title: Accuracy of Turbidimetric Limulus Amebocyte Lysate Assay for the Recovery of Endotoxin Interacted with Commonly Used Antimicrobial Agents of Endodontic Therapy.
abstract: BACKGROUND
  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 (O55: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.
authors.affiliation: Department of Restorative Dentistry, Endodontic Division, Piracicaba Dental School-State University of Campinas, Piracicaba, São Paulo, Brazil.
authors.forename: Ariane C S
authors.initials: AC
authors.lastname: Marinho
authors.affiliation: Lonza do Brasil Especialidades Químicas, Brooklin Novo, São Paulo, Brazil.
authors.forename: Ana R O
authors.initials: AR
authors.lastname: Polay
authors.affiliation: Department of Restorative Dentistry, Endodontic Division, Piracicaba Dental School-State University of Campinas, Piracicaba, São Paulo, Brazil. Electronic address: bpgomes@fop.unicamp.br.
authors.forename: Brenda P F A
authors.initials: BP
authors.lastname: Gomes
mesh_terms: D000890:Anti-Infective Agents / Q000008:administration & dosage / Q000737:chemistry*; D004731:Endotoxins / Q000032:analysis*; D006801:Humans; D008033:Limulus Test; D012388:Root Canal Irrigants / Q000008:administration & dosage / Q000737:chemistry*; D018915:Root Canal Preparation / Q000379:methods
chemical_list: D000890:Anti-Infective Agents; D004731:Endotoxins; D012388:Root Canal Irrigants
keywords: Endotoxin; intracanal medicaments; irrigants; limulus amebocyte lysate methods; root canal