Clinical Evaluation of a Dried Blood Spot Assay for Atazanavir

Abstract

Current procedures for obtaining and measuring plasma concentrations of HIV protease inhibitors (PIs) are technically challenging. Dried blood spot (DBS) assays offer a way to overcome many of the obstacles. We sought to develop a DBS assay for quantitation of the PI atazanavir (ATV) and to compare this method with a previously validated plasma assay. We prospectively enrolled 48 patients with well-controlled HIV disease who had been on ATV for at least 7 days. ATV was quantified from plasma by use of high-performance liquid chromatography (HPLC). A reversed-phase ultrahigh-performance liquid chromatography (UPLC) assay was utilized for DBS samples. The concentrations of ATV quantified in a DBS matrix showed very strong agreement with those measured in plasma (r 2 ‫؍‬ 0.988). The mean difference in ATV concentration between the two methods was ؊10.8% (95% confidence interval [95% CI], ؊7.65% to ؊13.95%), indicating that the DBS method has a slight negative bias. A majority (97.8%) of the differences in concentration between the two assays fell within ؎2 standard deviations. ATV concentrations were lower in subjects who had detectable HIV RNA in plasma (mean, 543 ng/ml) than in those with HIV RNA of <50 copies/ml (mean, 1,582 ng/ml) (P ‫؍‬ 0.03, Wilcoxon rank-sum test). In conclusion, our study demonstrated that ATV quantitation in a DBS matrix is feasible and accurate. DBS use offers a convenient alternative for measuring plasma concentrations of ATV and may have utility in monitoring of drug concentrations in clinical practice and in future studies.Presently, approximately 33 million persons are living with human immunodeficiency virus (HIV) disease (35). Treatment options for HIV disease have expanded over the last 15 years, particularly with the introduction of protease inhibitors (PIs) as a component of combination antiretroviral therapy (ART). Use of these agents has been associated with significant decreases in morbidity and mortality (13,16,29). Despite the efficacy of PIs, a substantial number of patients still experience virological failure (23). PIs show significant interindividual pharmacokinetic variability for identical dosing regimens (7,14,31,33). High PI concentrations have been associated with toxicity, while subtherapeutic concentrations have been associated with virologic failure (2,3,9,11,12,28,30,31,32). These findings have led to interest in the use of therapeutic drug monitoring (TDM), which individualizes therapy to maximize outcomes and minimize toxicity (1, 7, 10). Currently, the literature does not support and guidelines do not recommend routine use of TDM in HIV-infected adults (8, 21).Atazanavir (ATV) is an azapeptide PI approved for use in both treatment-naïve and treatment-experienced patients (18). It has the advantage of being dosed once a day and can be used with or without ritonavir (RTV), although coadministration of RTV is preferred (8). The current techniques for quantitation of ATV (as well as all other PIs) are plasma-or serum-based analytical procedures. These procedures ...

Use of dried blood spots for the determination of plasma concentrations of nevirapine and efavirenz

“…Previously, four studies evaluated the correlation between antiretroviral drug concentrations in DBS and plasma. Van Schooneveld et al 16 demonstrated in 48 individual patient samples that atazanavir DBS concentrations correlated well with plasma concentrations (r 2 ¼0.988), although DBS concentrations were slightly lower (210.8%) than plasma concentrations. Koal et al 17 evaluated 70 patient samples (lopinavir, atazanavir, ritonavir, saquinavir and efavirenz) for DBS and plasma concentrations.…”

Adherence Measurements in HIV: New Advancements in Pharmacologic Methods and Real-Time Monitoring

“…Recently, the use of DBS for the pharmacokinetic analysis of antiretroviral drugs has become a novel and attractive approach in the research and clinical settings. Early on, multiple DBS methods to measure drug concentrations of various antiretroviral drugs were developed using liquid chromatography, with a primary focus on the quantification of parent drug as an alternative to plasma drug concentrations[37–46]. A recent example of this approach is the ENCORE1 study, a multicenter, randomized clinical trial that evaluated the safety and efficacy of a reduced dose of efavirenz (EFV) in resource-limited settings.…”

Use of dried clinical samples for storing and detecting influenza RNA

“…Dried specimens, in general, have been employed in other areas of clinical and laboratory research. DBS have been used for over 20 years for newborn screening programs 18 and have been used for therapeutic drug monitoring 19,20 . Advances in RNA extraction methods and molecular tests have allowed dried blood or plasma spots to be used for diagnosis and monitoring of HIV infection, 8–10 assessment of drug resistance mutations, 7,8,21 HCV prevalence by antibody screening or RNA detection, 22,23 hepatitis A virus monitoring, 24 and detection of measles virus RNA 25 .…”

“…DBS have been used for over 20 years for newborn screening programs 18 and have been used for therapeutic drug monitoring. 19,20 Advances in RNA extraction methods and molecular tests have allowed dried blood or plasma spots to be used for diagnosis and monitoring of HIV infection, 8-10 assessment of drug resistance mutations, 7,8,21 HCV prevalence by antibody screening or RNA detection, 22,23 hepatitis A virus monitoring, 24 and detection of measles virus RNA. 25 While a typical Guthrie card contains 50-100 ll of blood or plasma per spot (250-500 ll total), an ST can hold up to 1 ml of sample, allowing for multiple analyses from a single specimen.…”