Análise comparativa entre alvos moleculares para detecção do Helicobacter pylori

KEYWORDS: Helicobacter pylori. Polymerase Chain Reaction. Molecular Diagnostic TechniquesDESCRITORES: Helicobacter pylori. Reação em Cadeia da Polimerase. Técnicas de Diagnóstico Molecula

Análise genotípica do Helicobacter pylori pelo método Multiple-Locus Variable-Number Tandem-Repeats no sul do Brasil

Background and objectives: Despite its use as a genotyping method for different microorganisms, few studies have reported the use of Multiple-Locus Variable Number of Tandem Repeats Analysis (MLVA) for the analysis of the clonal diversity of Helicobacter pylori. The aim of this study was to determine the genetic variability of H. pylori strains by MLVA in southern Brazil. Methods: 95 H. pylori DNA samples were obtained from the gastric biopsies of H. pylori-positive patients from two cities in southern Brazil and their clonal analysis was evaluated by MLVA method using electrophoresis in agarose gel. For selection of loci to be analyzed in this study, an in silico analysis of 12 loci previously described in the literature was performed. Results: From the in silico analysis, only four loci were viable for the genotypic analysis of these H. pylori strains, resulting in 90 strains distributed in eight different groups and five orphan strains. Conclusion: Despite MLVA method allow make inferences about the genotypic diversity of a population, our results showed that H. pylori genotyping methods should be critically evaluated prior to their use in this region of Brazil.Justificativa e objetivos: Apesar de ser utilizado como um método de genotipagem para diferentes microrganismos, poucos estudos relatam a utilização de Multiple-Locus Variable Number of Tandem Repeats Analysis (MLVA) para análise da diversidade clonal do Helicobacter pylori. O objetivo deste estudo foi determinar a variabilidade genética de cepas de H. pylori pelo MLVA no sul do Brasil. Métodos: 95 amostras de DNA de H. pylori foram obtidas a partir de biópsias gástricas de pacientes H. pylori-positivos provenientes de duas cidades do sul do Brasil e a diversidade genética das cepas foi avaliada pelo método MLVA utilizando eletroforese em gel de agarose. Para a seleção dos loci a serem analisados neste estudo, foi realizada uma análise in silico de 12 loci previamente descritos na literatura. Resultados: A partir da análise in silico, apenas quatro loci foram considerados viáveis para a análise genotípica das cepas, resultando em 90 cepas distribuídas em oito grupos diferentes e cinco cepas órfãs. Conclusões: Apesar de o método MLVA permitir fazer inferências acerca da diversidade genética de uma população, nossos resultados mostraram que os métodos de genotipagem do H. pylori devem ser criticamente avaliados antes de serem utilizados nessa região do Brasil

Análise genotípica do Helicobacter pylori pelo método Multiple-Locus Variable-Number Tandem-Repeats no sul do Brasil

Justificativa e Objetivos: Apesar de ser utilizado como um método de genotipagem para diferentes microrganismos, poucos estudos relatam a utilização de Multiple-Locus Variable Number of Tandem Repeats Analysis (MLVA) para análise da diversidade clonal do Helicobacter pylori. O objetivo deste estudo foi determinar a variabilidade genética de cepas de H. pylori pelo MLVA no sul do Brasil. Métodos: 95 amostras de DNA de H. pylori foram obtidas a partir de biópsias gástricas de pacientes H. pylori-positivos provenientes de duas cidades do sul do Brasil e a diversidade genética das cepas foi avaliada pelo método MLVA utilizando eletroforese em gel de agarose. Para a seleção dos loci a serem analisados neste estudo, foi realizada uma análise in silico de 12 loci previamente descritos na literatura. Resultados: A partir da análise in silico, apenas quatro loci foram considerados viáveis para a análise genotípica das cepas, resultando em 90 cepas distribuídas em oito grupos diferentes e cinco cepas órfãs. Conclusões: Apesar de o método MLVA permitir fazer inferências acerca da diversidade genética de uma população, nossos resultados mostraram que os métodos de genotipagem do H. pylori devem ser criticamente avaliados antes de serem utilizados nessa região do Brasil.Background and Objectives: Despite its use as a genotyping method for different microorganisms, few studies have reported the use of Multiple-Locus Variable Number of Tandem Repeats Analysis (MLVA) for the analysis of the clonal diversity of Helicobacter pylori. The aim of this study was to determine the genetic variability of H. pylori strains by MLVA in southern Brazil. Methods: 95 H. pylori DNA samples were obtained from the gastric biopsies of H. pylori-positive patients from two cities in southern Brazil and their clonal analysis was evaluated by MLVA method using electrophoresis in agarose gel. For selection of loci to be analyzed in this study, an in silico analysis of 12 loci previously described in the literature was performed. Results: From the in silico analysis, only four loci were viable for the genotypic analysis of these H. pylori strains, resulting in 90 strains distributed in eight different groups and five orphan strains. Conclusion: Despite MLVA method allow make inferences about the genotypic diversity of a population, our results showed that H. pylori genotyping methods should be critically evaluated prior to their use in this region of Brazil

Helicobacter pylori infection and associated factors

Background and Objectives: Helicobacter pylori is linked to gastroduodenal pathologies. To determine the frequency and potential risk factors of the H. pylori infection. Material and methods: A cross-sectional study was conducted, including 227 patients, submitted to upper gastrointestinal endoscopy. A questionnaire was applied to the patients, before endoscopy. The biopsy specimens were obtained from the antrum and gastric body for histology and PCR. The chi-square test was used for the categorical data analysis. P-values0.05). However, we observed a significant association between the number of persons per household and presence of H. pylori (p=0.04). A statistically significant relation also was found between H. pylori and the patient’s age (p=0.04) and between the histological and endoscopic diagnoses and the H. pylori infection (p≤0.01). Conclusions: We found a significant relation between household crowding and presence of H. pylori, which seems facilitate the person-to-person transmission H. pylori within families. Our results also suggest a cohort phenomenon. The increase in the frequency of H. pylori infection according to age may be due the acquisition of bacterium predominantly in childhood, when the sanitary conditions were deficient, and not during adulthood. Once acquired and untreated, the persistent H. pylori infection might have led to the development of severe gastroduodenal diseases

Potential antimycobacterial naphthoquinones, and compounds of a lipid nanocarrier

Tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium tuberculosis and although curable, is still considered a problem of public health worldwide. Prolonged treatment combined with its toxic effects and pharmacokinetic interactions with other compounds can hamper patient adherence to therapy, besides selecting resistant bacilli. These factors reinforce the need to search for new drugs and substances that can enhance the action of other drugs already established. Therefore, the aim of this study was to evaluate the antimycobacterial potential of naphthoquinones, a lipid nanocarrier and its ingredients (castor oil, soy lecithin, PEG stearate 660 and rifampicin (RIF)), by Resazurin Microtiter Assay, by determining the minimum inhibitory concentration (MIC). The naphthoquinones were evaluated against three strains in vitro: a pan-susceptible H37Rv (ATCC 27294); one monoresistant isoniazid (ATCC 35822) with mutation in the katG gene (S315T); and a monoresistant to RIF (ATCC 35338) with a mutation in rpoB gene (H526T), while RIF-NE and the excipients have been evaluated against other three strains: a pan-susceptible H37Rv (ATCC 27294); a susceptible clinical isolate and a multidrug-resistant (MDR) clinical isolate, with mutations in the katG gene (S315T) and rpoB (S531L). All naphthoquinones were active against the three strains of M. tuberculosis, with MIC between 234 and 12.5 μM. However, the molecules which have an amine grouping in their composition have shown promising results related to the first stage of a new drug development, due to the antimycobacterial activity and low toxicity against the J774A.1 macrophage lineage. About lipid nanocarriers, it was observed that the encapsulation of RIF was not able to enhance its in vitro activity for susceptible strains, however, increased 130x the susceptibility of the MDR strain to RIF. Based on this result, the antimycobacterial activity of the nanoemulsion without rifampicin (UN-NE) and excipients used in the formulation was evaluated. The PEG-660 stearate was active against the three strains of M. tuberculosis, while castor oil and soy lecithin showed no antimycobacterial activity. Considering the proportional concentrations of each component in the formulation of nanoemulsions and MIC of these compounds separately, it can infer that the antimicrobial activity of UN-NE for the three strains and RIF-NE for MDR strain can be related to PEG-660 stearate activity. Thus, some compounds evaluated in this study showed antimycobacterial activity and may be promising in the development of new therapeutic alternatives for TB, serving as prototypes for new drugs or new carriers of anti-TB drugs.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESA tuberculose (TB) é uma doença infecciosa causada, principalmente, pelo Mycobacterium tuberculosis e, apesar de curável, ainda é considerada um problema de saúde pública mundial. O tratamento prolongado, combinado com seus efeitos tóxicos e interações farmacocinéticas com outros compostos, pode dificultar a adesão do paciente à terapia, além de selecionar bacilos resistentes. Esses fatores reforçam a necessidade da busca por novos fármacos e por substâncias que possam potencializar a ação de outros fármacos já estabelecidos. Logo, o objetivo deste estudo foi avaliar o potencial antimicobacteriano de naftoquinonas, um nanocarreador lipídico e seus excipientes (óleo de rícino, lecitina de soja, estearato de PEG 660 e rifampicina (RIF)), pelo método Resazurin Microtiter Assay, através da determinação da concentração mínima inibitória (CMI). As naftoquinonas foram avaliadas in vitro frente a três cepas de M. tuberculosis: uma pan-suscetível H37Rv (ATCC 27294); uma monoresistente à isoniazida (INHr - ATCC 35822) com mutação no gene katG (S315T); e outra monoresistente à rifampicina (RIFr - ATCC 35338) com mutação no gene rpoB (H526T), enquanto a RIF-NE e seus excipientes foram avaliados frente a outras três cepas: uma pan-suscetível H37Rv (ATCC 27294); um isolado clínico suscetível e um isolado clínico multidroga resistente (MDR), com mutações nos genes katG (S315T) e rpoB (S531L). Todas as naftoquinonas foram ativas frente às três cepas de M. tuberculosis, com CMI entre 234 e 12,5 μM. Entretanto, as moléculas que apresentam um grupamento amina em sua composição, apresentaram resultados promissores relacionados à primeira etapa de desenvolvimento de um novo fármaco, devido à atividade antimicobacteriana e à baixa toxicidade frente a macrófagos da linhagem J774A.1. Em relação aos nanocarreadores lipídicos, foi possível observar que o encapsulamento da RIF não foi capaz de potencializar sua atividade in vitro para as cepas suscetíveis, porém, aumentou 130x a suscetibilidade da cepa MDR à RIF. Com base nesse resultado, foi avaliada a atividade antimicobacteriana da nanoemulsão sem rifampicina (UN-NE) e dos excipientes utilizados na sua formulação. O estearato de PEG 660 foi ativo frente às três cepas de M. tuberculosis, enquanto o óleo de rícino e a lecitina de soja não apresentaram atividade antimicobacteriana. Considerando as concentrações proporcionais de cada componente na formulação das nanoemulsões e a CMI desses compostos separadamente, pode-se inferir que a atividade antimicrobiana da UN-NE para as três cepas e da RIF-NE para a cepa MDR pode estar relacionada à atividade do estearato de PEG 660. Dessa forma, alguns compostos avaliados neste estudo apresentaram potencial antimicobacteriano e podem ser promissores no desenvolvimento de novas alternativas terapêuticas para a TB, servindo como protótipos para novos fármacos ou novos carreadores de fármacos anti-TB

In vitro susceptibility of Sporothrix spp. to complexes coordinated with Co(II) and cobalt chloride hexahydrate

Sporothrix spp. are the major dimorphic fungus associated with a type of subcutaneous mycosis, sporotrichosis. The limitation of antifungal availability and the past reports of in vitro resistance of Sporothrix spp. clinical isolates makes it important to search for new compounds with antifungal activities. In this study, we therefore evaluate the in vitro activities of complexes coordinated with Co(II) and cobalt chloride hexahydrate against clinical isolates of Sporothrix spp. Broth microdilution test was performed as per M38-A2 from CLSI (2008) in duplicate for 31 clinical isolates of Sporothrix spp. (27 S. brasiliensis e 04 S. schenckii stricto sensu). The antifungal activities of the complexes coordinated with Co(II) and cobalt chloride hexahydrate were detected at a concentration range of 32-128 µg/mL for all isolates. None of the compounds demonstrated any cytotoxicity (to macrophage cells) at the concentration of 200 µg/mL. The activity against Sporothrix spp. recorded in this study instigate the continuity of experimental studies with Co(II) to search for the mechanisms of antifungal action as well as to evaluate its interaction with the commercial antifungal drugs

Toxicity evaluation of nanocrystalline silver-impregnated coated dressing on the life cycle of worm Caenorhabditis elegans

In recent times, however, due to the emergence of bacterial strains with resistance to conventional antibiotics, silver has again gained attention as an alternative for developing new efficient bactericides, including the use of silver nanoparticles (AgNPs). However, the improper disposal of these items after use may cause toxicological effects on organisms in the environment. To evaluate the potential environmental hazard of nanosilver-coated dressings, the nematode Caenorhabditis elegans was chosen as a test organism. The assays were conducted in 24-well plates that contain four different sizes of coated dressing to obtain different concentrations. L1 and L4 C. elegans larval stages were exposed to these nanosilver concentrations. Dressing cutouts were arranged between two layers of agar for 3 days and Escherichia coli (OP 50 strain) was added as food source for the worms. After the exposure period, growth, reproduction, fertility, silver concentration in the medium and the concentration of reactive oxygen species (ROS) in the worms were evaluated. Scanning and transmission electron microscopy analyses were performed on the coated dressings, as well as analyses of zeta potential, ionic release and antibacterial power in two bacterial strains (Pseudomonas aeruginosa and Staphylococcus aureus). It was verified the antibacterial power of the coated dressing, in both bacteria strains tested. Characterization of the coated dressing indicated heterogeneous nanoparticles, as well as distinct zeta potentials for the medium in water and saline medium (0.9% NaCl). L1 larval worms exposed to nanosilver-coated dressing showed a high ROS concentration and reductions in growth, fertility and reproduction. Worms exposed to the coated dressing during the L4 stage showed almost no response. Overall, the obtained results indicate the potential environmental hazard of nanosilver-coated dressings.Fil: Ayech, Alinne. Universidade Federal do Rio Grande; BrasilFil: Josende, M. E.. Universidade Federal do Rio Grande; BrasilFil: Ventura Lima, J.. Universidade Federal do Rio Grande; BrasilFil: Ruas, Caroline Pires. Universidade Federal do Rio Grande; BrasilFil: Gelesky, M. A.. Universidade Federal do Rio Grande; BrasilFil: Ale, Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; ArgentinaFil: Cazenave, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; ArgentinaFil: Galdopórpora, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Desimone, Martín Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Duarte, Miriam. Universidade Federal do Rio Grande; BrasilFil: Halicki, Priscila Cristina Bartolomeu. Universidade Federal do Rio Grande; BrasilFil: Ramos, Daniela Fernandes. Universidade Federal do Rio Grande; BrasilFil: Carvalho, L. M.. Universidade Federal de Santa Maria; BrasilFil: Leal, Gabriela Camera. Universidade Federal de Santa Maria; BrasilFil: Monserrat, José María. Universidade Federal do Rio Grande; Brasi