In vitro study of structural and mechanical properties of latex and non-latex intermaxillary orthodontic elastics.

PURPOSE: We evaluated bacterial endotoxin adhesion, superficial micromorphology and mechanical properties of latex and non-latex intermaxillary orthodontic elastics. METHODS: To quantify the adhered bacterial endotoxin, elastics were divided into 5 groups: experimental (n = 12) latex and non-latex elastics, previously contaminated by an endotoxin solution, negative control (n = 6) latex and non-latex elastics without contamination, and positive control (n = 6) stainless steel specimens (metallic replicas), contaminated by an endotoxin solution. In parallel, the structural micromorphology (n = 6) and surface roughness of latex and non-latex intermaxillary orthodontic elastics were assessed using confocal laser microscopy. Force degradation (g) and deformation of the internal diameter change (mm) were also evaluated. Structural micromorphology, surface roughness (µm), force degradation (g) and internal diameter (mm) change were evaluated at time 0 and after 24 and 72 h in a deformation test. Data were analyzed by the Shapiro-Wilk, Kruskal-Wallis, Dunn, ANOVA and Bonferroni tests (α = 5%). RESULTS: Endotoxin adhered similarly to both types of elastics with scores of 3 (> 1.0 EU/mL). The surface microstructure of both types of elastics showed irregularities and porosities at all times. Initially, the latex elastics had a higher surface roughness (p < 0.001) than the non-latex ones. After 24 h loading, surface roughness of the latex elastics was significantly reduced (p < 0.001), while after 72 h, the values were similar for both types (p > 0.05). The non-latex elastics had significantly higher force generation values (p < 0.05) at 0, 24 and 72 h compared with the latex elastics, although there was a significant reduction (p < 0.001) in force over time for both elastics. Despite similar initial values, non-latex elastics had a significantly larger internal diameter (p < 0.001) after the loading periods of 24 and 72 h compared with the latex elastics. CONCLUSION: Both elastics showed high affinity with endotoxin and microstructural irregularities of their surface. The non-latex elastics generated higher force values but demonstrated greater deformation of the internal diameter after loading.

Standardized pyrogen testing of medical products with the bacterial endotoxin test (BET) as a substitute for rabbit Pyrogen testing (RPT): A scoping review.

The Bacterial Endotoxin Test (BET) is a method for exclusion of endotoxin-related pyrogen contamination in pharmaceutical products, as an alternative to the Rabbit Pyrogen Test (RPT). However, BET does not detect a broad range of biologically relevant pyrogens, and interferences can limit its practical use for different medical products. This work aimed to scope the evidence in the scientific literature for case-by-case validity assessments of BET in different uses for medical products. A search strategy was conducted in PubMed, Scopus, and Web of Science in April 2020, according to the PRISMA-ScR statement. Twenty-two references were included, evaluating medical products for endotoxin contamination through both BET and RPT according to standardized protocols. A critical appraisal was performed through ToxRTool, followed by data extraction and qualitative synthesis of outcomes and methodological issues. Four classes of products assessed by BET were identified, including nanoparticles, drugs, blood and biological products. A considerable variation was observed on the BET methods used. Collectively, the evidence indicates different factors influencing the outcome of BET, including the chemical nature of samples that may cause interference depending on the selected method. While some applications to medical products appear adequate, others, such as nanoparticles, may require the use of different in vitro pyrogen testing methods, reinforcing the need for case-by-case validation for each BET method and type of medical product.

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

Endometritis in Infertile Couples: The Role of Hysteroscopy and Bacterial Endotoxin.

OBJECTIVE: The role of endometritis on infertility is still controversial. The aim of our prospective controlled study was to select infertile couples and to analyze: hysteroscopic findings of endometritis, bacterial endotoxin level in the menstrual blood, histological pattern of endometrial biopsy and to determine the effect of antibiotic therapy on endometritis resolution. METHODS: 100 infertile couples of University Hospital waiting for in vitro fertilization program. We evaluated the incidence of endometritis in infertile population by hysteroscopy, endometrial biopsy and bacterial endotoxin levels in the menstrual samples. Moreover, we verified the effectiveness of antimicrobial treatment on the endometritis resolution by hysteroscopic control. RESULTS: Out of 100 women enrolled, 15 showed hysteroscopic evidence of endometritis and 12 of these patients presented higher bacterial endotoxin in the menstrual samples. The histological results were positive for endometritis in 9 cases, 1 woman had no adequate samples and 5 patients had negative result. After antibiotic therapy, 9 patients had a complete hysteroscopic endometritis resolution, 4 patients had an improvement and in 2 cases there was no endometrial change. Concerning histological findings after therapy, we had negative results in 12 patients and persistent inflammatory findings in 2 patients. CONCLUSION: Our study demonstrated a high incidence of endometritis in infertile couples (15%). The endometrial biopsy was in agreement with the hysteroscopic findings in 60% of the cases, and the bacterial endotoxin level in the menstrual samples was higher in 12 patients with suspected endometritis, then its measurement can be helpful to confirm an endometrial infection but its influence must be confirmed with further researches. The antibiotic therapy can improve the hysteroscopic endometrial inflammatory aspect in over 80% of cases.

Intralaboratory validation of kinetic chromogenic Limulus amebocyte lysate assay for bacterial endotoxin determination in anti-bothropic serum.

Over the years, substituting in vitro biological methods for in vivo tests has posed an ever increasing challenge for researchers, including those who study the applications for snake antivenom. In the quality control of antivenons, the only official test recommended by pharmacopoeias for detecting pyrogenicity is the rabbit pyrogen test. In the present study, we propose intralaboratory validation of a method to replace the rabbit pyrogen test: in vitro determination of bacterial endotoxin in anti-bothropic serum (ABS) with quantitative kinetic chromogenic limulus amebocyte lysate (LAL) assay. The kinetic chromogenic LAL assay is specific to the detection of gram-negative bacterial endotoxin. The validation of the test involved the determination of performance parameters required by the Agência Nacional de Vigilância Sanitária Brasileira (ANVISA, the Brazilian National Health Surveillance Agency), the United States Food and Drug Administration (FDA) and the United States Pharmacopeia (USP) 35. In all experiments, the correlation coefficient of the curve obtained with the control standard endotoxin (CSE; Escherichia coli 055:B5 strain, range, 0.005-50 EU/mL) was between -0.998 and -1.000; and the recovery of endotoxin added to the sample of ABS (0.5 EU/mL) at the working dilution (1:10) followed the recuperation criteria (i.e., 50-200%). We performed six determinations, in each of which the coefficient of variation for the intermediate precision was between 5.6% and 13.8% (below the 15% threshold) and the accuracy was between 90.7% and 114.3% (within the acceptable range of 80-120%). The endotoxin concentration limit for the ABS was determined to be ≤ 2.9 EU/mL. The intralaboratory validation of the methodology was considered to have been successful because it met the criteria for all of the performance parameters.

Comparative evaluation of pyrogens tests in pharmaceutical products

A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL) was standardized with gel-clot and chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good agreement. The use of beta-glucan-reactive and non-reactive LAL reagents identified some products with false-positive results. The interference test was carried out and the specifications validated for some new products as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended for the evaluation of purity and quality control of parenteral medicinal products, improving the existing methodologies in the context of reduction and replacement in the use of animal models.

Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a beta-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.

Comparative evaluation of pyrogens tests in pharmaceutical products

A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL) was standardized with gel-clot and chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good agreement. The use of beta-glucan-reactive and non-reactive LAL reagents identified some products with false-positive results. The interference test was carried out and the specifications validated for some new products as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended for the evaluation of purity and quality control of parenteral medicinal products, improving the existing methodologies in the context of reduction and replacement in the use of animal models.

Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a beta-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.