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Dose Dependent Antimicrobial Cellular Cytotoxicity-Implications for ex vivo Diagnostics

and diagnostics, such as interferon-γ (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) and flow cytometry, are increasingly employed in the research and diagnostic setting for severe T-cell mediated hypersensitivity. Despite an increasing use of IFN-γ release ELISpot for drug causality assessment...

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Published in:Frontiers in pharmacology 2021-08, Vol.12, p.640012-640012
Main Authors: Copaescu, Ana, Choshi, Phuti, Pedretti, Sarah, Mouhtouris, Effie, Peter, Jonathan, Trubiano, Jason A
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Language:English
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Summary:and diagnostics, such as interferon-γ (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) and flow cytometry, are increasingly employed in the research and diagnostic setting for severe T-cell mediated hypersensitivity. Despite an increasing use of IFN-γ release ELISpot for drug causality assessment and utilization of a range of antimicrobial concentrations , data regarding antimicrobial-associated cellular cytotoxicity and implications for assay performance remain scarcely described in the literature. Using the measurement of lactate dehydrogenase (LDH) and the 7-AAD cell viability staining, we aimed via an exploratory study, to determine the maximal antimicrobial concentrations required to preserve cell viability for commonly implicated antimicrobials in severe T-cell mediated hypersensitivity. After an 18-h incubation of patient peripheral blood monocytes (PBMCs) and antimicrobials at varying drug concentrations, the cell cytotoxicity was measured in two ways. A colorimetric based assay that detects LDH activity and by flow cytometry using the 7-AAD cell viability staining. We used the PBMCs collected from three healthy control participants with no known history of adverse drug reaction and two patients with a rifampicin-associated drug reaction with eosinophilia and systemic symptoms (DRESS), confirmed on IFN-γ ELISpot assay. The PBMCs were stimulated for the investigated drugs at the previously published drug maximum concentration ( ), and concentrations 10- and 100-fold above. In a human immunodeficiency virus (HIV) negative and a positive rifampicin-associated DRESS with positive IFN-γ ELISpot assay, use of 10- and 100-fold drug concentrations decreased spot forming units/million cells by 32-100%, and this corresponded to cell cytotoxicity of more than 40 and 20% using an LDH assay and 7-AAD cell viability staining, respectively. The other antimicrobials (ceftriaxone, flucloxacillin, piperacillin/tazobactam, and isoniazid) tested in healthy controls showed similar dose-dependent increased cytotoxicity using the LDH assay, but cytotoxicity remained lower than 40% for all and 10-fold drug concentrations except flucloxacillin. All 100-fold concentrations resulted in cell death >40% (median 57%), except for isoniazid. 7-AAD cell viability staining also confirmed an increase in lymphocyte death in PBMCs incubated with 10-fold and 100-fold above for ceftriaxone, and flucloxacillin; however, piperacillin/tazobactam and isoniazid indicated no differences in
ISSN:1663-9812
1663-9812
DOI:10.3389/fphar.2021.640012