Therapeutic drug monitoring of cefepime with micellar electrokinetic capillary chromatography: Assay improvement, quality assurance, and impact on patient drug levels

The improvement and performance of a micellar electrokinetic capillary chromatography assay for cefepime in human serum and plasma with a 50 μm id fused-silica capillary elongated from 40 to 60 cm is reported. Sample preparation with dodecylsulfate protein precipitation at pH 4.5, the pH 9.1 separation medium and the applied voltage were as reported previously[16]. The change resulted in a significant lower current, higher resolution and increased detection time intervals. The performance of the assay with multi-level internal calibration was assessed with calibration and control samples. Quality assurance data of a two year period assessed under the new conditions demonstrated the robustness of the assay. In serum samples of patients who received both cefepime and sulfamethoxazole, cefepime could not be detected due to the inseparability of the two compounds. The presence of an interference can be recognized by an increased peak width (width > 0.2 min), the appearance of a shoulder or an unresolved double peak. The patient data gathered during a three year period reveal that introduction of therapeutic drug monitoring led to a 50% reduction of the median drug level. The data suggest that therapeutic drug monitoring can help to minimize the risk of major adverse reactions and to increase drug safety on an individual basis. This article is protected by copyright. All rights reserved.

Use of a novel DNA melting profile assay for the identification of PCR-amplified genomic sequences encoding for variant-specific surface proteins from the clonal GS/M-83-H7 line of Giardia lamblia.

During infections, Giardia lamblia undergoes a continuous change of its major surface antigens, the variant-specific surface proteins (VSPs). Many studies on antigenic variation have been performed using G. lamblia clone GS/M-83-H7, which expresses surface antigen VSP H7. The present study was focused on the identification and characterization of vsp gene sequences within the genome of the clonal G. lamblia GS/M-83-H7 line. For this purpose, we applied a PCR which specifically amplified truncated sequences from the 3'-terminal region of the vsp genes. Upon cloning, most of the vsp gene amplification products were shown to be approximately identical in size and thus could not be distinguished from each other by conventional gel electrophoresis. In order to pre-estimate the sequence complexity within the large panel of vsp clones isolated, we elaborated a novel concept which facilitated our large-scale genetic screening approach: PCR products from cloned DNA molecules were generated and then subjected to a DNA melting profile assay based on the use of the LightCycler Instrument. This high-throughput assay system proved to be well suited to monitor sequence differences between the amplification products from closely related vsp genes and thus could be used for the primary, sequence-related discrimination of the corresponding clones. After testing 50 candidates, vsp clones could be divided into five groups, each characterized by an individual DNA melting profile of the corresponding amplification products. Sequence analysis of some of these 50 candidates confirmed data from the aforementioned assay in that clones were demonstrated to be identical within, but different between, the distinct groups. The nucleotide and deduced amino acid sequences of five representative vsp clones showed high similarities both among each other and also with the corresponding gene segment of the variant-specific surface antigen (VSP H7) expressed by the original GS/M-83-H7 variant type. Furthermore, three of the genomic vsp sequences turned out to be identical to vsp sequences that represented previously characterized transcription products from in vivo- or in vitro-switched GS/M-83-H7 trophozoites. In conclusion, the DNA melting profile assay seems to be a versatile tool for the PCR-based genotyping of moderately or highly diversified sequence orthologues.

Zebrafish Caudal Fin Angiogenesis Assay-Advanced Quantitative Assessment Including 3-Way Correlative Microscopy.

BACKGROUND Researchers evaluating angiomodulating compounds as a part of scientific projects or pre-clinical studies are often confronted with limitations of applied animal models. The rough and insufficient early-stage compound assessment without reliable quantification of the vascular response counts, at least partially, to the low transition rate to clinics. OBJECTIVE To establish an advanced, rapid and cost-effective angiogenesis assay for the precise and sensitive assessment of angiomodulating compounds using zebrafish caudal fin regeneration. It should provide information regarding the angiogenic mechanisms involved and should include qualitative and quantitative data of drug effects in a non-biased and time-efficient way. APPROACH & RESULTS Basic vascular parameters (total regenerated area, vascular projection area, contour length, vessel area density) were extracted from in vivo fluorescence microscopy images using a stereological approach. Skeletonization of the vasculature by our custom-made software Skelios provided additional parameters including "graph energy" and "distance to farthest node". The latter gave important insights into the complexity, connectivity and maturation status of the regenerating vascular network. The employment of a reference point (vascular parameters prior amputation) is unique for the model and crucial for a proper assessment. Additionally, the assay provides exceptional possibilities for correlative microscopy by combining in vivo-imaging and morphological investigation of the area of interest. The 3-way correlative microscopy links the dynamic changes in vivo with their structural substrate at the subcellular level. CONCLUSIONS The improved zebrafish fin regeneration model with advanced quantitative analysis and optional 3-way correlative morphology is a promising in vivo angiogenesis assay, well-suitable for basic research and preclinical investigations.

Application of a human tumor colony forming assay to new drug screening.

Mode of access: Internet.

Routine testing of samples for radioactivity in mills and assay offices in the United States : a progress report /

"August 1953."

Illustrated history of the United States mint with short historical sketches and illustrations of the branch mints and assay offices, and a complete description of American coinage ...

Mode of access: Internet.

Chaffers' Handbook to hall marks on gold & silver plate, with revised tables of the annual date letters employed in the assay offices of England, Scotland and Ireland.

"First issued in 1897."

Ionization chamber assay of radioactive gases /

Contract No. W-7405-eng-48.

The vitamin directory; a complete, authoritative description and trade listing of all vitamin materials, assay methods and instruments, experimental animals, reference materials and reagents.

Cover-title.

A philosophical and practical essay on the gold and silver mines of Mexico and Peru : containing the nature of the ore, and the manner of working the mines, the qualities and use of quicksilver, the cleansing and refining these metals : with many useful observations concerning the assay of metals, the manner of conveying them to Europe, and remarks on the gold mines in Hungary, and those of Asia and Africa /

No Spanish ed. in: NUC pre-1956, BLC, Sabin, Palau y Dulcet (2nd ed.).

A tissue enzyme assay for chlorinated hydrocarbon insecticides.

"Project 16030 ELZ; Program element 1BA027."

Comparison of bioassay and high performance liquid chromatographic assay of artesunate and dihydroartemisinin in plasma

The study was a comparison of bioassay and HPLC analysis of artesunate (ARTS) and dihydroartemisinin (DHA) in plasma. ARTS and DHA in plasma samples from patients treated with ARTS were quantified by HPLC and expressed as DHA. DHA-equivalents in the same plasma samples were measured using a standardised parasite culture technique. DHA concentrations estimated by both methods were highly correlated (bioassay = 0.96 x HPLC + 11.0; r(2) = 0.92). At high concentrations ( > 12 000 nmol/l) bioassay sometimes overestimated DHA. Bioassay of active drug in plasma correlates well with specific chemical analysis by HPLC. ARTS and DHA appear to account for the total antimalarial activity in plasma after ARTS administration. (C) 2003 Elsevier Science B.V. All rights reserved.

Simple In Vitro Assay for Determining the Sensitivity of Plasmodium vivax Isolates from Fresh Human Blood to Antimalarials in Areas where P. vivax Is Endemic

The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P.falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB(+) serum. We successfully carried out 34 in vitro drug assays on 39 P. vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean percentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% +/- 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P. vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC50] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5 ng/ml, IC50 = 0.47 ng/ml). The poor response of P. vivax to both tafenoquine (MIC = 14,000 ng/ml, IC50 = 9,739 ng/ml) and sulfadoxine (MIC = 500,000 ng/ml, IC50 = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P. vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assessing the antimalarial sensitivity of P. vivax populations and for screening new antimalarials in the absence of long-term P. vivax cultures.

A simplified endogenous erythroid colony assay for the investigation of polycythaemia

The in vitro growth of erythroid colonies in the absence of erythropoietin, known as endogenous erythroid colonies (EEC) forms part of the diagnostic criteria for polycythaemia vera (PV). The availability of EEC culture in routine laboratory setting is limited as culture methods are technically demanding, difficult to standardize, expensive and laborious. In this study, we assessed the performance characteristics of a simplified method using ammonium chloride red cell lysis followed by culture on commercially available, batch-tested, methylcellulose media. Seventy-six patients were included; four were secondarily excluded on the basis of culture failure. Of the 14 patients with PV, 13 (93%) were positive for EEC on at least one occasion: 90% (nine of 10) of bone marrow and 67% (six of nine) of peripheral blood specimens were positive. All 30 patients with secondary polycythaemia (n = 12) or apparent polycythaemia (n = 18) were negative for EEC. The incidence of EEC in idiopathic erythrocytosis was 40% (eight of 28); 50% (five of 10) in those who met one of the minor criteria for PV and 17% (three of 18) in those who did not. We conclude that our EEC assay yield results comparable with that of more elaborate methods.