Association between socioeconomic factors and origin of hospital referrals among patients with oral cancer.

BACKGROUND: The Brazilian Unified Health System (SUS) is responsible for offering free assistance to more than 100 million Brazilians, including treatment of oral cancer lesions. Considering that the Brazilian public system aids the most vulnerable population, this study analyzed whether the origin of hospital referrals of patients with oral cancer is associated with socioeconomic factors. MATERIAL AND METHODS: A cross-sectional study was carried out from cancer hospital records of the National Cancer Institute (RHC-INCA), considering the primary locations (C00 to C06) diagnosed between 2016 and 2019. Data on gender, skin color (white and non-white), education (no schooling, incomplete or complete elementary education; high school; incomplete and complete higher education) and origin of referral (SUS and non-SUS) were analyzed by multiple logistic regression (p<0.05). RESULTS: Higher referral rates by the SUS were observed in 2017 (OR=1.27; 95% CI=1.098-1.480) and 2018 (OR=1.28; 95% CI=1.101-1.490); no differences were found between the years 2016 and 2019. Regarding gender, men were 40% more likely to have the SUS as the source of referral (OR=1.40; 95% CI=1.233-1.600). Non-white individuals were 34% more likely to have the SUS as the source of the referral (OR=1.34; 95% CI=1.190-1.512). Illiterate individuals or individuals who only attended elementary school were 6.38 times more likely to be referred by the SUS than individuals with higher education (OR=6.38; 95% CI=5.228-7.796). CONCLUSIONS: It is concluded that the origin of hospital referrals via SUS of patients with oral cancer is associated with socioeconomic factors.

Trend of hospitalized cases of oral cancer in Brazil and its relationship with oral health coverage in public health system between 2009 and 2017.

BACKGROUND: This study aimed to analyze the trend in the number of hospitalized cases of oral cancer in Brazil, according to the coverage of oral health services in public health system, and also investigate the influence of healthcare and clinical characteristics on the severity of oral cancer cases. MATERIAL AND METHODS: This retrospective study considered the period between 2009 and 2017. Data from the Hospital Registry of Cancer from the National Cancer Institute were used, considering the primary locations C00 to C06. Detailed information including sex, age, alcohol and tobacco use, year of first consultation, and the clinical stage of the cases were also collected. The frequency of hospitalized cases was correlated with the coverage of Primary Care Oral Health Teams (ESB) and the number of Dental Specialty Centers (CEO). It was also estimated the chance of advanced oral cancer cases, according to healthcare and clinical characteristics. Data were analyzed using Tweedie's multiple regression and multiple binary logistic regression (α<0.05). RESULTS: There was an increasing trend in the number of hospitalized cases of oral cancer in Brazil between 2009 and 2017 (B=0.043, p<0.001, PR=1.044). The increase in ESB coverage was associated with small increase in the number of hospitalized cases of oral cancer (B=0.001, p=0.003, PR=1.001). The increase in the number of CEO was associated with decrease in the number of hospitalized cases of oral cancer (B=-0.085, p<0.001, PR =0.918). The increase of ESB (OR=0.998) and CEO (OR=0.974) contributed for reducing the number of stage IV cases, whilst the history of alcohol and tobacco use (OR=1.574) was associated with an increase in the number of stage IV cases. CONCLUSIONS: Although an increasing trend was detected, the expansion of the public health system reduced the number of hospitalized cases and the frequency of advanced oral cancer cases in Brazil.

Multiresidue analysis and evaluation of the matrix effect on 20 pesticides in Brazilian maize (Zea mays L.) flour.

Maize consists of a cereal widely used in the preparation of different food products. Brazil is one of the world's largest maize producers. Several types of pesticides have been applied in maize crop, which can lead to the contamination of the derived products. The present work aims at the validation of multiresidue method to analyze the matrix effect and level of pesticides in maize flour. Twenty residues were investigated in samples commercialized in the state of Ceará, Brazil. The method was satisfactorily validated, according to parameters recommended by European Union. About 55% of the pesticides had an intense negative matrix effect. Multiresidue analyzes showed the presence of traces of fenitrotion in 20% of maize flour samples. Detected levels were below maximum residue limits recommended for maize. The results indicate that maize products need continuous monitoring to ensure food security.

Osteopenia-osteoporosis discrimination in postmenopausal women by 1H NMR-based metabonomics.

This is a report on how 1H NMR-based metabonomics was employed to discriminate osteopenia from osteoporosis in postmenopausal women, identifying the main metabolites associated to the separation between the groups. The Assays were performed using seventy-eight samples, being twenty-eight healthy volunteers, twenty-six osteopenia patients and twenty-four osteoporosis patients. PCA, LDA, PLS-DA and OPLS-DA formalisms were used. PCA discriminated the samples from healthy volunteers from diseased patient samples. Osteopenia-osteoporosis discrimination was only obtained using Analysis Discriminants formalisms, as LDA, PLS-DA and OPLS-DA. The metabonomics model using LDA formalism presented 88.0% accuracy, 88.5% specificity and 88.0% sensitivity. Cross-Validation, however, presented some problems as the accuracy of modeling decreased. LOOCV resulted in 78.0% accuracy. The OPLS-DA based model was better: R2Y and Q2 values equal to 0.871 (p<0.001) and 0.415 (p<0.001). LDA and OPLS-DA indicated the important spectral regions for discrimination, making possible to assign the metabolites involved in the skeletal system homeostasis, as follows: VLDL, LDL, leucine, isoleucine, allantoin, taurine and unsaturated lipids. These results indicate that 1H NMR-based metabonomics can be used as a diagnosis tool to discriminate osteoporosis from osteopenia using a single serum sample.

Non-toxigenic strain of Clostridioides difficile Z31 reduces the occurrence of C. difficile infection (CDI) in one-day-old piglets on a commercial pig farm.

Neonatal porcine diarrhea (NPD) is a current problem on pig farms and is caused by several enteropathogens. Among them, Clostridioides difficile stands out due to its importance in piglets and zoonotic potential. A non-toxigenic strain of C. difficile (NTCD), named Z31, was previously tested in hamster and piglet experimental models as a strategy to prevent C. difficile infection (CDI). To evaluate the capacity of the strain Z31 to prevent CDI and NPD in one-day-old piglets on a commercial farm, 90 piglets from 16 litters received 1 × 106 spores of Z31 while 84 animals from the same litters served as controls. Animals were clinically evaluated, and fecal samples were collected 24 h after administration and submitted to A/B toxin detection and isolation of C. difficile. Stool samples were also submitted to rotavirus, Escherichia coli, and Clostridium perfringens detection. Administration of Z31 reduced the incidence of CDI in treated animals (7.8%) when compared to the control group (25.0%; P = 0.003). In animals that developed CDI, the intensity of diarrhea was lower in those that received Z31 than in the control group. Neonatal porcine diarrhea was reduced in treated animals when compared to untreated animals (P < 0.001). The present study suggests that Z31 can potentially be used to prevent CDI in piglets on commercial farms.

Extender Supplementation with Antioxidants Selected after the Evaluation of Sperm Susceptibility to Oxidative Challenges in Goats.

This study aimed to detect the most deleterious ROS for goat sperm and then supplemented the extender with a proper antioxidant. For this, 12 adult goats (aged 1-7) were used. Fresh samples were submitted to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) and malondialdehyde (MDA-toxic product of lipid peroxidation). After experiment 1, sperms were cryopreserved in extenders supplemented to glutathione peroxidase (Control: 0 UI/mL; GPx1: 1 UI/mL; GPx5: 5 UI/mL, and GPx10: 10 UI/mL) and catalase (Control: 0 UI/mL; CAT60: 60 UI/mL; CAT120: 120 UI/mL, and CAT240: 240 UI/mL). Each sample was evaluated by motility, plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, assay of the sperm chromatin structure, mitochondrial activity (3,3-diaminobenzidine), and measurement of lipid peroxidation (thiobarbituric acid reactive substances [TBARS]). It was possible to observe a mitochondrial dysfunction (DAB-Class IV) and low membrane integrity after hydrogen peroxide action. However, the high rates of TBARS were observed on hydroxyl radical. CAT240 presents the lower percentage of plasma membrane integrity. It was possible to attest that hydrogen peroxide and hydroxyl radical are the more harmful for goat sperm. Antioxidant therapy must be improving perhaps using combination between antioxidants.

Role of TRPV1 receptor in inflammation and impairment of esophageal mucosal integrity in a murine model of nonerosive reflux disease.

BACKGROUND: Microscopic inflammation and impairment of the esophageal epithelial barrier are considered relevant for perception of symptoms in patients with nonerosive reflux disease (NERD). In these patients, the receptor transient receptor potential vanilloid 1 (TRPV1) is overexpressed in the esophageal mucosa, but its role is not yet fully understood. We evaluated the role of TRPV1 in esophageal inflammation and mucosal barrier impairment in a murine model of NERD. METHODS: Nonerosive reflux disease was surgically induced in Swiss mice by pyloric substenosis and ligature of the gastric fundus, and the mice were killed 7 days post surgery. The experimental groups were: I, sham surgery (negative control); II, NERD untreated; III and IV, NERD + SB366791 or capsazepine (TRPV1 antagonists); and V, NERD + resiniferatoxin (for long-term desensitization of TRPV1). The esophagus was collected for western blotting and histopathology and for evaluation of wet weight, myeloperoxidase (MPO), keratinocyte-derived chemokine (KC), transepithelial electrical resistance (TEER), and basal permeability to fluorescein. KEY RESULTS: Compared to sham, NERD mice had increased esophageal wet weight and MPO and KC levels. The mucosa had no ulcers but exhibited inflammation. NERD mice showed mucosal TRPV1 overexpression, a more pronounced decrease in TEER at pH 0.5 (containing pepsin and taurodeoxycholic acid), and increased basal permeability. Pharmacological modulation of TRPV1 prevented esophageal inflammation development, TEER changes by acidic exposure, and increase in esophageal permeability. CONCLUSIONS & INFERENCES: The TRPV1 receptor has a critical role in esophageal inflammation and mucosal barrier impairment in NERD mice, suggesting that TRPV1 might be a pharmacological target in patients with NERD.

Isolation of Clostridium perfringens and C. difficile in crab-eating fox ( Cerdocyon thous - Linnaeus 1776) from Northeastern Brazil / Isolamento de Clostridium perfringens e C. difficile em cachorro-do-mato (Cerdocyon thous - Linnaeus 1776) da região Nordeste do Brasil

The aim of the present study was to isolate Clostridium perfringens and C. difficile in crab-eating fox (Cerdocyon thous) from Northeastern Brazil. Stool samples of 18 captive crab-eating foxes from four states of Northeastern Brazil (Alagoas, Bahia, Paraíba e Pernambuco) were collected and subjected to C. perfringens and C. difficile isolation. Suggestive colonies of C. perfringens were then analyzed for genes encoding the major C. perfringens toxins (alpha, beta, epsilon and iota), beta-2 toxin (cpb2), enterotoxin (cpe), and NetB- (netB) and NetF- (netF) encoding genes. C. difficile strains were analyzed by multiplex-PCR for a housekeeping gene (tpi), toxins A (tcdA) and B (tcdB) and a binary toxin gene (cdtB). Unthawed aliquots of stool samples positive for toxigenic C. difficile were subjected to a commercial ELISA to evaluate the presence of A/B toxins. Clostridium perfringens (type A) was isolated from five (27%) samples, and only one sample was positive for beta-2 enconding gene (cpb2). Two (11%) stool samples were positive for C. difficile, but negative for A/B toxins. These two wild canids were also positive for C. perfringens type A. This is the first report of C. difficile in crab-eating fox.(AU)

O objetivo deste estudo foi isolar Clostridium perfringens e C. difficile em cachorro-do-mato (Cerdocyon thous) da região Nordeste do Brasil. Amostras de fezes de 18 cachorros-do-mato mantidos em cativeiro e oriundos de quatro estados da região Nordeste do Brasil (Alagoas, Bahia, Paraíba e Pernambuco) foram coletadas e submetidas a isolamento de C. perfringens e C. difficile. As colônias sugestivas de C. perfringens foram analisadas para os genes que codificam as principais toxinas de C. perfringens (alfa, beta, épsilon e iota), toxina beta-2 (cpb2), enterotoxina (cpe) e NetB- (netB) e NetF- (netF). As cepas de C. difficile foram analisadas por PCR-multiplex para o gene tpi, toxinas A (tcdA) e B (tcdB) e um gene de toxina binária (cdtB). Alíquotas de amostras de fezes positivas para C. difficile toxigênico foram submetidas a um ELISA comercial para avaliar a presença de toxinas A/B. Clostridium perfringens (tipo A) foi isolado de cinco (27%) amostras, e apenas uma amostra foi positiva para o gene da toxina beta-2 (cpb2). Duas (11%) amostras de fezes foram positivas para C. difficile, mas negativas para toxinas A/B. Estes dois canídeos silvestres também foram positivos para C. perfringens tipo A. Este é o primeiro relato de C. difficile em cachorro-do-mato.(AU)

ADME studies and preliminary safety pharmacology of LDT5, a lead compound for the treatment of benign prostatic hyperplasia.

This study aimed to estimate the absorption, distribution, metabolism and excretion (ADME) properties and safety of LDT5, a lead compound for oral treatment of benign prostatic hyperplasia that has previously been characterized as a multi-target antagonist of α1A-, α1D-adrenoceptors and 5-HT1A receptors. The preclinical characterization of this compound comprised the evaluation of its in vitro properties, including plasma, microsomal and hepatocytes stability, cytochrome P450 metabolism and inhibition, plasma protein binding, and permeability using MDCK-MDR1 cells. De-risking and preliminary safety pharmacology assays were performed through screening of 44 off-target receptors and in vivo tests in mice (rota-rod and single dose toxicity). LDT5 is stable in rat and human plasma, human liver microsomes and hepatocytes, but unstable in rat liver microsomes and hepatocytes (half-life of 11 min). LDT5 is highly permeable across the MDCK-MDR1 monolayer (Papp ∼32×10-6 cm/s), indicating good intestinal absorption and putative brain penetration. LDT5 is not extensively protein-bound and is a substrate of human CYP2D6 and CYP2C19 but not of CYP3A4 (half-life >60 min), and did not significantly influence the activities of any of the human cytochrome P450 isoforms screened. LDT5 was considered safe albeit new studies are necessary to rule out putative central adverse effects through D2, 5-HT1A and 5-HT2B receptors, after chronic use. This work highlights the drug-likeness properties of LDT5 and supports its further preclinical development.

The anti-inflammatory and antinociceptive effects of proteins extracted from Acacia farnesiana seeds / Efeito antiiflamatório e antinociceptivo de proteínas extraídas de sementes de Acacia farnesiana

ABSTRACT Seeds of Acacia farnesiana are commonly sold in the local markets of northeastern Brazil as a therapeutic agent. The present work aimed to evaluate the anti-inflammatory and analgesic activities of proteins obtained from A. farnesiana seeds. Five different protein fractions (albumin, globulin, prolamin, acidic and basic glutelins) were obtained and investigated for the protein pattern, the presence of hemagglutinating and proteolytic activities. The globulin fraction (GLB) was also evaluated for anti-inflammatory and analgesic activities. Globulins reduced the paw edema induced by carrageenan in a dose-dependent manner, which was accompanied by a reduction of myeloperoxidase activity (p < 0.05). Additionally, GLB reduced the neutrophil peritoneal migration induced by carrageenan. However, GLB was not able to inhibit the edema triggered by dextran. Pre-treatment with globulins reduced the abdominal constrictions induced by acetic acid as well as the paw licking time induced by formalin (69.1% at first phase). However, it did not produce a significant antinociceptive effect in the hot plate test (55-56 °C). Treating the GLB with heat (at 100 °C for 30 min) abolished its anti-edematogenic and hemagglutinating activities. Our results showed that seeds from A. farnesiana are a source of proteins with anti-inflammatory and analgesic properties.

RESUMO Sementes de Acacia farnesiana são comumente vendidas em feiras locais no nordeste do Brasil como agente terapêutico. O presente trabalho objetivou avaliar as atividades antiinflamatória e antinociceptiva de proteínas obtidas de sementes de A. farnesiana. Cinco frações protéicas distintas (albuminas, globulinas, prolaminas, glutelinas ácidas e básicas) foram obtidas e investigadas quanto o perfil de proteínas, presença de atividade hemaglutinante e proteolítica. A fração globulina (GLB) também foi avaliada quanto a presença de atividade antiinflamatória e analgésica. Globulinas reduziram o edema de pata induzido por carragenina de modo dependente da dose que foi acompanhada da redução da atividade da mieloperoxidase (p < 0,05). Em adição, GLB reduziu a migração de neutrófilos para cavidade peritoneal induzida por carragenina. Entretanto, GLB não foi capaz de inibir o edema induzido por dextrana. O pré-tratamento com globulinas reduziu as contorções abdominais induzidas por ácido acético, bem como o tempo de lambedura da pata induzida por formalina (69.1% na primeira fase). Por outro lado, GLB não produziu um efeito antinociceptivo significante no teste de placa quente (55-56 °C). O pré-tratamento de GLB com calor (100 °C por 30 min) aboliu sua atividade anti-edematogênica e hemaglutinante. Nossos resultados mostraram que sementes de A. farnesiana são fonte de proteínas com propriedades antiinflamatórias e analgésicas.

ADME studies and preliminary safety pharmacology of LDT5, a lead compound for the treatment of benign prostatic hyperplasia

This study aimed to estimate the absorption, distribution, metabolism and excretion (ADME) properties and safety of LDT5, a lead compound for oral treatment of benign prostatic hyperplasia that has previously been characterized as a multi-target antagonist of α1A-, α1D-adrenoceptors and 5-HT1A receptors. The preclinical characterization of this compound comprised the evaluation of its in vitro properties, including plasma, microsomal and hepatocytes stability, cytochrome P450 metabolism and inhibition, plasma protein binding, and permeability using MDCK-MDR1 cells. De-risking and preliminary safety pharmacology assays were performed through screening of 44 off-target receptors and in vivo tests in mice (rota-rod and single dose toxicity). LDT5 is stable in rat and human plasma, human liver microsomes and hepatocytes, but unstable in rat liver microsomes and hepatocytes (half-life of 11 min). LDT5 is highly permeable across the MDCK-MDR1 monolayer (Papp ∼32×10-6 cm/s), indicating good intestinal absorption and putative brain penetration. LDT5 is not extensively protein-bound and is a substrate of human CYP2D6 and CYP2C19 but not of CYP3A4 (half-life >60 min), and did not significantly influence the activities of any of the human cytochrome P450 isoforms screened. LDT5 was considered safe albeit new studies are necessary to rule out putative central adverse effects through D2, 5-HT1A and 5-HT2B receptors, after chronic use. This work highlights the drug-likeness properties of LDT5 and supports its further preclinical development.

Latent structure of the symptomatology of hospitalized patients with bipolar mania.

Several studies have attempted to understand the dimensions of psychiatric symptoms in manic episodes, but only a few have been able to model the latent structure of mania in bipolar disorder patients using confirmatory factor analysis. The objective of the present study was to search for the best model of the symptomatology of hospitalized manic patients. To achieve this goal, 117 manic inpatients during a manic crisis participated in this research. Exploratory factor analysis was conducted followed by confirmatory factor analysis using an exploratory factor analysis solution and three other theory-based models. The exploratory factor analysis results revealed a six-factor structure: depression, suicide, insomnia, mania, psychosis, and anxiety. This solution also presented the best fit to the data when tested with confirmatory factor analysis. A five-factor solution, without suicide as a separate dimension, appeared to be more theoretically suitable. Another important finding was that anxiety was an independent dimension in mania. Some hypotheses are discussed in light of contemporary theories, and future studies should investigate this aspect further.

The hydrogen sulfide donor, Lawesson's reagent, prevents alendronate-induced gastric damage in rats.

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1ß], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg, ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35 ± 9.8 mm(2)); increased levels of TNF-α, IL-1ß, and MDA (2311 ± 302.3 pg/mL, 901.9 ± 106.2 pg/mL, 121.1 ± 4.3 nmol/g, respectively); increased MPO activity (26.1 ± 3.8 U/mg); and reduced GSH levels (180.3 ± 21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77 ± 5.3 mm(2)); reduced TNF-α, IL-1ß, and MDA formation (1502 ± 150.2 pg/mL, 632.3 ± 43.4 pg/mL, 78.4 ± 7.6 nmol/g, respectively); lowered MPO activity (11.7 ± 2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9 ± 40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.

The hydrogen sulfide donor, Lawesson's reagent, prevents alendronate-induced gastric damage in rats

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1β], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg, ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35±9.8 mm2); increased levels of TNF-α, IL-1β, and MDA (2311±302.3 pg/mL, 901.9±106.2 pg/mL, 121.1±4.3 nmol/g, respectively); increased MPO activity (26.1±3.8 U/mg); and reduced GSH levels (180.3±21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77±5.3 mm2); reduced TNF-α, IL-1β, and MDA formation (1502±150.2 pg/mL, 632.3±43.4 pg/mL, 78.4±7.6 nmol/g, respectively); lowered MPO activity (11.7±2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9±40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.

Detection of A/B toxin and isolation of Clostridium difficile and Clostridium perfringens from foals.

REASONS FOR PERFORMING THE STUDY: Toxin detection and screening could contribute to knowledge of the transmission patterns, risk factors and epidemiology of Clostridium difficile and Clostridium perfringens. OBJECTIVE: To isolate C. difficile and C. perfringens and to detect A/B toxins in faecal samples from diarrhoeic and nondiarrhoeic foals. STUDY DESIGN: Cross-sectional observational study. METHODS: A total of 153 samples from foals were collected: 139 samples from farms and 14 samples from diarrhoeic foals admitted to a veterinary hospital. The A/B toxins were detected by cytotoxicity assay. All suspected colonies of C. perfringens were subjected to polymerase chain reaction for detection of the major toxin genes (α, ß, ε and ι) and for detection of ß2-, NetB- and enterotoxin-encoding genes. Furthermore, C. difficile and C. perfringens isolates were evaluated for in vitro antimicrobial susceptibility. RESULTS: Seven of 153 (4.6%) samples, all from diarrhoeic foals, were positive for C. difficile A/B toxin. Of these, 5 of 14 (35.7%) were from hospitalised foals, and only 2 of 63 (3.2%) diarrhoeic foal samples were from farms (P = 0.002). Clostridium perfringens was isolated from 31 (20.3%) foals, of which 21 of 76 (27.6%) were diarrhoeic and 10 of 76 (13.2%) were nondiarrhoeic, demonstrating a difference between these 2 groups (P = 0.045). Only 4 strains were positive for the ß2-encoding gene (cpb2). All C. difficile and C. perfringens isolates were susceptible to metronidazole and vancomycin. CONCLUSIONS: The present report highlights the need for laboratory diagnostics to differentiate C. difficile-associated infection in foals from other causes of diarrhoea to facilitate adequate antimicrobial therapy. POTENTIAL RELEVANCE: More studies are needed to clarify the role of C. perfringens as a primary agent of diarrhoea in foals.

In vitro evaluation of Clostridium septicum alpha toxoid / Avaliação in vitro de toxoide alfa de Clostridium septicum

Aiming to investigate in vitro alternatives, a test for neutralizing antibody detection using cell culture was developed. This test was more sensitive than previous animal models, allowing for detection of substantially lower alpha toxin and anti-alpha toxin titers. Titers observed during in vivo and in vitro seroneutralization had a correlation of 99.12 percent, indicating that cell culture is a viable alternative in the evaluation of vaccine potency, screening of vaccinal seeds, and Clostridium septicum alpha toxin titration.

Padronizou-se um teste para detecção de anticorpos neutralizantes in vitro, em cultura de células. O modelo in vitro mostrou-se mais sensível que os testes com animais, permitindo a detecção de títulos de toxina e antitoxina alfa mais baixos. Os títulos observados na soroneutralização in vivo e in vitro, apresentaram correlação de 99,12 por cento, demonstrando ser a cultura de células uma alternativa viável na avaliação da potência de vacinas, triagem de sementes vacinais e titulação de toxina alfa de Clostridium septicum.

Mionecroses clostridiais bovinas / Bovine clostridial myonecrosis

As mionecroses são enfermidades altamente fatais, representadas pelo carbúnculo sintomático e edema maligno. Com o objetivo de determinar a ocorrência dos diferentes clostrídios envolvidos nas mionecroses em bovinos no Brasil, foi realizado um estudo retrospectivo a partir de 30 casos suspeitos de carbúnculo sintomático e edema maligno. Empregou-se a técnica de estreptavidina biotina peroxidase em tecidos de bovinos fixados em formol e incluídos em parafina de arquivos de patologia de diferentes estados do país. Foram detectados 21 Clostridium chauvoei (70%), cincoClostridium septicum(17%), seguidos de doisC. chauvoeiassociado aoC. septicum (7%), um C. chauvoei mais Clostridium sordellii (3%) e um C. chauvoei mais Clostridium novyi tipo A (3%). Este é o primeiro diagnóstico de C. novyi tipo A no Brasil, e o primeiro relato da ocorrência de C. chauvoei associado à lesão no miocárdio. Os achados da maior ocorrência de C. chauvoei e C. septicum corroboram com estudos anteriores no país. Estes dados sobre a ocorrência dos agentes responsáveis pelas mionecroses em diferentes estados do país são de extrema importância frente às decisões relativas ao controle dessas enfermidades.

Clostridial myonecroses are highly fatal diseases, characterized by black leg and malignant oedema. In order to determine the occurrence of the different agents involved in Brazilian clostridial myonecrosis, a retrospective study was conducted concerning 30 suspected cases of blackleg and gas gangrene using a streptavidin-biotin peroxidase technique in formalin-fixed, paraffin-embedded tissues of cattle from the archives of pathology of different states of Brazil. The clostridia predominantly detected wereClostridium chauvoei(70%), followed by Clostridium septicum (5 cases or 17%), C. chauvoei plus C. septicum (2 cases or 7%), C. chauvoei plus Clostridium sordellii (1 case or 3%), C. chauvoei plus Clostridium novyi type A (1 case or 3%). This is the first diagnosis of C. novyi type A in Brazil. The findings of the higher occurrence of C. chauvoei and C. septicum concur with previous works in Brazil. This article also includes the first report in Brazil of the occurrence of C. chauvoei associated with a lesion in the myocardium. Thus, this study supplies important data about the occurrence of the agents responsible for clostridial myonecrosis in different states of Brazil, these data being important for decisions relative to control of these diseases.

Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization.

Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-epsilon) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the toxin (as an antigen), rabbits were immunized with 50, 100 or 200 microg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-epsilon is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D.

Hepatitis C virus infection diagnosis using metabonomics.

Metabonomics based on nuclear magnetic resonance (NMR) can reveal the profile of endogenous metabolites of low molecular weight in biofluids related to disease. The profile is identified a 'metabolic fingerprint' like from the pathological process, why this metabonomics has been used as a diagnostic method. The aim of the present study was to apply metabonomics to identify patients infected with the hepatitis C virus (HCV) through an analysis of ¹H NMR spectra of urine samples associated with multivariate statistical methods. A pilot study was carried out for the diagnostic test evaluation, involving two groups: (i) 34 patients positive for anti-HCV and HCV-RNA and negative for anti-HBc (disease group); and (ii) 32 individuals positive for anti-HBc and negative for HBsAg and anti-HCV. The urine samples were analyzed through ¹H NMR, applying principal component analysis and discriminant analysis for classification. The metabonomics model was capable of identifying 32 of the 34 patients in the disease group as positive and 31 of the 32 individuals in the control group as negative, demonstrating 94% sensitivity and specificity of 97% as well as positive and negative predictive values of 97% and 94%, respectively, and 95% accuracy (P < 0.001). In conclusion, the metabonomics model based on ¹H NMR spectra of urine samples in this preliminary study discriminated patients with HCV infection with high sensitivity and specificity, thereby demonstrating this model to be a potential tool for use in medical practice in the near future.

Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens / Sensibilidade antimicrobiana de estirpes de Clostridium perfringens isoladas de aves de corte

Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant.

Clostridium perfringens é um habitante normal da microbiota intestinal de frangos, sendo um agente potencialmente patogênico, causador de enterite necrótica e colangio-hepatite.A concentração inibitória mínima (CIM) de sete drogas utilizadas na terapêutica, como agentes promotores de crescimento ou na prevenção de coccidiose foi determinada pelo método de diluição em ágar para 55 estirpes de C. perfringens isoladas do intestino de frangos de corte. Todas as estirpes revelaram alta sensibilidade à penicilina, avilamicina, narasin e monensina, apenas 7,3% demonstraram CIM intermediário para lincomicinae 89.1% foram consideradas sensíveis. Para tetraciclina e bacitracina, 41,8% e 47.3% das amostras, respectivamente, foram consideradas resistentes.