http://hdl.handle.net/10400.18/80 Sat, 11 May 2013 02:42:46 GMT 2013-05-11T02:42:46Z http://hdl.handle.net/10400.18/1564 Title: Analysis of longitudinal nef sequence variation throughout HIV-2 infection Authors: Remédios, M.; Paixão, E.; Feliciano, H.; Silva-Graça, A.; Pádua, E. Abstract: Background: Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2) may case a severe immunodeficiency in humans (AIDS). However HIV-2 is more frequently associated with lower levels of transmission and disease progression, compared with HIV-1 infections. The role of nef gene in vivo during the development of AIDS has been clearly demonstrated in simian immunodeficiency virus infected Rhesus macaques model, but the determinants which play a role in the pathogenesis of HIV are relatively poorly understood. However, even less is known about the role of nef in HIV-2 infections. Methods: In this study, it was analyzed the variation of 48 nef gene sequences, obtained from samples taken between 1994 and 2009, corresponding to 17 HIV-2-infected individuals with different clinical stages of infection. The sequences obtained by Nested PCR were classified by phylogenetic analysis and the functional protein motifs, described as important in CD4 and MHC-I downregulation and in viral infectivity were also analyzed. Results: In all individuals were identified nef sequences from group A of HIV-2, which encoded possible functional and complete protein. There was a greater conservation of residues in the Nef sequences of individuals in the symptomatic stage (63%), comparatively to individuals in the asymptomatic stage (19%). While some functional motifs (MGxxxS1, DDDD93, RR137 and DD205) and also residues (G128, I141 and L142) remained conserved, others (YSRF39, LRAR21, PxxP101, EE185) revealed changes during the follow-up period. The PxxP motif exhibited wide inter-individual variation in vivo from an HIV-1-like tetra-proline motif (PxxP)3 to disruption of the minimal core PxxPLR motif. The disruption was observed in 11 sequences exclusively from asymptomatic individuals (p=0.021). The sequence motif variation towards tetra-proline configuration was observed in 2 symptomatic individuals during time of infection. The results also revealed the existence of a negative selective pressure, as well as codons under positive pressure in the sequences. Conclusion: In this HIV-2-infected individuals studied, it was observed a need for a greater degree of Nef protein conservation in a symptomatic phase. Sequences altered and potentially critical for the Nef function in vivo, in earlier stages of infection, may contribute at some level to a different pattern in viral pathogenesis and disease progression. Description: Abstract publicado em: International Journal of Infectious Diseases. 2010;14(Supl 1). Thu, 11 Mar 2010 00:00:00 GMT http://hdl.handle.net/10400.18/1564 2010-03-11T00:00:00Z http://hdl.handle.net/10400.18/1562 Title: Contribution of freely available databases for the accurate classification of emergent pathogens Authors: João, Inês; Cristóvão, Paula; Jordao, Luisa Abstract: OBJECTIVES: Nontuberculous mycobacteria (NTM) are a heterogeneous group of microorganisms. Their clinical relevance and treatment differs significantly upon NTM species, which makes identification at this level crucial. Additionally, inaccurate diagnosis can lead to therapeutic approaches consistent with Mycobacterium tuberculosis infection which are usefulness.In the present study different molecular methods were used in the identification of NTM. The aim was to evaluate and compare the performance of freely available databases in the accurate identification of NTM. METHODS: Partial sequencing of hsp65 and 16S rRNA genes of 54 strains of NTM. The resulting sequences were compared with the nucleotide sequences from GenBank, RIDOM and EzTaxon databases for 16S rRNA gene. For hsp65 gene GenBank and Web Databases were used. RESULTS: Seventy-two percent of NTM were identified to species level by 16SrRNA analysis. The remaining 28% failed this goal. Thirty-three obtained an unique outcome in all databases and 6 isolates (15%) obtained at least one different resultAnalysis of hsp65 gene sequences identified unambiguously 76% of the strains, for the remaining 22% the analysis led to the possibility of classification into more than one species. Of all the strains analyzed, this approach failed to identify only 2%. CONCLUSION: The obsolescence of databases, including the GenBank and RIDOM, made the identification of emerging pathogens, as NTM, very complex. If instead of allowing free deposit it was mandatory to update the taxonomy of mycobacteria and asserting the quality of sequences prior to deposit, e.g. in GenBank, several problems would be avoided. Among the shortcomings, we highlight the existence of non-validated "species" names, poorly classified species and even classification limited to genera. As there is no control of sequences submited to this database, identical sequences could give different percentages of similarity. Furthermore, this database contains an enormous amount of flawed sequences, which can lead to identification errors. On the other hand controlled databases which contain high quality and properly characterized sequences, have a limited usefulness due to lagging updating (RIDOM), and the absence of sequences other than from reference strains (EzTaxon and Web Database). Mon, 01 Apr 2013 00:00:00 GMT http://hdl.handle.net/10400.18/1562 2013-04-01T00:00:00Z http://hdl.handle.net/10400.18/1561 Title: Exploitation of a link between antibacterial agent-resistance and biofilm-formation by classical and emergent pathogens Authors: João, Inês; Reis, Lúcia; Carvalho, Patrícia; Duarte, Aida; Jordão, Luisa Abstract: Objectives: In recent years nosocomial infections have gained growing importance. Among their etiological agents are “classical” pathogens such as S.aureus and also emergent pathogens, previously neglected, such as nontuberculous mycobacteria (MTM). The ability to resist to antibacterial agents, such as antibiotics and disinfectants, is shared by all of them. Here we aim to establish a link between bacterial virulence, antibacterial agents’ resistance and biofilm formation. Methods: Bacterial reference strains and clinical isolates were grown in adequate medium. Among the “classical” pathogens used are E.coli, K. pneumoniae, S. aureus and P. aeroginosa. The group of emergent pathogens includes M.fortuitum, M.abcessus, M.chelonae, M.avium etc. NTM susceptibility test to antibiotics was evaluated by broth based microdilution method and interpreted according to NCCLS guidelines. The desinfectant (oxygen peroxide, ammonium quaternary salts [AQS] and glutaraldehyde [GA) efficacy was performed according to the approved guidelines. The susceptibility was performed by two different methods: broth microdilution and diffusion in solid medium. In order to evaluate the effect of these agents in bacteria a scanning electron microscopy (SEM) study was performed. Biofilm forming ability was evaluated by the microtiter-plate test. The assay was performed at 25ºC and 37ºC in optimal growth media, phosphate saline buffer pH 7.4 and water during for different periods of time. Fast growing bacteria were assayed for 3 days while slow growers were assayed for 15 days.; Results: The results of the antibiotic susceptibility test showed, with no surprise, that the resistant strains are the most prevalent. The resistance spectrum ranged from 1 to 5 antibiotics currently used in therapeutic schemes. Oxygen peroxide was the most effective disinfectant followed by QAS and GA. Nevertheless, among NTM we identified one isolate resistant to all disinfectants tested. The SEM analysis showed that different disinfectants caused different effects on bacteria suggesting different action mechanisms. The ability to form biofilms was time, medium and temperature dependent.; Conclusion: Bacteria resistance to antibiotics and disinfectants vary in the same manner. The mechanisms involved in the resistance are not fully elucidated and more studies are needed to provide effective conclusions. Biofilm formation can be part of the mechanism involved both in resistance development and propagation of infections by these agents. Mon, 01 Apr 2013 00:00:00 GMT http://hdl.handle.net/10400.18/1561 2013-04-01T00:00:00Z http://hdl.handle.net/10400.18/1498 Title: First autochthonous dengue virus infections in Madeira Island, Portugal, in 2012. Authors: Zé-Zé, L.; Amaro, F.; Osório, H.C.; Parreira, P.; Luz, T.; Andrade, G.; Fernandes, P.L.; Alves, M.J. Fri, 15 Feb 2013 00:00:00 GMT http://hdl.handle.net/10400.18/1498 2013-02-15T00:00:00Z