Accuracy of Turbidimetric Limulus Amebocyte Lysate Assay for the Recovery of Endotoxin Interacted with Commonly Used Antimicrobial Agents of Endodontic Therapy

Author(s)

Marinho, Ariane C. S. ^[1] ; Polay, Ana R. O. ^[2] ; Gomes, Brenda P. F. A. ^[1]

Affiliation

^[1] Univ Estadual Campinas, Piracicaba Dent Sch, Endodont Div, Dept Restorat Dent, BR-13414903 Piracicaba, SP - Brazil

^[2] Lonza Brasil Especialidades Quim, Sao Paulo - Brazil

• Document type: Journal article
• Source: JOURNAL OF ENDODONTICS; v. 41, n. 10, p. 1653-1659, OCT 2015.
• Web of Science Citations: 6

Abstract

Introduction: This study was conducted to investigate whether the interaction between the turbidimetric limulus amebocyte lysate (LAL) substrate for endotoxin measurement and the substances/antimicrobial agents used in endodontic therapy can lead to the inhibition/enhancement of endotoxin recovery. Methods: Ten microliters of a suspension of Escherichia coli endotoxin (055:B55) was inoculated and kept in contact for 1 hour with different substances categorized as follows: group 1: auxiliary chemical substances: 5.25% and 2.5% sodium hypochlorite solutions, 2% chlorhexidine (CHX) (gel and solution), 1% Natrosol gel (Drogal Chemicals and Pharmaceuticals Ltd, Piracicaba, SP, Brazil), 17% EDTA, 10% citric acid, 3% hydrogen peroxide, 5% sodium thiosulfate, and 0.5% Tween 80 associated with 0.07% soy lecithin (Drogal Chemicals and Pharmaceuticals Ltd) and group 2: intracanal medications: neomycin/polymyxin B/hydrocortisone (Otosporin; Glaxo Wellcome, Rio de Janeiro, RJ, Brazil); calcium hydroxide (Ca{[}OH](2)); Ca(OH)(2) + 2% CHX gel; Ca(OH)(2) + 2% CHX gel + zinc oxide eugenol; Ca(OH)(2) + camphorated paramonochlorophenol (Calen; S.S. White, Rio de Janeiro, RJ, Brazil); triple antibiotic paste; mineral trioxide aggregate (MTA); and iodoform. Positive and negative controls consisted of root canal hemorrhagic exudate and pyrogen-free sterile water, respectively. All samples were diluted up to a 10:4 dilution. Each dilution was individually examined by the turbidimetric LAL assay. Collected data were analyzed through performance characteristics of the LAL assay such as linearity, coefficient of variation percentage, and product positive control (PPC) values. Results: Correlation coefficient (>= 0.980) and coefficient of variation percentage (<10%) of the standard curve in triplicate showed the tests' linearity. Spike recovery of auxiliary chemical substances achieved PPC values ranging from 50%-197%, showing no interferences with LAL substrate. Conversely, 3% hydrogen peroxide achieved product inhibition in which endotoxin values were underestimated even after the 10:4 dilutions. Regarding intracanal medicaments, neomycin/polymyxin B/hydrocortisone also inhibited endotoxin detection in all dilutions investigated (PPC values <50%). In contrast, Ca(OH)(2) + 2% CHX gel + ZOE as well as triple antibiotic paste led to the enhancement of endotoxin detection in which endotoxin values could not be validated by the turbidimetric kinetic LAL assay (PPC value >200%). Conclusions: The performance characteristics of the kinetic turbidimetric assay for endotoxin measurement, such as precision and reproducibility, are modulated by the interaction of the LAL substrate with the substances/antimicrobial agents used in endodontic therapy. (AU)

• FAPESP's process: 10/19136-1 - Microbiological and endotoxins analysis in primarily infected root canals with apical periodontitis and their antigenic potencial against macrophages to produce IL1-beta e TNF-alfa during endodontic treatment
• Grantee: Brenda Paula Figueiredo de Almeida Gomes
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/02402-9 - Microbiological and LPS investigation and their antigenic activity in teeth with pulpal necrosis and irreversible pulpitis in pro-inflammatory cytokine production
• Grantee: Ariane Cássia Salustiano Marinho
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 10/17877-4 - Microbiological analysis and quantification of endotoxins and proinflammatory cytokines from primarily infected root canals with apical periodontitis
• Grantee: Brenda Paula Figueiredo de Almeida Gomes
• Support Opportunities: Regular Research Grants