Use of Deep-Amplicon Sequencing (DAS), Real-Time PCR and In Situ Hybridization to Detect H. pylori and Other Pathogenic Helicobacter Species in Feces from Children.

BACKGROUND: Detecting Helicobacter pylori in fecal samples is easier and more comfortable than invasive techniques, especially in children. Thus, the objective of the present work was to detect H. pylori in feces from children by molecular methods as an alternative for diagnostic and epidemiological studies. METHODS: Forty-five fecal samples were taken from pediatric patients who presented symptoms compatible with H. pylori infection. HpSA test, culture, real-time quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), direct viable count associated with FISH (DVC-FISH), and Illumina-based deep-amplicon sequencing (DAS) were applied. RESULTS: No H. pylori colonies were isolated from the samples. qPCR analysis detected H. pylori in the feces of 24.4% of the patients. In comparison, DVC-FISH analysis showed the presence of viable H. pylori cells in 53.3% of the samples, 37% of which carried 23S rRNA mutations that confer resistance to clarithromycin. After DAS, H. pylori-specific 16S rDNA sequences were detected in 26 samples. In addition, DNA from H. hepaticus was identified in 10 samples, and H. pullorum DNA was detected in one sample. CONCLUSION: The results of this study show the presence of H. pylori, H. hepaticus, and H. pullorum in children's stools, demonstrating the coexistence of more than one Helicobacter species in the same patient. The DVC-FISH method showed the presence of viable, potentially infective H. pylori cells in a high percentage of the children's stools. These results support the idea that fecal-oral transmission is probably a common route for H. pylori and suggest possible fecal-oral transmission of other pathogenic Helicobacter species.

Development of Optical Label-Free Biosensor Method in Detection of Listeria monocytogenes from Food.

The present work describes an alternative method for detecting and identifying Listeria monocytogenes in food samples by developing a nanophotonic biosensor containing bioreceptors and optical transducers. The development of photonic sensors for the detection of pathogens in the food industry involves the implementation of procedures for selecting probes against the antigens of interest and the functionalization of the sensor surfaces on which the said bioreceptors are located. As a previous step to functionalizing the biosensor, an immobilization control of these antibodies on silicon nitride surfaces was carried out to check the effectiveness of in plane immobilization. On the one hand, it was observed that a Listeria monocytogenes-specific polyclonal antibody has a greater binding capacity to the antigen at a wide range of concentrations. A Listeria monocytogenes monoclonal antibody is more specific and has a greater binding capacity only at low concentrations. An assay for evaluating selected antibodies against particular antigens of Listeria monocytogenes bacteria was designed to determine the binding specificity of each probe using the indirect ELISA detection technique. In addition, a validation method was established against the reference method for many replicates belonging to different batches of meat-detectable samples, with a medium and pre-enrichment time that allowed optimal recovery of the target microorganism. Moreover, no cross-reactivity with other nontarget bacteria was observed. Thus, this system is a simple, highly sensitive, and accurate platform for L. monocytogenes detection.

Programa educativo para la prevención del cáncer bucal desde la adolescencia / Educational program for the prevention of oral cancer since adolescence

RESUMEN Fundamento: el cáncer bucal aporta un número considerable en las cifras de morbilidad las que aumentarán en los decenios venideros. Informar sobre el tema pudiera ser el punto de partida si se pretende combatir este flagelo de la vida moderna. Objetivo: diseñar un programa educativo sobre cáncer bucal para adolescentes según necesidades identificadas. Métodos: se realizó una investigación de desarrollo con enfoque mixto, de intervención educativa, prexperimental, prospectiva y de corte longitudinal en el municipio de Santa Clara en el período comprendido entre febrero/2016 a noviembre/2017. Se utilizaron métodos teóricos y empíricos que permitieron la fundamentación bibliográfica y la recogida de información. Resultados: en el diagnóstico aplicado predominaron factores de riesgos como mala higiene bucal, consumo de dieta no protectora e insuficiencias de conocimientos sobre el cáncer bucal entre los adolescentes; y práctica de hábitos tóxicos por padres u otros familiares, por lo que se diseñó un programa educativo el cual fue valorado por criterio de especialistas y aplicado con resultados muy favorables en la solución de los problemas detectados. Conclusiones: los especialistas lo consideraron pertinente, factible, útil, con adecuada estructura y valor científico y pedagógico, y se comprobó su efectividad a partir de su aplicación en el grupo de adolescentes para el cual fue diseñado.

ABSTRACT Background: oral cancer contributes with a considerable number in the morbidity figures which will increase in the coming decades. Reporting on this issue could be the starting point in the battle against this scourge of modern life. Objective: to design an educational program on oral cancer for adolescents according to identified needs. Methods: a development research was carried out with a mixed approach, educational intervention, pre-experimental, prospective and longitudinal study in the municipality of Santa Clara in the period from February/ 2016 to November / 2017. Theoretical and empirical methods were used that allowed the bibliographic foundation and the collection of information. Results: in the applied diagnosis, risk factors predominated, such as poor oral hygiene, non-protective diet consumption and insufficiency of knowledge about oral cancer among adolescents; and practice of toxic habits by parents or other family members, that´s why an educational program was designed, which was assessed by specialists and applied with very favorable results in the solution of the problems detected. Conclusions: the specialists considered it pertinent, feasible, useful, with adequate structure and with scientific and pedagogical values, and its effectiveness was proven from its application in the adolescent group for which it was designed.

High prevalence of Salmonella spp. in wastewater reused for irrigation assessed by molecular methods.

Salmonella spp. is one of the most important causal agents of food-borne illness in developed countries and its presence in irrigation water poses a risk to public health. Its detection in environmental samples is not easy when culture methods are used, and molecular techniques such as PCR or ribosomal rRNA probe hybridization (Fluorescent in situ Hybridization, FISH) are outstanding alternatives. The aim of this work was to determine the environmental risk due to the presence of Salmonella spp. in wastewater by culture, PCR and FISH. A new specific rDNA probe for Salmonella was designed and its efficiency was compared with the rest of methods Serotype and antibiotic resistance of isolated strains were determined. Forty-five wastewater samples (collected from two secondary wastewater treatment plants) were analysed. Salmonella strains were isolated in 24 wastewater samples (53%), two of them after disinfection treatment. Twenty-three Salmonella strains exhibited resistance to one or more antimicrobial agent. Analysis of wastewater samples yielded PCR positive results for Salmonella in 28 out of the 45 wastewater samples (62%). FISH analysis allowed for the detection of Salmonella in 27 (60%) samples. By using molecular methods, Salmonella was detected in four samples after disinfection treatment. These results show the prevalence of Salmonella in reclaimed wastewater even after U.V. disinfection, what is a matter of public health concern, the high rates of resistance to antibiotics and the adequacy of molecular methods for its rapid detection. FISH method, with SA23 probe developed and assayed in this work provides a tool for detecting Salmonella in water within few hours, with a high rate of effectiveness.