Limulus Amebocyte Lysate Test Research Articles

Visual Detection of LPS at the Femtomolar Level Based on Click Chemistry-Induced Gold Nanoparticles Electrokinetic Accumulation.

Lipopolysaccharide (LPS) presents a significant threat to human health. Herein, a novel method for detecting LPS was developed by coupling hybridization chain reaction (HCR), gold nanoparticles (AuNPs) agglutination (AA) triggered by a Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry (CuAAC), and electrokinetic accumulation (EA) in a microfluidic chip, termed the HCR-AA-EA method. Thereinto, the LPS-binding aptamer (LBA) was coupled with the AuNP-coated Fe3O4 nanoparticle, which was connected with the polymer of H1 capped on CuO (H1-CuO) and H2-CuO. Upon LPS recognition by LBA, the polymers of H1- and H2-CuO were released into the solution, creating a "one LPS-multiple CuO" effect. Under ascorbic acid reduction, CuAAC was initiated between the alkyne and azide groups on the AuNPs' surface; then, the product was observed visually in the microchannel by EA. Finally, LPS was quantified by the integrated density of AuNP aggregates. The limit of detections were 29.9 and 127.2 fM for water samples and serum samples, respectively. The levels of LPS in the injections and serum samples by our method had a good correlation with those from the limulus amebocyte lysate test (r = 0.99), indicating high accuracy. Remarkably, to popularize our method, a low-cost, wall-power-free portable device was developed, enabling point-of-care testing.

Soy Protein Concentrate Diets Inversely Affect LPS-Binding Protein Expression in Colon and Liver, Reduce Liver Inflammation, and Increase Fecal LPS Excretion in Obese Zucker Rats.

Dietary soy protein and soy isoflavones have anti-inflammatory properties. Previously, we reported that feeding soy protein concentrate diet (SPC) with low or high isoflavone (LIF or HIF) to young (seven-week-old) obese (fa/fa) Zucker rats inhibits lipopolysaccharide (LPS) translocation and decreases liver inflammation compared to a casein control (CAS) diet. The current study investigated whether SPC-LIF and SPC-HIF diets would reduce liver inflammation in adult obese Zucker rats fed a CAS diet. A total of 21 six-week-old male obese (fa/fa) Zucker rats were given CAS diet for 8 weeks to develop obesity then randomly assigned to CAS, SPC-LIF, or SPC-HIF (seven rats/group) diet for an additional 10 weeks. The expression of LPS-translocation, inflammation, and intestinal permeability markers were quantified by qPCR in liver, visceral adipose tissue (VAT), and colon. LPS concentration was determined in both the colon content and fecal samples by a Limulus amebocyte lysate (LAL) test. SPC-LIF and SPC-HIF diets significantly decreased liver LPS-binding protein (LBP) expression compared to CAS diet (p < 0.01 and p < 0.05, respectively). SPC-HIF diet also significantly decreased liver MCP-1 and TNF-α expression (p < 0.05) and had a trend to decrease liver iNOS expression (p = 0.06). In the colon, SPC-HIF diet significantly increased LBP expression compared to CAS diet (p < 0.05). When samples from all three groups were combined, there was a negative correlation between colon LBP expression and liver LBP expression (p = 0.046). SPC diets did not alter the expression of intestinal permeability markers (i.e., occludin, claudin 3, and zonula occludens-1) in the colon or inflammation markers (i.e., TNF-α and iNOS) in VAT or the colon. LPS levels in the colon content did not differ between any groups. Fecal LPS levels were significantly higher in the SPC-LIF and SPC-HIF groups compared to the CAS group (p < 0.01). In conclusion, SPC, particularly SPC with HIF, reduces liver LBP expression and inflammation makers (i.e., TNF-α and MCP-1 expression) in adult obese Zucker rats, likely by reducing LPS translocation.

Analysis of commercially available snake antivenoms reveals high contents of endotoxins in some products

As injectable therapeutics, snake antivenoms must meet specifications for endotoxin content. The Limulus amebocyte lysate (LAL) test was used to evaluate the endotoxin content in several commercially available antivenoms released for clinical use. It was found that some products have endotoxin concentrations higher than the accepted limit for these contaminants. These results emphasize the need to include endotoxin determination as part of the routine evaluation of antivenoms by manufacturers and regulatory agencies.

Pre-Clinical Safety and Immunogenicity Study of a Coronavirus Protein-Based Subunit Vaccine for COVID-19.

Creating an effective and safe vaccine is critical to fighting the coronavirus infection successfully. Several types of COVID-19 vaccines exist, including inactivated, live attenuated, recombinant, synthetic peptide, virus-like particle-based, DNA and mRNA-based, and sub-unit vaccines containing purified immunogenic viral proteins. However, the scale and speed at which COVID-19 is spreading demonstrate a global public demand for an effective prophylaxis that must be supplied more. The developed products promise a bright future for SARS-CoV-2 prevention; however, evidence of safety and immunogenicity is mandatory before any vaccine can be produced. In this paper, we report on the results of our work examining the safety, toxicity, immunizing dose choice, and immunogenicity of QazCoVac-P, a Kazakhstan-made sub-unit vaccine for COVID-19. First, we looked into the product's safety profile by assessing its pyrogenicity in vaccinated rabbit models and using the LAL (limulus amebocyte lysate) test. We examined the vaccine's acute and sub-chronic toxicity on BALB/c mice and rats. The vaccine did not cause clinically significant toxicity-related changes or symptoms in our toxicity experiments. Finally, we performed a double immunization of mice, ferrets, Syrian hamsters, and rhesus macaques (Macaca mulatta). We used ELISA to measure antibody titers with the maximum mean geometric titer of antibodies in the animals' blood sera totaling approximately 8 log2. The results of this and other studies warrant recommending the QazCoVac-P vaccine for clinical trials.

Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.

Yeast Expressed Hybrid Peptide CLP Abridged Pro-Inflammatory Cytokine Levels by Endotoxin Neutralization.

The aim of this study was to apply a strategy to express a recombinant CLP peptide and explore its application as a product derived from natural compounds. The amphiphilic CLP peptide was hybridized from three parent peptides (CM4, LL37, and TP5) and was considered to have potent endotoxin-neutralizing activity with minimal cytotoxic and hemolytic activity. To achieve high secretion expression, an expression vector of pPICZαA-HSA-CLP was constructed by the golden gate cloning strategy before being transformed into Pichia pastoris and integrated into the genome. The recombinant CLP was purified through the Ni-NTA affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. The Limulus amebocyte lysate (LAL) test exhibited that the hybrid peptide CLP inhibited lipopolysaccharides (LPS) in a dose-dependent manner and was significantly (p < 0.05) more efficient compared to the parent peptides. In addition, it essentially diminished (p < 0.05) the levels of nitric oxide and pro-inflammatory cytokines (including TNF-α, IL6, and IL-1β) in LPS-induced mouse RAW264.7 macrophages. As an attendant to the control and the parental peptide LL37, the number of LPS-induced apoptotic cells was diminished compared to the control parental peptide LL37 (p < 0.05) with the treatment of CLP. Consequently, we concluded that the hybrid peptide CLP might be used as a therapeutic agent.

Endotoxins Affecting Human Health during Agricultural Practices: An Overview

Agricultural operations and the processing sector generate dust laden with endotoxin in the workplace. Endotoxin, a pro-inflammatory agent, has adverse effects on health, especially in the lungs, as exposure to endotoxin reduces lung function capacity. Endotoxin exposure to workers and its harmful impact on the health of agricultural workers needs to be studied in detail for future interventions to reduce exposure to endotoxin. The review can help to identify the analytical methods used to determine endotoxin exposure in agriculture. A detailed study of the research articles published in the last two decades related to agriculture and allied fields was carried out. In the agricultural sector, Pantoea agglomerans, a Gram-negative bacterium, was predominantly present. The filters were stored at a temperature of −20 °C, and E. coli 055: B5 was the predominately used standard to analyze the endotoxin. The quantitative kinetic Limulus Amebocyte Lysate test was the most common detection method for quantifying endotoxin. Control strategies to reduce endotoxin exposure are also emphasized in this review.

Effect of peracetic acid–ultraviolet combination treatment on microbial and endotoxin levels in a pharmaceutical water system

Abstract In the pharmaceutical industry, the microbiological quality of water is vital. This research investigated how combining peracetic acid (0.1% V/V) and UV light (&amp;gt;150 mJ/cm2) as wide-spectrum disinfectants affect microbial and endotoxin levels in a pharmaceutical water system. Water samples were taken aseptically from 12 points across the system. The pour plate technique and membrane filtration were used for microbial counts. The presence of endotoxin in distilled water samples was investigated by the Limulus amebocyte lysate (LAL) test gel-clot method. After peracetic acid–UV combination treatment, microbial counts of samples significantly decreased (P &amp;lt; 0.05) compared with UV treatment alone, and they were lower than the action limits specified by the European Pharmacopeia (100 CFU/ml for purified water and 10 CFU/100 ml for water for injection). In addition, water samples were mainly LAL-negative (10 negative weekly reports out of 12 total reports). It is concluded that disinfection of all stages of the water system with peracetic acid–UV combination remarkably improved the microbial quality of the water system. Therefore, rotation between more than one disinfectant policy and periodic disinfection of the water system by peracetic acid–UV combination is recommended to minimize contamination of the water system and pharmaceutical products as well as bacterial infections in product consumers.

Monomeric lipopolysaccharide (LPS) activity affected by dimerization of receptors is the key for the similarity between the Limulus amebocyte lysate (LAL) test and the rabbit pyrogen test

Endotoxin or lipopolysaccharide (LPS) triggers inflammatory cytokine production of mammal cells through Toll-like receptor 4 (TLR4) activation. This reaction causes pyrogenicity in rabbits. The rabbit pyrogen test has been used for the pyrogen detection in parenteral drugs and medical devices. Limulus amebocyte lysate (LAL) test, adopted as an alternative test for the rabbit pyrogen test in pharmacopoeias, has been safely used for more than 40 years. Even though the mechanisms of these biological assays are different, there should be similarity between the LAL test and the rabbit pyrogen test. Biological activity relates to the aggregation states of LPS, and there is still controversy whether the biologically active form of LPS is monomers or aggregates. Previous studies showed inconsistent results in biological activities of monomeric LPS. This suggests the difficulty to prepare monomeric LPS. Therefore, a simulation can be helpful to understand the activity of different status of LPS. In this study, the TLR4 activation was simulated by using simplified models, and compared the activity of LPS in different forms. The activation of TLR4 and the LAL activation require dimerization of receptors, which reduces the activity of monomeric LPS. The inert monomeric LPS caused by the dimerization of the receptors is the key to the similarity between the LAL test and the rabbit pyrogen test.

Monomeric lipopolysaccharide (LPS) activity affected by dimerization of receptors is the key for the similarity between the Limulus amebocyte lysate (LAL) test and the rabbit pyrogen test

Monomeric lipopolysaccharide (LPS) activity affected by dimerization of receptors is the key for the similarity between the Limulus amebocyte lysate (LAL) test and the rabbit pyrogen test

A Route to Translate a Silk-Based Medical Device from Lab to Clinic: The Silk Biomaterials Srl Experience.

Simple SummarySilk has always been a source of inspiration for textile designers to create very disruptive fashion products. More recently, silk has stimulated the creativity of scientists, as it emerged as a promising biomaterial for the development of next generation medical devices. Although silk has been clinically used as a suture for decades, only the development of novel processing approaches paved the way for the production of a plurality of regenerated silk-based materials, i.e., films, hydrogels, sponges, powder, nano- and microparticles, nanofibers, etc., which have been recognized as scaffolds-of-choice in different medical applications. However, the translation of research achievements into medical products used in clinical settings is complex from manufacturing, quality, and regulatory perspectives. The aim of this paper is to disclose how the clinical translation route works using, as a case study, a silk-based medical device recently developed by the Italian start up Silk Biomaterials srl. The results reported here will cover some fundamental aspects of the regulatory and quality path, from the demonstration of the robustness of the manufacturing process up to the evaluation of the biocompatibility, and of the functional performance of the device.The medical device is a nerve conduit entirely made of Bombyx mori silk fibroin. It is a tubular scaffold used for repairing peripheral nerve gaps, whose function is to protect the severed nerves and to favor their natural healing process. As any implantable medical device, the conduit must perform its function without causing adverse effects to the patient, meaning that it must be compliant with a range of regulations aimed at evaluating the risks related to the constituent materials and the manufacturing process, the toxicological impact of the processing aids, the biological safety, the functional performance, and the ability to sustain tissue regeneration processes. An exhaustive on-bench testing plan has been performed for the determination of the morphological, geometrical, physical, structural, and mechanical properties. For the toxicological analysis, the device was extracted with solvent and the number of leachable substances was determined by suitable chromatographic techniques. The biological safety was assessed by means of a set of tests, including cytotoxicity, delayed hypersensitivity, intracutaneous reactivity, pyrogen test, LAL (Limulus Amebocyte Lysate) test, acute systemic toxicity, and genotoxicity. Overall, the accumulated results demonstrated the suitability of the device for the intended use and supported the starting of a first-in-human clinical trial.

Blood Biomarkers and Serologic Immunological Profiles Related to Periodontitis in Abdominal Aortic Aneurysm Patients.

BackgroundPeriodontitis is a chronic inflammatory gum disease associated with systemic diseases such as cardiovascular diseases.AimTo investigate the association of systemic blood biomarkers, C-reactive protein (CRP), levels of lipopolysaccharide (LPS), and IgG levels against periodontal pathogens Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) with the stability, based on the aortic diameter, the growth rate and the eligibility for surgical intervention, of patients with abdominal aortic aneurysm (AAA).MethodsPatients with stable AAA (n = 30) and unstable AAA (n = 31) were recruited. The anti-A. actinomycetemcomitans and anti-P. gingivalis IgG levels were analyzed by ELISA, the LPS analysis was performed by using the limulus amebocyte lysate (LAL) test, and plasma levels of CRP were determined using an immune turbidimetric method. The association between these blood systemic biomarkers, AAA features, periodontal clinical parameters and oral microbial profiles were explored. Regression models were used to test the relationship between variables.ResultsThe presence of antibodies against Pg and Aa, LPS and high CRP concentrations were found in all AAA patients. The IgG levels were similar in patients with stable and unstable AAA (both for Aa and Pg). Among investigated blood biomarkers, only CRP was associated with AAA stability. The amount of LPS in saliva, supra, and subgingival plaque were significantly associated with the systemic LPS (p <0.05).ConclusionsThis post-hoc study emphasizes the presence of antibodies against Pg and Aa, LPS and high CRP concentrations in all AAA patients. The presence of Pg in saliva and subgingival plaque was significantly associated with the blood LPS levels. For further studies investigating periodontitis and systemic diseases, specific predictive blood biomarkers should be considered instead of the use of antibodies alone.

Quantitation of endotoxin by gas chromatography-mass spectrometry in Neisseria meningitidis serogroups A, C, W, Y and X during polysaccharide purification used in conjugate vaccine

Bacterial lipopolysaccharide (LPS) responsible for endotoxin effect induces inflammatory reactions. The endotoxins are difficult to separate from the gram-negative polysaccharide (PS) during polysaccharide purification. The most common method to quantify LPS is the limulus amebocyte lysate (LAL) test which interferes with the agents used during PS purification. The gas chromatography-mass spectrometry (GC-MS) provides a suitable alternative by estimating lipid-A chain anchored 3-hydroxy fatty acid methyl ester (FAME) to estimate LPS however, there are no reports of its application in natural polysaccharides used for vaccine preparation. The transesterification of LPS and meningococcal PS yielded primary target 3-O-acetylated myristic acid which was detected by GC-MS and provided quantitative estimation of endotoxin. The GC-MS method was found in agreement with the LAL values showing lower endotoxin content< 10Eu/µg in meningococcal C and Y serogroup polysaccharides in comparison to higher endotoxin 177–523 Eu/µg in meningococcal A, W and X serogroups. The high endotoxin content in purified polysaccharide was attributed to it being detected in its intermediate stage by GC-MS unlike the LAL test. Thus GC-MS serves as a valuable method for endotoxin monitoring and quantitation in gram-negative meningococcal intermediate and purified PS during vaccine preparation.

Methodical aspects of lipopolysaccharides obtaining and characterization from Escherichia coli cells

In this study, a procedure was applied to purify lipopolysaccharides from Escherichia coli based on a hot phenolic extraction protocol. The purity of the extracted lipopolysaccharides was assessed by HPLC-UV. Pyrogenic activity was determined using the Limulus Amebocyte Lysate test and used to monitor the functionality of the purified lipopolysaccharides. HPLC analysis showed a high degree of purity comparable to commercial lipopolysaccharide. Pyrogenic activity confirmed the functional activity of purified lipopolysaccharides. The presented protocol can be used to isolate lipopolysaccharides with high purity and functional activity.

Improved bioluminescence-based endotoxin measurement method using a salt-resistant luciferase mutant

Endotoxin is a component of the cell wall of gram-negative bacteria and causes fever and shock symptoms upon entering the bloodstream. We previously demonstrated that the bioluminescence-based Limulus amebocyte lysate test is highly sensitive and rapid for measuring endotoxin. However, as the firefly luciferase reaction is inhibited in the presence of sodium chloride, the endotoxin detection method did not meet the validation guidelines under medical dialysis conditions (range of 75–125% of the measured values tested in water). Here, we used a salt-resistant luciferase mutant, which met the criteria for validation of endotoxin measurement.

Effect of Twinlight Laser on the Attachment of Human Gingival Fibroblasts to the Root Surface In Vitro.

BackgroundThis study aimed to compare the effectiveness of subgingival scaling and root planing with the Twinlight laser, Er: YAG laser, and hand instrumentation on the removal of endotoxin and attachment of human gingival fibroblasts (HGFs) to cementum surfaces in vitro.Material/MethodsSingle-rooted teeth extracted for periodontal disease were collected and divided into 3 groups: group A, root planing with Gracey curet no. 5/6; group B, irradiation with Er: YAG laser; group C, irradiation with Er: YAG laser and Nd: YAG laser. Endotoxins were determined by the limulus amebocyte lysate test. Cell attachment and proliferation of HGFs on root specimens were evaluated by cell counting kit-8 assay. The root surface and cell morphology were observed by scanning electron microscope.ResultsA flat root surface with scratches was found in group A, Group B had a homogeneous rough morphology without carbonization, and group C had a non-homogeneous rough morphology with ablation. The endotoxin concentration was highest in group A (P<0.05) and lowest in group C (P>0.05). HGFs cultured in group B showed significantly increased adhesion and proliferation compared with groups A and C (P<0.05). HGFs in group B were well attached, covered densely by pseudopodia. HGFs in group A were round with poor extension and short pseudopodia, while the cells in the group C were in narrow, triangular, or polygonal shapes.ConclusionsTwinlight laser-assisted periodontal treatment effectively improved the biocompatibility of root surface and promoted the attachment and proliferation of fibroblasts by removing calculus and reducing the concentration of endotoxins.

SEM Evaluation of the Effects of Laser-Mediated Implant Surface Decontamination: An In Situ Human Pilot Study.

Successful treatment of peri-implantitis requires decontamination of implant surfaces exposed to biofilms and byproducts of tissue inflammation. In this regard, dental lasers may provide a clinical benefit. While the inherent characteristics of specific laser wavelengths may damage titanium implant surfaces, in vitro and animal studies have shown that damage to the target surface can be avoided with the selection of appropriate laser parameters. In this in situ human study, five hopeless implants were irradiated, each by one of the following lasers: Nd:YAG (1,064 nm), Er,Cr:YSGG (2,780 nm), Er:YAG (2,940 nm), CO2 (9,300 nm), and CO2 (10,600 nm) at their recommended settings. All implants were then removed and examined under scanning electron microscopy for the presence of residual bacteria and to assess the extent of damage to the implant surface. An additional implant (implant no. six) was irradiated and evaluated by the Limulus Amebocyte Lysate test for the presence of residual lipopolysaccharide endotoxin. The results showed that while there were localized areas of heat-related damage to an implant surface following laser irradiation, residual bacteria were rarely noted. Additionally, the Limulus Amebocyte Lysate test indicated a nearly complete removal of endotoxin. With the use of appropriate settings, all current dental lasers can be utilized for implant surface decontamination in a human.

Refinement of an open-microcavity optical biosensor for bacterial endotoxin test

The Limulus Amebocyte Lysate (LAL) test is an in vitro assay widely used in the pharmaceutical and biotechnology industries to detect bacterial endotoxins. Endotoxin is a structural component of the cell wall of Gram-negative bacteria, which has serious pathogenic effects in the body and may cause dysfunction of multiple organ systems and increased risk of mortality. To address the growing need for LAL assays due to the increased demand from drug and vaccine manufacturers, we have developed a new LAL assay approach. Our detection mechanism is different and improved from those currently used in the industry, leading to increased test sensitivity and reduced assay time. Our study utilizes an open-microcavity photonic-crystal biosensor to quantify endotoxin concentrations. It has demonstrated an improved LAL assay sensitivity by 10 fold compared to the commercial standard methods and reduced the time needed for the assay by more than half. In addition, this approach requires as little as 5 μL of LAL reagent per test, thereby decreasing costs and conserving horseshoe crabs. The results reported in this paper indicate the possibility of using the photonic-crystal biosensor based approach for significant enhancements of endotoxin testing.

Outstanding Contributions of LAL Technology to Pharmaceutical and Medical Science: Review of Methods, Progress, Challenges, and Future Perspectives in Early Detection and Management of Bacterial Infections and Invasive Fungal Diseases.

The blue blood of the horseshoe crab is a natural, irreplaceable, and precious resource that is highly valued by the biomedical industry. The Limulus amebocyte lysate (LAL) obtained from horseshoe crab blood cells functions as a surprisingly sophisticated sensing system that allows for the extremely sensitive detection of bacterial and fungal cell-wall components. Notably, LAL tests have markedly contributed to the quality control of pharmaceutical drugs and medical devices as successful alternatives to the rabbit pyrogen test. Furthermore, LAL-based endotoxin and (1→3)-β-D-glucan (β-glucan) assay techniques are expected to have optimal use as effective biomarkers, serving as adjuncts in the diagnosis of bacterial sepsis and fungal infections. The innovative β-glucan assay has substantially contributed to the early diagnosis and management of invasive fungal diseases; however, the clinical significance of the endotoxin assay remains unclear and is challenging to elucidate. Many obstacles need to be overcome to enhance the analytical sensitivity and clinical performance of the LAL assay in detecting circulating levels of endotoxin in human blood. Additionally, there are complex interactions between endotoxin molecules and blood components that are attributable to the unique physicochemical properties of lipopolysaccharide (LPS). In this regard, while exploring the potential of new LPS-sensing technologies, a novel platform for the ultrasensitive detection of blood endotoxin will enable a reappraisal of the LAL assay for the highly sensitive and reliable detection of endotoxemia.

Identification of Culturable and Nonculturable Microorganisms, Lipopolysaccharides, and Lipoteichoic Acids From Root Canals of Teeth With Endodontic Failure

AimTo elucidate the presence of apical periodontitis in the root canal of teeth with secondary/persistent infection, including composition of microbiota, levels of endotoxins and lipoteichoic acid (LTA), and clinical implications of these findings. MethodSamples were collected from root canals of 50 patients who needed endodontic retreatment and had radiographic evidence of apical periodontitis. Microorganisms were identified by using the culture technique and biochemical tests. Nested–polymerase chain reaction (nested-PCR) was used to identify 17 species of specific bacteria. Lipopolysaccharides (LPS) and LTAs were quantified by using, respectively, limulus amebocyte lysate and enzyme-linked immunosorbent assay tests. ResultsBacteria were detected in all samples by culture and molecular methods. A total of 154 gram-positive strains, of 188 strains isolated, were found in the root canals by culture. Enterococcus faecalis and Gemella morbillorum were the most prevalent species identified by the biochemical tests, whereas molecular analyses (nested-PCR) showed a high frequency of P. gingivalis, E. faecalis, and Fusobacterium nucleatum. LPS and LTA were detected in all samples, with mean values being 3.52 EU/mL and 597.83 pg/mL, respectively. Significant statistical correlations were found between levels of LTA and clinical features. ConclusionDespite the prevalence of gram-positives, the microbiota present in secondary/persistent infections showed a large variety of species. Within this diversity, associations were found between specific bacteria and clinical features. In addition, higher levels of LTA were statistically associated with larger periapical radiolucent areas, but no correlation between this feature and LPS was found.