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- ItemAcesso aberto (Open Access)Different Assay Conditions for Detecting the Production and Release of Heat-Labile and Heat-Stable Toxins in Enterotoxigenic Escherichia coli Isolates(Mdpi Ag, 2013-12-01) Rocha, Leticia B.; Ozaki, Christiane Y.; Horton, Denise S. P. Q.; Menezes, Caroline A.; Silva, Anderson; Fernandes, Irene; Magnoli, Fabio C.; Vaz, Tania M. I.; Guth, Beatriz Ernestina Cabilio [UNIFESP]; Piazza, Roxane M. F.; Butantan Inst; São Paulo Trop Med Inst; Fleury Med & Hlth; Adolfo Lutz Inst; Universidade Federal de São Paulo (UNIFESP)Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. for this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. the presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work.
- ItemAcesso aberto (Open Access)Health and economic outcomes of the detection of Klebsiella pneumoniae-produced extended-spectrum beta-lactamase (ESBL) in a hospital with high prevalence of this infection(Elsevier B.V., 2006-01-01) Marra, Alexandre Rodrigues; Castelo Filho, Adauto [UNIFESP]; Carmo Filho, José Rodrigues do; Cal, Ruy Guilherme Rodrigues [UNIFESP]; Sader, Helio Silva [UNIFESP]; Wey, Sergio Barsanti [UNIFESP]; Pereira, Carlos Alberto Pires [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introduction: Klebsiella pneumoniae is of high prevalence in hospital infections, mainly in bloodstream infections (BSI), and some produce extended-spectrum beta-lactamase (ESBL). for hospitals with a high prevalence of strains producing this enzyme, there is no reference material to show whether the use of the E-test method for their detection, which can be quite expensive, is actually required.Objective: To evaluate the cost-benefit of the disk diffusion and E-test methods for the detection of ESBL-producing K. pneumoniae strains in hospitals where a high prevalence of this resistance mechanism in BSI is found.Methods: One hundred and eight patients with K. pneumoniae BSI were evaluated retrospectively. ESBL-producing strains were identified by the disk diffusion method and by the E-test method. We estimated the costs of both diagnostic methods based on antimicrobial therapy adequacy.Results: Fifty-two percent of K. pneumoniae infections were due to ESBL-producing strains. the disk diffusion method yielded a positive predictive value (PPV) of 94.7% (95% Cl: 88.9-100%) and a negative predictive value (NPV) of 96.1% (CI 95%: 90.8-101.4%) in relation to the E-test. We evaluated cost-effectiveness, i.e., we analyzed the cost of both E-test and disk diffusion methods with carbapenem and cephalosporins, and found that the use of the disk diffusion method accounts for approximately US$3300.Conclusions: in hospitals with a high prevalence of ESBL-producing strains, the disk diffusion method can be used to detect ESBL-producing K. pneumoniae without compromising the clinical progression of patients with BSI. the E-test showed higher accuracy but this method was more expensive than the disk diffusion method. However, the use of the E-test method was demonstrated to be more cost-effective, as we evaluated cost based on antimicrobial therapy adequacy. (C) 2005 International Society for Infectious Diseases. Published by Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Interaction between Shiga Toxin and Monoclonal Antibodies: Binding Characteristics and in Vitro Neutralizing Abilities(Mdpi Ag, 2012-09-01) Rocha, Leticia B.; Luz, Daniela E.; Moraes, Claudia T. P.; Caravelli, Andressa; Fernandes, Irene; Guth, Beatriz Ernestina Cabilio [UNIFESP]; Horton, Denise S. P. Q.; Piazza, Roxane M. F.; Butantan Inst; Universidade Federal de São Paulo (UNIFESP)Monoclonal antibodies (MAbs) have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC), numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. the selected clones designated 3E2 (anti-Stx1) and 2E11 (anti-Stx2) were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 x 10(-10) M, detected as little as 6.2 ng of Stx1 and was stable up to 50 degrees C. in contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 degrees C, had a high dissociation constant of 6.1 x 10(-10) M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 mu g 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. in conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.
- ItemAcesso aberto (Open Access)Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli(Instituto Oswaldo Cruz, Ministério da Saúde, 2006-12-01) Menezes, Caroline A; Imamura, Sergio Y; Trabulsi, Luiz Rachid; Fernandes-Filho, Antônio; Martinez, Marina B; Guth, Beatriz Ernestina Cabilio [UNIFESP]; Girão, Dennys M; Piazza, Roxane Mf; Instituto Butantan Laboratório de Bacteriologia; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Universidade Federal do Rio de Janeiro Instituto Prof. Paulo de Góes Departamento de Microbiologia MédicaStrains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.