Dose Dependent Antimicrobial Cellular Cytotoxicity—Implications for ex vivo Diagnostics

Abstract

Introduction:Ex vivo and in vitro 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 ex vivo, 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.Method: 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 (Cmax), and concentrations 10- and 100-fold above.Results: In a human immunodeficiency virus (HIV) negative and a positive rifampicin-associated DRESS with positive ex vivo IFN-γ ELISpot assay, use of 10- and 100-fold Cmax 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 Cmax and 10-fold Cmax drug concentrations except flucloxacillin. All 100-fold Cmax 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 Cmax for ceftriaxone, and flucloxacillin; however, piperacillin/tazobactam and isoniazid indicated no differences in percentages of viable lymphocytes across concentrations tested.Conclusion: The LDH cytotoxicity and 7-AAD cell viability staining techniques both demonstrate increased cell death corresponding to a loss in ELISpot sensitivity, with use of higher antimicrobial drug concentrations for ex vivo diagnostic IFN-γ ELISpot assays. For all the antimicrobials evaluated, the use of Cmax and 10-fold Cmax concentrations impacts cell viability and potentially affects ELISpot performance. These findings inform future approaches for ex vivo diagnostics such as IFN-γ release ELISpot.

Eumelanin-Inspired Antimicrobial with Biocidal Activity against Methicillin-Resistant Staphylococcus aureus

“…A maximal cytotoxicity of 50% was seen at concentrations above 62.5 μg/mL (66.8 μM) for EIPE-1 , whereas 30% cytotoxicity was seen for DBI at 250 μg/mL (617 μM). Cytotoxicity increased in a dose-dependent manner with increasing EIPE-1 concentrations, similar to what has been observed in other antimicrobials . However, DBI exhibited no cytotoxicity in concentrations up to 125 μg/mL (Figure ).…”

“…Cytotoxicity increased in a dose-dependent manner with increasing EIPE-1 concentrations, similar to what has been observed in other antimicrobials. 29 However, DBI exhibited no cytotoxicity in concentrations up to 125 μg/mL (Figure 2). The cytotoxicity seen for EIPE-1 required 4−5fold greater concentrations and prolonged exposure (24 h) than the MIC determined against S. aureus, demonstrating that the amount of EIPE-1 needed to inhibit MRSA growth is well below concentrations that induce host cell cytotoxicity.…”

“…DBI was synthesized according to a reported procedure. 29 Et 3 N was freshly distilled before use. Unless otherwise specified, all reactions were conducted in oven-dried glassware under a nitrogen atmosphere.…”

“…All commercial reagents were used as received. DBI was synthesized according to a reported procedure . Et 3 N was freshly distilled before use.…”

Time to reinvent the wheel for drug causality diagnostic approaches to severe cutaneous adverse reactions

“…2 Historically, these were performed using the patients' peripheral blood mononuclear cells (PBMCs), but new research has shown increased sensitivity using blister fluid cells (BFCs), 4 varied cut-offs and utilising non-cytotoxic drug concentrations. 5,6 Both assays have acceptable sensitivity with ELISpots demonstrating a sensitivity of 52% in SCAR with a specificity of 100% and LTT reported sensitivity of 27%-74% and specificity of 85%-100%. 3 Further work is required to understand drug metabolites to increase the sensitivity of these assays, as shown with oxypurinol for allopurinol and 4-nitrosulfamethoxazole for TMP-SMX.…”