripheral blood mononuclear cell, and urine PK measurements over 24 h after an observed DTG + FTC/TAF dose.
Plasma trough (Cmin) concentrations of TAF, tenofovir (TFV), FTC, and DTG were 0.05, 164, 1,006, and 718 ng/mL, respectively. Intracellular trough concentrations of TFV-DP and FTC-TP were 1142 and 11,201 fmol/million cells, respectively. Compared to published mean trough concentrations in PWH with normal kidney function, observed TFV and FTC trough concentrations were 15.5- and 20-fold higher, while intracellular trough concentrations of TFV-DP and FTC-TP were 2.2-fold and 5.4-fold higher, respectively. TFV and FTC urine levels were 20 times lower than in people with normal GFR.
In a single ESKD PWH on PD, daily TAF was associated with plasma TFV and intracellular TFV-DP trough concentrations 15-fold and 2-fold higher than those of people with uncompromised kidney function, potentially contributing to nephrotoxicity. This suggests that TFV accumulates on PD; thus, daily TAF in PD patients may require dose adjustment or regimen change to optimize treatment, minimize toxicity, and preserve residual kidney function.
A relationship between the vaginal microbiota and tenofovir (TFV) concentrations and activity after topical administration has been previously reported.
CONRAD A15-138 was a randomized, ...placebo-controlled Phase I study aimed at characterizing the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of TFV and levonorgestrel (LNG) administered through a vaginal ring (IVR) for 90 days. Herein, we describe changes from baseline in the vaginal microbiota with IVR use and the impact of the vaginal microbiota on mucosal TFV PK.
The study screened 68 participants and randomized 47 (37 TFV/LNG, 10 placebo), assessing the vaginal microbiota by sequencing the V3-V4 regions of 16S rRNA genes prior to IVR insertion and monthly for 3 months. Concentrations of TFV in vaginal fluid (VF), and TFV and TFV-diphosphate (TFV-DP) in vaginal tissue, and modeled PD against HIV-1
were measured before and after treatment.
There were no clinically significant changes in relative abundance of vaginal bacterial phylotypes from pre-insertion baseline at any month among active and placebo IVR users. There were no significant changes in community state type (CST) with IVR use. Participants with diverse, anaerobic CST IVA/B microbiota had higher
release of TFV from the IVR compared to women with
-dominated (LbD) microbiota, who had expected
TFV release rates. Median VF TFV concentrations were significantly higher among women with CST IVA/B microbiota in months 1 (3,135 ng/mg VF) and 2 (3,800 ng/mg). Women with LbD microbiota had significantly higher median VF TFV concentration (1,423 ng/mg) and median TFV (103 ng/mg) and TFV-DP (5,877 fmol/mg) tissue concentrations versus women with CST IVA/B microbiota at month 3. All women demonstrated a significant increase from pre-insertion baseline of
HIV-1 inhibition by VF (p values <0.05). PD differences in tissue according to CST, however, were not statistically significant.
TFV/LNG IVR use did not change the vaginal microbiota nor increase the incidence of CST IVA/B. Vaginal microbiota, and in particular CST IVA/B, possibly through increased vaginal pH, impacted
TFV release and cervicovaginal (CV) PK, but both PK and PD data suggest CV protection against HIV-1.
ClinicalTrials.gov (#NCT03279120).
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