In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect ...H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA) O-methyl RNA probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.
Gastric cancer is the 5th most common cancer in the world, with an estimated 950,000 new cases diagnosed in 2012. It is also the 3rd leading cause of cancer death in both worldwide. Helicobacter ...pylori (H. pylori) is a gastric pathogen that causes chronic inflammation and significantly increases the risk of developing gastric diseases, including gastric cancer. The global prevalence of H. pylori infection in humans is of more than 50%, and therefore it represents a public health issue. H. pylori can be detected after endoscopy by histology, culture and urease tests, but all biopsy-based methods are liable to sampling errors. For that reason, the development of novel, fast and efficient diagnostic methods is extremely important. Confocal Laser Endoscopy is a recent technology that has been tested to identify specific cellular and subcellular changes on the surface of the gastric mucosa. However, there is no specific staining to detect H. pylori in vivo. Fluorescence in situ hybridization (FISH) using rRNA-targeted probes is a molecular technique that allows the specific identification of bacteria in different types of samples without prior cultivation. The in vivo use of FISH, also called fluorescence in vivo hybridization (FlVH), has proved to be a challenge, in part due to the need of highly resistant oligonucleotides able to hybridize in human body conditions. The development of nucleic acid chemistry allowed the synthesis of new nucleic acid mimics that possess advantages comparatively with the typical DNA or RNA probes. These new mimics may be used to design FISH probes and increase the efficiency of the detection. Locked nucleic acid (LNA) has been developed as a novel RNA derivative nucleotide analog being able to hybridize with DNA or RNA according to Watson-Crick base-pairing rules with higher selectivity. The general aim of this thesis was to explore the applicability of nucleic acid mimics for the in vivo diagnostic of H. pylori . The validation of the study was achieved using fluorescence in vivo hybridization (FIVH) for the detection of H. pylori SS1 in infected C57BL/6 mice. After 15 days of post-infection FIVH was performed using 0.5 μM and 2 μM of 10_HyP_LNA/2’OMe PS probe. Fluorescence were analysed ex vivo by microscopy in mucus samples, cryosections and paraffin-embedded tissue slides. Results showed that the 10_HyP_LNA/2’OMe PS probe displayed high specificity in vivo allowing direct observation of the location and distribution of the H. pylori SS1 within the mice stomach mucosa. In summary, the 10_HyP_LNA/2’OMe PS probe could efficiently detect H. pylori not only in varying in vitro conditions but also directly in vivo in the gastric mucosa. This work offers a set of new LNA-based FISH protocols and studies that can be used for other researches in the microbiology field. Furthermore, a new approach is also presented for the diagnostic of H. pylori. These studies should be extended to other emergent microbes for the development of methods to identify important bacteria directly in vivo, not only in a diagnostic perspective but also to understand the interaction mechanisms involving the microbiome within the human body.
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