Investigación y desarrollo de nuevos métodos moleculares y bioinformáticos en salud pública para la detección de microorganismos y el estudio de la respuesta inmune humoral

Resumo

Microarray technologies have widely spread and have become a common tool in genomic applications and studies. Although they have been mostly used in gene expression analyses, their high throughput capabilities make them a valuable technology to be applied in other fields such as clinical diagnosis or microbiology. Recent events such as the Anthrax alerts after Sept. 11th, or the currently active influenza A/H1N1 epidemics have shown the necessity of fast and accurate techniques for the detection and identification of infectious diseases, and microarrays are a good candidate to be used in this context. The work presented hereby addresses the issue of how best microarray and bioinformatics technologies (Lopez-Campos et al., 2008) can be jointly applied for the study and detection of emerging infectious diseases of interest in public health as well as for the study of the humoral immune response generated after the infection by different subtypes of VIH. Development of microarray based techniques for the detection of pathogens includes the optimization of microarray manufacture and probe design processes. Poly-l-lysine and isothiocyanate modified-glass provide good surface properties for the attachment of oligonucleotide probes and other relevant molecules. A major topic for microarray success is probe design; therefore, it is necessary to develop tools that support this complex process. BUSSUB has been developed as a tool that helps in the selection of the sequence areas of interest for the design of probes as well as for performing electronic PCR. Microarrays have also been designed and tested showing high sensitivity and specificity for the simultaneous detection of bioterrorism class A and B agents. To test the feasibility of the application of microarray technologies in public health laboratories, a microarray for the detection and characterization of human respiratory adenoviruses was developed. It was also successfully assayed with clinical samples from patients with respiratory infections. This study shows that microarrays can be effectively used in clinical environments and may become a powerful method for the study and diagnosis of infectious diseases (Lopez-Campos et al., 2007). Lastly, protein microarrays have been used to study and characterize the humoral response against different HIV subtypes. This approach shows that it is possible to analyse simultaneously in a quantitative way the reactivity of sera against HIV antigens coming from different HIV subtypes. This analysis enabled the characterization and identification of different response profiles against the different subtypes. These results facilitated also the comparison of microarrays with ELISA tests, showing differences between the results obtained with each of the technologies. The simultaneous detection of different dilutions was also useful for the analysis of the humoral response taking into account the affinity of the sera against the different immobilised antigens (Lopez-Campos et al., 2006).