Dose Dependent Antimicrobial Cellular Cytotoxicity-Implications for ex vivo Diagnostics

Area of Science:

• Immunology
• Pharmacology
• Diagnostic Assay Development

Background:

• Interferon-gamma (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) assays are vital for diagnosing severe T-cell mediated hypersensitivity.
• Limited data exists on antimicrobial-associated cellular cytotoxicity and its impact on ex vivo diagnostic assay performance.
• Understanding drug concentrations that preserve cell viability is essential for accurate drug causality assessment.

Purpose of the Study:

• To determine maximal antimicrobial concentrations that maintain cell viability for ex vivo IFN-γ ELISpot assays.
• To investigate the impact of antimicrobial concentrations on cell cytotoxicity using lactate dehydrogenase (LDH) and 7-AAD staining.
• To assess the implications of drug concentrations on ELISpot assay performance in hypersensitivity diagnostics.

Main Methods:

• Peripheral blood mononuclear cells (PBMCs) from healthy controls and patients with drug reactions were incubated with antimicrobials at varying concentrations (Cmax, 10x Cmax, 100x Cmax).
• Cell cytotoxicity was measured using lactate dehydrogenase (LDH) assay and 7-AAD cell viability staining via flow cytometry.
• IFN-γ ELISpot assay was used to assess drug-specific T-cell responses.

Main Results:

• Higher antimicrobial concentrations (10x and 100x Cmax) significantly increased cell cytotoxicity and reduced IFN-γ ELISpot sensitivity.
• LDH assay showed dose-dependent cytotoxicity for most tested antimicrobials, with >40% cell death at 100x Cmax for most drugs.
• 7-AAD staining confirmed increased lymphocyte death with higher drug concentrations for ceftriaxone and flucloxacillin, but not piperacillin/tazobactam or isoniazid.

Conclusions:

• Lactate dehydrogenase (LDH) and 7-AAD cell viability assays demonstrate that elevated antimicrobial concentrations induce cell death, compromising ELISpot assay sensitivity.
• Antimicrobial concentrations at Cmax and 10-fold Cmax negatively impact cell viability and ELISpot assay performance.
• Findings guide the selection of appropriate antimicrobial concentrations for ex vivo IFN-γ ELISpot assays to ensure diagnostic accuracy.