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.