Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

Location analysis of city sections: socio-demographic segmentation and restaurant potentiality estimation : a case study of Lisbon

One of the objectives of this study is to perform classification of socio-demographic components for the level of city section in City of Lisbon. In order to accomplish suitable platform for the restaurant potentiality map, the socio-demographic components were selected to produce a map of spatial clusters in accordance to restaurant suitability. Consequently, the second objective is to obtain potentiality map in terms of underestimation and overestimation in number of restaurants. To the best of our knowledge there has not been found identical methodology for the estimation of restaurant potentiality. The results were achieved with combination of SOM (Self-Organized Map) which provides a segmentation map and GAM (Generalized Additive Model) with spatial component for restaurant potentiality. Final results indicate that the highest influence in restaurant potentiality is given to tourist sites, spatial autocorrelation in terms of neighboring restaurants (spatial component), and tax value, where lower importance is given to household with 1 or 2 members and employed population, respectively. In addition, an important conclusion is that the most attractive market sites have shown no change or moderate underestimation in terms of restaurants potentiality.