Validation and Application of a Dried Blood Spot Assay for Biofilm-Active Antibiotics Commonly Used for Treatment of Prosthetic Implant Infections

Abstract

d Dried blood spot (DBS) antibiotic assays can facilitate pharmacokinetic (PK)/pharmacodynamic (PD) studies in situations where venous blood sampling is logistically difficult. We sought to develop, validate, and apply a DBS assay for rifampin (RIF), fusidic acid (FUS), and ciprofloxacin (CIP). These antibiotics are considered active against organisms in biofilms and are therefore commonly used for the treatment of infections associated with prosthetic implants. A liquid chromatography-mass spectroscopy DBS assay was developed and validated, including red cell partitioning and thermal stability for each drug and the rifampin metabolite desacetyl rifampin (Des-RIF). Plasma and DBS concentrations in 10 healthy adults were compared, and the concentration-time profiles were incorporated into population PK models. The limits of quantification for RIF, Des-RIF, CIP, and FUS in DBS were 15 g/liter, 14 g/liter, 25 g/liter, and 153 g/liter, respectively. Adjusting for hematocrit, red cell partitioning, and relative recovery, DBS-predicted plasma concentrations were comparable to measured plasma concentrations for each antibiotic (r > 0.95; P < 0.0001), and Bland-Altman plots showed no significant bias. The final population PK estimates of clearance, volume of distribution, and time above threshold MICs for measured and DBS-predicted plasma concentrations were comparable. These drugs were stable in DBSs for at least 10 days at room temperature and 1 month at 4°C. The present DBS antibiotic assays are robust and can be used as surrogates for plasma concentrations to provide valid PK and PK/PD data in a variety of clinical situations, including therapeutic drug monitoring or studies of implant infections. P harmacokinetic (PK)/pharmacodynamic (PD) studies of infectious diseases explore the triangular relationship between antibiotic exposure, the antibiotic susceptibility of the infecting organism (taken as the MIC), and predefined clinical outcomes (1). This allows estimation of microbiological susceptibility "breakpoints," facilitates the design of optimal dosing regimens (2), and provides data relating to the emergence of drug-resistant organisms (3, 4). Most PK/PD studies are performed in animal models or in highly selected samples of adults, such as patients in intensive care units, for whom sequential sampling of sufficient volumes of venous blood is both feasible and ethical. In contrast, PK/PD studies are rarely performed on other hospitalized or ambulatory patient populations due to difficulties associated with blood sampling. Relevant examples include patients receiving treatment for implant infections, young children, and patients treated in nonurban or resource-poor settings.Measurement of drug concentrations in dried blood spots (DBSs) represents a new approach to overcome the limitations of traditional therapeutic drug monitoring and PK/PD studies. DBSs are a convenient and inexpensive means of sampling and storage of whole blood for subsequent assays. Low-volume finger prick samples (10 to 20 l) are taken on f...

Validation of a Dried Blood Spot Ceftriaxone Assay in Papua New Guinean Children with Severe Bacterial Infections

“…However, there is no guidance on the optimal populations, sample size, and reportable outcome requirements for clinical validation studies, sometimes termed bridging studies. Our approach is that, in addition to demonstration of in vitro feasibility and suitability of a microsampling assay, population-specific PK bridging studies should ideally be performed in healthy volunteers (7,8) and accessible patients with the infection of interest (15). This would confirm that, in addition to adequate correlations between plasma and DBS concentrations, important PK parameters also accord in clinical settings of interest.…”

“…One pertinent clinical situation is severely ill children in the tropics. Our published data suggest that studies using DBS could be performed with sampling blood volumes that were well within accepted limits and without the need for low temperature storage and transportation of samples (7,8). Children in tropical countries are, however, at high risk of anemia from a variety of genetic, nutritional, and infective causes.…”

Penicillin Dried Blood Spot Assay for Use in Patients Receiving Intramuscular Benzathine Penicillin G and Other Penicillin Preparations To Prevent Rheumatic Fever

“…Chromatographic data were processed using LAB Solution (Version 5.56; Shimadzu, Japan). Matrix effects (ion suppression/enhancement), absolute recovery, and process efficiency were determined at 0.1, 1, 10, and 100 mg/liter using our established method (13,14). The lower limit of quantification (LOQ) and lower limit of detection (LOD) were determined at signal-to-noise ratios of 10:1 and 3:1, respectively.…”

“…The matrix effect and process efficiency and recovery were assessed as described above for plasma. The blood-to-plasma partition ratio was determined at 0.1, 1, 10, and 100 mg/liter using a method established in our laboratory (13). Penicillin G stability in dried blood spots.…”

“…We have previously demonstrated the accuracy and feasibility of this method for assay of other antibiotics (13,14). Very low (20-l) volumes of whole blood can be collected from finger or heel prick samples and impregnated into filter paper, which is then stored in foil bags without the need for processing of plasma and immediate frozen storage.…”