Potentially pathogenic culturable bacteria in hemodialysis waters.

BACKGROUND: Hemodialysis patients are at risk of acquiring healthcare-related infections due to using non-sterile water to prepare hemodialysis fluid. Therefore, microbiological control and monitoring of used water are of crucial importance. MATERIALS AND METHODS: In this work, we identified bacterial populations occupying a hemodialysis water distribution system for almost a 6-month period in Ahvaz city, southwest of Iran. A total of 18 samples from three points were collected. We found high colony counts of bacteria on R2A agar. 31 bacteria with different morphological and biochemical characteristics were identified by molecular-genetic methods based on 16 S rRNA gene sequencing. Endotoxin concentrations were measured, using Endosafe® Rapid LAL Single-Test Vials. RESULTS: A diverse bacterial community was identified, containing predominantly Gram-negative bacilli. The most frequently isolated genus was Sphingomonas. Five species including M. fortuitum, M. lentiflavum, M.szulgai, M. barrassiae, and M. gordonae was identified .Despite the presence of Gram-negative bacteria the endotoxin analysis of all samples revealed that their endotoxin values were below the detection limit. CONCLUSION: The members of Sphingomonas genus along with Bosea and mycobacteria could be regarded as pioneers in surface colonization and biofilm creation. These bacteria with others like Pelomonas, Bradyrhizobium, staphylococcus, and Microbacterium may represent a potential health risk to patients under hemodialysis treatment.

Characterization of airborne endotoxin in personal exposure to fine particulate matter (PM2.5) and bioreactivity for elderly residents in Hong Kong.

The heavy metals and bioreactivity properties of endotoxin in personal exposure to fine particulate matter (PM2.5) were characterized in the analysis. The average personal exposure concentrations to PM2.5 were ranged from 6.8 to 96.6 µg/m3. The mean personal PM2.5 concentrations in spring, summer, autumn, and winter were 32.1±15.8, 22.4±11.8, 35.3±11.9, and 50.2±19.9 µg/m3, respectively. There were 85 % of study targets exceeded the World Health Organization (WHO) PM2.5 threshold (24 hours). The mean endotoxin concentrations ranged from 1.086 ± 0.384-1.912 ± 0.419 EU/m3, with a geometric mean (GM) varied from 1.034 to 1.869. The concentration of iron (Fe) (0.008-1.16 µg/m3) was one of the most abundant transition metals in the samples that could affect endotoxin toxicity under Toll-Like Receptor 4 (TLR4) stimulation. In summer, the interleukin 6 (IL-6) levels showed statistically significant differences compared to other seasons. Spearman correlation analysis showed endotoxin concentrations were positively correlated with chromium (Cr) and nickel (Ni), implying possible roles as nutrients and further transport via adhering to the surface of fine inorganic particles. Mixed-effects model analysis demonstrated that Tumor necrosis factor-α (TNF-α) production was positively associated with endotoxin concentration and Cr as a combined exposure factor. The Cr contained the highest combined effect (0.205-0.262), suggesting that Cr can potentially exacerbate the effect of endotoxin on inflammation and oxidative stress. The findings will be useful for practical policies for mitigating air pollution to protect the public health of the citizens.

Endotoxin, a novel biomarker for the rapid risk assessment of faecal contamination of coastal and transitional waters.

Current methods for testing water for faecal contamination rely on the culture of faecal indicator bacteria (FIB; Escherichia coli and Enterococci) that take 24-48 h, which leads to delays in taking proactive measures and poses a risk to public health. More rapid methods are therefore required. Here, we have tested a rapid, portable assay (Bacterisk) that detects the bacterial biomarker endotoxin in 30 min to quantify the bacterial biomass present, to evaluate 159 coastal water samples and to compare the results with the traditional culture of FIB. There was a significant correlation between the Bacterisk data given in endotoxin risk (ER) units and FIB culture that could accurately distinguish between poor and sufficient or good quality bathing water using the EU bathing directive values. Receiver operating characteristic analysis was used to determine the optimal ER threshold for coastal water samples, and the area under the curve was 0.9176 with a p-value of <0.0001. The optimal threshold was 7,300 ER units with a sensitivity of 95.45% and a specificity of 83.48%. In conclusion, we have shown that the Bacterisk assay provides a rapid and easy-to-use in situ method to assess bathing water quality.

Perspective on the influence of suspension manufacturing unit operations on bioburden viability and selection of sampling points at the pilot scale.

The suspension wet media milling manufacturing process is a complex multi-unit operation, resulting in drug substance comminution to a target particle size. As a result of this complexity, microbial contamination is of paramount concern, particularly for suspensions dosed for parenteral use. This perspective sought to review the influence of (4) critical manufacturing unit operations using a quality risk management approach to better identify and articulate impact of each unit operation on bioburden viability. The manufacturing unit operations in scope included slurry compounding, deaeration, milling, and filling. Bow tie risk analysis was used as a visual gap analysis tool to evaluate if conventional controls were appropriate to detect and mitigate potential for microbial contamination. A deep dive into these unit operations clarified that mechanisms such as turbohypobiosis, cavitation during deaeration, high energy milling, and inert overlay may have an appreciable influence on bioburden viability and proliferation. The resultant analysis also explicated that endotoxin oversight must be closely monitored through barriers (input material controls, water quality controls) to minimize impact to the product and patient. The identified manufacturing unit operations were not appropriate as mitigating controls for endotoxin. The output of this article relates risk intersections for microbial contamination during wet media milling and offers insights in critical areas for intervention.

Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination.

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

Novel aptasensor based on polyaniline functionalized carboxylated dobby carbon nanotubes and molybdenum disulfide for endotoxin detection.

Endotoxins, also known as lipopolysaccharides (LPS), are present within the cell walls of Gram-negative bacteria and are released upon cellular death, which can pose a significant risk to human and animal health. Due to the minimal amount of endotoxin required to trigger an inflammatory response in human body, the demand for sensitive methods with low endotoxin detection limits is essential necessary. This paper presents a straightforward aptamer sensor which can enhance the conductivity and specific surface area of molybdenum disulfide (MoS2) by incorporating carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) and polyaniline (PANI). Doping with gold nanoparticles (AuNPs) improves biocompatibility and sensitivity while providing binding sites for thiolated endotoxin-binding aptamers (LBA). This biosensor achieved a remarkable detection limit as low as 0.5 fg mL-1, enabling trace-level identification of LPS. It also exhibits excellent repeatability, selectivity, and stability, facilitating rapid and accurate LPS detection. Moreover, this method demonstrates high recovery rates and specificity for LPS analysis in food samples, showcasing its promising application prospects in trace-level LPS detection within the food industry.

Risk of hypersensitivity pneumonitis and other interstitial lung diseases following organic dust exposure.

BACKGROUND: Organic dust is associated with hypersensitivity pneumonitis, and associations with other types of interstitial lung disease (ILD) have been suggested. We examined the association between occupational organic dust exposure and hypersensitivity pneumonitis and other ILDs in a cohort study. METHODS: The study population included all residents of Denmark born in 1956 or later with at least 1 year of gainful employment since 1976. Incident cases of hypersensitivity pneumonitis and other ILDs were identified in the Danish National Patient Register 1994-2015. Job exposure matrices were used to assign individual annual levels of exposure to organic dust, endotoxin and wood dust from 1976 to 2015. We analysed exposure-response relations by different exposure metrics using a discrete-time hazard model. RESULTS: For organic dust, we observed increasing risk with increasing cumulative exposure with incidence rate ratios (IRR) per 10 unit-years of 1.19 (95% CI 1.12 to 1.27) for hypersensitivity pneumonitis and 1.04 (95% CI 1.02 to 1.06) for other ILDs. We found increasing risk with increasing cumulative endotoxin exposure for hypersensitivity pneumonitis and other ILDs with IRRs per 5000 endotoxin units/m3-years of 1.55 (95% CI 1.38 to 1.73) and 1.09 (95% CI 1.00 to 1.19), respectively. For both exposures, risk also increased with increasing duration of exposure and recent exposure. No increased risks were observed for wood dust exposure. CONCLUSION: Exposure-response relations were observed between organic dust and endotoxin exposure and hypersensitivity pneumonitis and other ILDs, with lower risk estimates for the latter. The findings indicate that organic dust should be considered a possible cause of any ILD. TRIAL REGISTRATION NUMBER: j.no.: 1-16-02-196-17.

A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products.

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.

Current Considerations and Practical Solutions for Overcoming Nanoparticle Interference with LAL Assays and Minimizing Endotoxin Contamination.

Monitoring endotoxin contamination in drugs and medical devices is required to avoid pyrogenic responses and septic shock in patients receiving these products. Endotoxin contamination of engineered nanomaterials and nanotechnology-based medical products represents a significant translational hurdle. Nanoparticles often interfere with an in vitro limulus amebocyte lysate (LAL) assay commonly used in the pharmaceutical industry for the detection and quantification of endotoxin. Such interference challenges the preclinical development of nanotechnology-formulated drugs and medical devices containing engineered nanomaterials. Protocols for the analysis of nanoparticles using LAL assays have been reported before. Here, we discuss considerations for selecting an LAL format and describe a few experimental approaches for overcoming nanoparticle interference with the LAL assays to obtain more accurate estimations of endotoxin contamination in nanotechnology-based products. The discussed approaches do not solve all types of nanoparticle interference with the LAL assays but could be used as a starting point to address the problem. This chapter also describes approaches to prevent endotoxin contamination in nanotechnology-formulated products.

Occupational exposure to endotoxins and small cell lung cancer: a systematic review with meta-analysis.

The relationship of occupational exposure to endotoxins with different histologic subtypes of lung cancer has not been established. Our objective was to conduct a systematic review with meta-analysis to assess the effect of exposure to endotoxins on the development of small cell lung cancer (SCLC). A bibliographic search was conducted using MEDLINE, Embase, CENTRAL, and Web of Science databases until December 2022, including all cohort and/or case-control studies that examined occupational exposure to endotoxins and SCLC. Risk of bias was assessed using the U.S. Office of Health Assessment and Translation tool. A random effects model was applied, publication bias were assessed, and a sensitivity analysis was conducted. Four papers were selected for meta-analysis purposes. A total of 144 incident cases of SCLC and 897 population or hospital controls were included. Occupational exposure to endotoxins was considered for textile/leather industry and agricultural sector workers exposed to endotoxins originating from wool, cotton, or leather dust. Except for one study, all investigations were classified as having a low probability of risk of biases. The results of the meta-analysis were not statistically significant (pooled OR: 0.86; 95% CI:0.69-1.08). In addition, neither between-study heterogeneity (I2=0%;p=0.92) nor publication bias was observed (p=0.49). The results of the sensitivity analysis, after including five studies that assessed the risk of SCLC among textile industry and crop/livestock farm workers (not specifically exposed to endotoxins), showed a negative statistically non-significant association and low between-study heterogeneity (pooled OR: 0.90; 95% CI:0.79-1.02; I2=22%;p=0.23). Subjects exposed to occupational exposure to endotoxins seem to exhibit a negative association with the development of SCLC, although the results are not conclusive.

Nanozyme with tailored selectivity and highly catalytic activity for efficient sensing of endotoxin and sourcing gram-negative bacteria.

Endotoxin detection is important for determining bacterial contamination and infection in fields of food, pharmaceutical and clinical disease diagnosis. The horseshoe crab deformed cell lysate analysis is regarded as the gold-standard method, but the endangered and high-cost horseshoe crab animals required in sensing process further raise animal ethical issues and hinder their applications. The colorimetric methods based on nanozymes are simple and economical, but the low selectivity and sensitivity are still the bottleneck for their further application. Herein, we successfully developed a phenylboronic acid functionalized iron-based nanozyme with higher selectivity and highly catalytic activity for endotoxin sensing. The as-prepared colorimetric sensor using the obtained nanozyme as sensing probes shows a good linear relationship for endotoxin sensing in the range of 1-20 µg mL-1, with a LOD = 0.42 µg mL-1, along with good selectivity and reproducibility. The sensor can also be well applied to detecting endotoxin in practical samples such as beer and serum. Moreover, the parameters including time and temperature which could affect the endotoxin release from E. coli were also studied and optimized, based on the relationship between endotoxin and Gram-negative bacteria, the as-prepared sensor achieves the qualitative and quantification of E. coli.

Bacterial contamination of coffee and personal exposure to inhalable dust and endotoxin in primary coffee processing factories in Ethiopia.

INTRODUCTION AND OBJECTIVE: Endotoxins from gram-negative bacteria might be released when the coffee cherries are processed and may cause respiratory health problems among workers in the coffee industry. The relationship between bacterial contamination and occupational exposure to endotoxin levels has not been thoroughly explored previously in primary coffee processing factories in Ethiopia, or elsewhere. The aim of this study was to characterize the level of personal endotoxin exposure and its relations with bacterial contamination of coffee cherries in such factories in Ethiopia. MATERIAL AND METHODS: A cross-sectional study was conducted from March 2020 - February 2021 in 9 primary coffee processing factories in 3 regions in Ethiopia. A total of 180 personal air samples were collected to analyze workers' exposure to inhalable dust and endotoxin. Correlation tests were performed to assess the relationship between total bacteria and endotoxin levels and between inhalable dust and endotoxin levels. RESULTS: The geometric mean (GM) of personal inhalable dust exposure among machine room workers and hand pickers were 9.58 mg/m3 and 2.56 mg/m3, respectively. The overall GM of endotoxin exposure among machine room workers and hand pickers were 10,198 EU/m3 and 780 EU/m3, respectively. Gram-negative bacteria were found in all 54 coffee samples. The correlation between inhalable dust and endotoxin exposure was significant (r=0.80; P <0.01). CONCLUSIONS: About 92% of the samples from hand pickers and all samples from machine room workers exceeded the occupational exposure limit of 90 EU/m3 recommended by the Dutch Expert Committee on Occupational Standards. Prevention and control of bacterial contamination of the coffee in primary coffee processing are suggested to reduce endotoxin exposure that might cause respiratory health problems among coffee workers.

Production of pro-inflammatory mediators stimulated by exposure of poultry house workers to airborne dust particulates.

INTRODUCTION AND OBJECTIVE: Poultry house employees spend a significant part of their work shift being exposed to airborne particulate pollutants. The aim of this study was to assess their exposure at different stages of chicken production cycle, based on quantification of pro-inflammatory mediators (IL-1ß, IL-6, IL-8, and TNFα) in nasal lavage (NAL) samples. MATERIAL AND METHODS: The concentrations of airborne dust at 3 different stages of the production cycle (i.e. empty poultry house, with 7- and 42-day-old chickens) were stationary measured using Grimm spectrometer, as well as CIS and Button samplers. The dust collected by the latter 2 samplers was analyzed for endotoxin and (1→3)-ß-D-glucan content. NAL samples were collected from employees after their work shift to determine the pro-inflammatory mediator levels. RESULTS: The maximum particulate aerosol, endotoxin, and (1→3)-ß-D-glucan concentrations at workplaces reached the levels of 4.12 mg/m3, 45.21 ng/m3, and 56.54 ng/m3, respectively. The IL-1ß, IL-6, and IL-8 concentrations in NAL samples ranged between 0.62-18.12 pg/mL, <0.70-25.37 pg/mL, and <3.50-259.5 pg/mL, respectively. All TNFα levels were below 4 pg/mL. There were no significant differences between these cytokine concentrations in NAL samples collected at different stages of chicken breeding in either 'winter' or 'summer' seasons. CONCLUSIONS: Inhalation stimulation with poultry dust containing endotoxins and (1→3)-ß-D-glucans resulted in the production of pro-inflammatory mediators, which proves the course of immunological processes in the exposed employees that may lead to adverse effects. The use of nasal lavage fluid in the control of such exposure confirms that NAL analysis is a reliable laboratory tool for assessing the impact of poultry dust on exposed farm workers.

Panorama da qualidade biológica da água destinada ao tratamento dialítico em pacientes sob cuidados críticos em Unidades de Terapia Intensiva / Overview of the biological quality of water intended for dialysis treatment in patients under critical care in Intensive Care Units

A incidência da lesão renal aguda caracteriza-se como evento frequente em pacientes críticos internados em Unidades de Terapia Intensiva e está associada ao aumento de mortalidade, causando grande impacto à Saúde Pública. As intercorrências clínicas são minimizadas com intervenções dialíticas, acarretando a exposição do paciente a volumes expressivos de água tratada durante a terapia renal em leito. As análises microbiológicas e de determinação de endotoxinas bacterianas em amostras de água tratada e em soluções de dialisato foram executadas em dois hospitais públicos do município de São Paulo, seguindo metodologias analíticas preconizadas em compêndios oficiais. A avaliação demonstrou que a porcentagem de resultados satisfatórios no período de 2010 a 2022 variou entre 35,2 a 100% e de 40 a 100% para as unidades hospitalares I e II para a água tratada, respectivamente; e, 100% para as soluções de dialisato para a unidade hospitalar I. A eficácia de ações delineadas pelas equipes técnicas das unidades hospitalares, na adequação da água destinada à terapia dialítica, aponta para a importância em estimular outras instituições hospitalares na padronização e implantação de melhoria contínua de seus sistemas de tratamento de água para uso em procedimento dialítico, prevenindo riscos adicionais aos pacientes expostos à terapia renal.

The incidence of acute kidney is high among critically ill patients admitted to Intensive Care Units and is associated with increased mortality, having a major impact on public health. Clinical complications are minimized with dialysis interventions, which expose patients to significant volumes of treated water during in-bed renal therapy. Microbiological analyzes and determination of bacterial endotoxins were performed on treated water samples and dialysate solutions in two public hospitals in São Paulo city, using analytical methodologies recommended in official compendia. The evaluation showed that the percentage of satisfactory results for treated water ranged from 35.2% to 100% in Hospital Unit I and from 40% to 100% in Hospital Unit II between 2010 and 2022. For dialysate solutions in Hospital Unit I, the percentage of satisfactory results was 100% during the same period. The effectiveness of actions implemented by the technical hospital teams, in adapting water for dialysis therapy, points to the importance of encouraging other hospital institutions to standardize and implement a program of continuous improvement for their water treatment systems used in dialysis procedures. This will help to prevent additional risks to patients exposed to renal therapy.

Effects of systemic inflammation due to hepatic ischemia-reperfusion injury upon lean or obese visceral adipose tissue

Purpose: To evaluate how the induction of liver damage by ischemia and reperfusion affects the adipose tissue of lean and obese mice. Methods: Lean and diet-induced obese mice were subjected to liver ischemia (30 min) followed by 6 h of reperfusion. The vascular stromal fraction of visceral adipose tissue was analyzed by cytometry, and gene expression was evaluated by an Array assay and by RT-qPCR. Intestinal permeability was assessed by oral administration of fluorescein isothiocyanate (FITC)-dextran and endotoxemia by serum endotoxin measurements using a limulus amebocyte lysate assay. Results: It was found that, after liver ischemia and reperfusion, there is an infiltration of neutrophils, monocytes, and lymphocytes, as well as an increase in the gene expression that encode cytokines, chemokines and their receptors in the visceral adipose tissue of lean mice. This inflammatory response was associated with the presence of endotoxemia in lean mice. However, these changes were not observed in the visceral adipose tissue of obese mice. Conclusions: Liver ischemia and reperfusion induce an acute inflammatory response in adipose tissue of lean mice characterized by an intense chemokine induction and leukocyte infiltration; however, inflammatory alterations are already present at baseline in the obese adipose tissue and liver ischemia and reperfusion do not injure further.

Validation and applicability of an alternative method for dialysis water and dialysate quality analysis / Validação e aplicabilidade de um método alternativo para análise da qualidade da água e do dialisato em diálise

ABSTRACT Introduction: In hemodialysis, patients are exposed to a large volume of water, which may lead to fatal risks if not meeting quality standards. This study aimed to validate an alternative method for monitoring microbiological quality of treated water and assess its applicability in dialysis and dialysate analysis, to allow corrective actions in real-time. Methods: Validation and applicability were analyzed by conventional and alternative methods. For validation, E. coli standard endotoxin was diluted with apyrogenic water in five concentrations. For the applicability analysis, treated water for dialysis was collected from different points in the treatment system (reverse osmosis, drainage canalization at the storage tank bottom, reuse, and loop), and dialysate was collected from four machines located in different rooms in the hemodialysis sector. Results: The validation results were in accordance with the Brazilian Pharmacopoeia acceptance criteria, except for the last two concentrations analyzed. In addition, the ruggedness criterion performed under the US Pharmacopoeia was in agreement with the results. Discussion: A limiting factor in the applicability analysis was the absence of the endotoxin maximum permitted level in dialysate by the Brazilian legislation. When comparing the analysis time, the alternative method was more time-consuming than the conventional one. This suggests that the alternative method is effective in the case of few analyses, that is, real-time analyses, favoring corrective actions promptly. On the other hand, it does not support the implementation of the alternative method in a laboratory routine due to the high demand for analyses.

RESUMO Introdução: Na hemodiálise, os pacientes são expostos a um grande volume de água, o que pode levar a riscos fatais se não cumprir com padrões de qualidade. Este estudo teve como objetivo validar um método alternativo para monitorar a qualidade microbiológica da água tratada e avaliar sua aplicabilidade em análises de diálise e dialisato, para permitir ações corretivas em tempo real. Métodos: A validação e aplicabilidade foram analisadas por métodos convencionais e alternativos. Para validação, a endotoxina padrão de E. coli foi diluída com água apirogênica em cinco concentrações. Para a análise de aplicabilidade, a água tratada para diálise foi coletada em diferentes pontos do sistema de tratamento (osmose reversa, canalização de drenagem no fundo do tanque de armazenamento, reutilização e circuito) e o dialisato foi coletado em quatro máquinas localizadas em diferentes salas do setor de hemodiálise. Resultados: Os resultados da validação obedeceram aos critérios de aceitação da Farmacopeia Brasileira, com exceção das duas últimas concentrações analisadas. Além disso, o critério de robustez realizado sob a Farmacopeia dos EUA estava de acordo com os resultados. Discussão: Um fator limitante na análise de aplicabilidade foi a ausência do nível máximo permitido de endotoxina no dialisato pela legislação brasileira. Ao comparar o tempo de análise, o método alternativo consumiu mais tempo que o convencional. Isso sugere que o método alternativo é eficaz no caso de poucas análises, ou seja, análises em tempo real, favorecendo ações corretivas imediatamente. Por outro lado, não suporta a implementação do método alternativo em uma rotina de laboratório devido à alta demanda por análises.

A Protocol to Perform Systemic Lipopolysacharide (LPS) Challenge in Rats / Protocolo para realizar reto sistémico con lipopolisacárido (LPS) en ratas

Abstract 19. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. In animals, intraperitoneal administration of LPS, stimulates innate immunity and the production of proinflammatory cytokines. LPS provides an inflammatory stimulus that activates the neuroimmune and neuroendocrine systems resulting in a set of responses termed sickness behavior. The purpose of this protocol is to describe step-by-step the preparation and procedure of application of intraperitoneal injection of LPS in rats, as a guide for those researchers that want to use this assay to mount an inflammatory response. LPS intraperitoneal challenge in rats has been widely used to evaluate antiinflammatory reagents and to address basic scientific questions.

Resumen 23. El lipopolisacárido (LPS) es un componente de la membrana externa de las bacterias Gram negativas. En animales, la administración intraperitoneal de LPS estimula la inmunidad innata y la producción de citoquinas proinflamatorias. El LPS proporciona un estímulo inflamatorio que activa el sistema neuroinmunológico y el sistema neuroendocrino, lo que da como resultado un conjunto de respuestas denominadas conductas de enfermedad. El propósito de este protocolo es describir paso a paso la preparación y el procedimiento de aplicación de la inyección intraperitoneal de LPS en ratas, como una guía para aquellos investigadores que desean utilizar este método para estimular una respuesta inflamatoria en el animal. La estimulación con LPS en ratas, aplicada intraperitonealmente, se ha utilizado ampliamente para evaluar reactivos antiinflamatorios y para abordar preguntas básicas de investigación científica.

Comparison of two rotary systems in bacteria/lps removal from endodontic infections: randomized clinical trial

Abstract: This clinical study compared the effectiveness of two rotary systems: HyFlex CM (Coltene-Whaledent, Altstetten, Switzerland) and ProTaper Next (Dentsply Sirona, Ballaigues, Switzerland) on the removal of cultivable bacteria and endotoxins from primarily infected root canals. This study was designed as a randomized single-blinded, 2-arm, clinical trial. Twenty-four primarily infected root canals were selected and randomly divided into 2 groups: HyFlex CM (n = 12); and ProTaper Next (n = 12). Samples were collected before and after the biomechanical preparation and inoculated in specific flasks. Irrigation was performed using 2.5% sodium hypochlorite. A kinetic turbidimetric lysate assay of limulus amoebocytes was used to quantify endotoxins. Microbiological culture technique was used to determine the count of bacterial colony forming units (CFU/mL). Data collected were statistically analyzed using SigmaPlot 12.0 for Windows. The Two-Way ANOVA statistical test was performed and the level of significance was 5%. In the samples before the biomechanical preparation, cultivable bacteria and endotoxins were evidenced in 100% of the cases. The culture analysis revealed that there was no statistically significant difference in the bacterial reduction between the two instrumentation systems. Endotoxins were present in 100% of the canals after instrumentation and there was no statistical difference between the two systems in endotoxin reduction. Thus, it was concluded that both instrumentation systems were effective in reducing root canal bacteria and endotoxins with primary endodontic infection and that there was no statistical difference between them. However, no system was able to eliminate 100% of the bacteria and their by-products.

Aplicabilidade de métodos alternativos no monitoramento da qualidade microbiológica da água tratada para diálise / Applicability of alternative methods for the monitoring microbiological quality of treated water for dialysis

A fim de garantir a qualidade final de produtos os laboratórios de análise microbiológica fornecem dados sobre a qualidade dos mesmos em todas as suas etapas de produção. A crescente preocupação com a saúde dos pacientes conduz à busca de métodos que forneçam resultados precisos e rápidos, pois possibilitam que ações corretivas sejam tomadas em tempo real. O presente trabalho teve por objetivo avaliar o potencial de tecnologia alternativa no monitoramento de endotoxina bacteriana na água tratada para diálise e dialisato e avaliar o potencial da citometria de fluxo na análise de água. Para isso utilizou-se Portable Test System (PTS®) como método alternativo para detecção de endotoxina bacteriana no monitoramento da água tratada para diálise e dialisato, o qual foi validado frente ao método convencional farmacopeico. Paralelamente realizou-se revisão narrativa da literatura a fim de avaliar a aplicabilidade da citometria de fluxo em análises de água. A análise dos diferentes parâmetros de validação para endotoxina bacteriana no método alternativo mostrou que, exceto para a menor diluição analisada, houve linearidade e precisão nos resultados. Por outro lado a concentração de 0,25 UE/mL foi a menor que apresentou exatidão e especificidade. Observou-se ainda, que o limite de detecção foi de 0,125UE/mL e o de quantificação de 0,25 UE/mL, portanto o intervalo foi de 0,25-1,0 UE/mL. Adicionalmente pela análise de resistência pode-se perceber que ao variar analistas não houve diferença significativa. Em relação ao tempo de análise em uma condição de rotina laboratorial com muitas amostras, o PTS® mostrou-se demorado. Ressalta ainda, que seria importante que a legislação vigente determinasse a análise mensal de endotoxinas no dialisato. A revisão da literatura evidencia o potencial da tecnologia de citometria de fluxo, pois a mesma mostrou-se satisfatória quando comparada a metodologias convencionais para análise de água. O trabalho desenvolvido permitiu concluir que o PTS®) mostrou-se adequado para analisar amostras in loco, permitindo análises em tempo real, que para as quais haja a expectativa de ausência de endotoxinas ou de concentração respeitando o intervalo de 0,25 UE/mL a 1,0 UE/mL. Quanto a citometria de fluxo, esta mostrou-se uma tecnologia promissora em analisar amostras de água, sendo portanto recomendável proceder a estudos de validação e aplicabilidade

In order to guarantee the final quality of products, the microbiological analysis laboratories provide data about their quality at all production stages. The growing concern for patients' health leads to the search for methods that provide accurate and fast results, as they enable corrective actions to be taken in real time. The present work aimed to evaluate the alternative technology potential in the monitoring of bacterial endotoxin in treated water for dialysis and dialysate and to evaluate the potential of flow cytometry in water analysis. The different validation parameters analysis for bacterial endotoxin in alternative method showed that, except for the lowest dilution analyzed, there was linearity and precision in the results. On the other hand, the concentration of 0.25 EU / mL was the lowest that presented accuracy and specificity. It was further observed that the detection limit was 0.125UE / mL and the quantification limit was 0.25 EU / mL, so the range was 0.25-1.0 EU / mL. Additionally by the ruggedness analysis it was possible to perceived that when varying analysts there was no significant difference. Regarding the analysis time in a laboratory routine condition with many samples, the PTS® was was time consuming. It was also observed that it would be important to determine monthly analysis of endotoxins in dialysate. The literature review evidence the flow cytometry technology potential of the because it was satisfactory when compared to conventional methodologies for water analysis. The research showed that the PTS® was suitable for analyzing samples in loco, allowing real-time analyzes, for which there is expectation of endotoxins absence or concentration respecting the range of 0.25 EU / mL to 1.0 EU / mL. For the flow cytometry, it was shown to be a promising technology for analyzing water samples, and it is therefore advisable to carry out validation and applicability studies