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- A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samplesPublication . Kox, L.F.; Rhienthong, D.; Miranda, A.M.; Udomsantisuk, N.; Ellis, K.; van Leeuwen, J.; van Heusden, S.; Kuijper, S.; Kolk, A.H.Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.
- Natural variation of the nef gene in human immunodeficiency virus type 2 infections in PortugalPublication . Pádua, E.; Jenkins, A.; Brown, S.; Bootman, J.; Paixão, M.T.; Almond, N.; Berry, N.Human immunodeficiency virus type 2 (HIV-2) infections cause severe immunodeficiency in humans, although HIV-2 is associated frequently with reduced virulence and pathogenicity compared to HIV-1. Genetic determinants that play a role in HIV pathogenesis are relatively poorly understood but nef has been implicated in inducing a more pathogenic phenotype in vivo. However, relatively little is known about the role of nef in HIV-2 pathogenesis. To address this, the genetic composition of 44 nef alleles from 37 HIV-2-infected individuals in Portugal, encompassing a wide spectrum of disease associations, CD4 counts and virus load, has been assessed. All nef alleles were subtype A, with no evidence of gross deletions, truncations or disruptions in the nef-encoding sequence; all were full-length and intact. HIV-2 long terminal repeat sequences were conserved and also indicated subtype A infections. Detailed analysis of motifs that mediate nef function in HIV-1 and simian immunodeficiency virus, such as CD4 downregulation and putative SH2/SH3 interactions, revealed significant natural variation. In particular, the central P(104)xxPLR motif exhibited wide interpatient variation, ranging from an HIV-1-like tetra-proline structure (PxxP)(3) to a disrupted minimal core motif (P(104)xxQLR). The P(107)-->Q substitution was associated with an asymptomatic phenotype (Fisher's exact test, P=0.026) and low virus loads. These data indicate that discrete differences in the nef gene sequence rather than gross structural changes are more likely to play a role in HIV-2 pathogenesis mediated via specific functional interactions.
- Workshop Report: Developing Pollution Source Tracking for Recreational and Shellfish WatersPublication . Pond, Kate; Rangdale, R.; Meijer, Wim; Brandão, João; Falcão, Leonor; Rince, Alain; Greaves, John; Gawler, Andy; Masterson, Bartholomew; McDonnell, E.; Cronin, A.; Pedley, S.The Environment Agency of England and Wales obtained European Community Initiative INTERREG IIIB funding for a project called ICReW—Improving Coastal and Recreational Waters. The project consists of seven pilot actions aiming to contribute to the reduction of pollution, to enhance water quality, and to ensure that land-use practices and recreational activities can exist side by side without impacting public health. One of these actions is to identify and develop a common methodology for source-tracking fecal pollution, for regulatory purposes, over a wide geographical area in Europe. In order to do this the Department for Environment, Food and Rural Affairs, UK, sponsored the first international workshop on the subject. Key researchers from around the world were invited to attend to recommend the most appropriate method(s) for development and field trial in Europe. The meeting concluded that for the specific requirements of the ICReW project two methods should be developed and trialled: bacteroides genotyping and F+RNA coliphage genotyping. This article summarizes the reasons why these methods were chosen as the most appropriate for the circumstances of this particular project. The inherent challenges of establishing a pilot program to test the methods are outlined and recommendations were provided for the trial.
- Prevalence and molecular characterization of human T cell leukemia virus type 2 in a group of intravenous drug users coinfected with HIV type 1 in PortugalPublication . Silva, A.F.; Almeida, C.; Cortes Martins, H.; Coutinho, R.; Leitão, E.; Silva, R.; Paixão, M.T.; Pádua, E.Human T cell lymphotropic virus type 2 infections can be found in the large urban areas of the United States and Europe, where Spain and Italy are the most affected countries. The population most affected by the epidemic is characterized by high-risk behavior groups, mainly the sharing of needles between intravenous drug users (IDUs) with contaminated cellular blood products. It is also described that HTLV-2 infection appears as a coinfection with HIV-1. We have selected samples corresponding to 583 IDUs infected with HIV and screened for the presence of HTLV-1/2 antibodies. We have performed the molecular characterization of HTLV-2 in three confirmed positive cases on the basis of the long terminal repeat region. We can observe the Portuguese sequences (PortHl, PortNn, and PortVs) in the HTLV-2b cluster, grouping with the Spanish sequences, showing close phylogenetic relatedness. We may assume that HTLV-2 infection was introduced in Portugal from Spain. These results update previous reports that mentioned Portugal as being free of HTLV- 2 infections, and allow the identification of the subtype that is present, giving a first-hand description of the prevalence of HTLV-2 infection in a particular high-risk behavior group and justifying the importance of epidemiological surveillance in order to prevent dissemination of the infection.
- Mortality in Portugal associated with the heat wave of August 2003: early estimation of effect, using a rapid methodPublication . Nogueira, Paulo; Marinho Falcão, José; Contreiras, Maria Teresa; Paixão, Eleonora; Brandão, João; Batista, InêsDuring the first two weeks of August 2003, Portugal was affected by a severe heat wave. Following the identification in Portugal of the influence of heat waves on mortality in 1981 and 1991 (estimated excess of about 1900 and 1000 deaths respectively), the Observatório Nacional de Saúde (ONSA) - Instituto Nacional de Saúde Dr. Ricardo Jorge, together with the Vigilância Previsão e Informação - Instituto de Meteorologia, created a surveillance system called ÍCARO, which has been in operation since 1999. ÍCARO identifies heat waves with potential influence on mortality [1]. Before the end of the 2003 heat waves, ONSA had produced a preliminary estimate of its effect on mortality. The results based on daily number of deaths from 1 June to 12 August 2003 were presented within 4 working days. Data was gathered from 31 National Civil registrars, covering the district capitals of all 18 districts of mainland Portugal, and representing approximately 40% of the mainland’s mortality. The number of deaths registered in the period 30 July to 12 August was compared with the ones registered during 3 comparison periods (of 2003): 1-14 July, 1-28 July, and 15-28 July. 15-28 July, the period best resembling the heat waves in time and characteristics, produced an estimation of 37.7% higher mortality rate then the value expected under normal temperature conditions. From this value, an estimate of 1316 death excess was obtained for mainland Portugal. The main purpose of this article is to present the method used to identify and assess the occurrence of an effect (excess mortality) during the heat waves of summer 2003
- Genetic analysis of human immunodeficiency virus type 1 nef in portugal: Subtyping, identification of mosaic genes, and amino acid sequence variabilityPublication . Parreira, R.; Pádua, E.; Piedade, J.; Venenno, T.; Paixão, M. T.; Esteves, A.Extending our previous genetic characterization of human immunodeficiency virus type 1 (HIV-1) strains circulating in Portugal, we here report the first phylogenetic and putative amino acid sequence variability analyses of nef accessory gene. Viral sequences (n = 53) were amplified by nested PCR from proviral DNA purified from peripheral blood mononuclear cells of HIV-1 infected individuals (n = 49). Phylogenetic inference analysis demonstrated a distribution of the viral sequences between subtypes A (sub-subtype A1), B, D, F (sub-subtype F1), G, H, and J, with subtypes G and B accounting altogether for more than half of the genotypes found. A significant number of the proviral DNA sequences analyzed (18.4%) were shown to correspond to intragenic nef recombinants, with the majority having the typical CRF02_AG nef structure. In addition, three novel intragenic recombinant structures were found (B/G/B, CRF02_AG/H, and D/G). From phylogenetic analysis, it was concluded that part of the non-recombinant nef genes might have actually been amplified from mosaic viruses: CRF06_cpx, CRF14_BG, and a new envA/nefJ recombinant. While comparing all the putative Nef sequences, significant amino acid sequence variability was observed. However, most of the described nef functional motifs were relatively well conserved in the majority of the sequences analyzed and numerous amino acid changes fell outside these regions. The results presented unambiguously endorse the high level of complexity of HIV-1 epidemics in Portugal.
- Molecular characterization of the HA gene of influenza type B virusesPublication . Pechirra, Pedro; Nunes, Baltazar; Coelho, Anabela; Ribeiro, Carlos; Gonçalves, Paulo; Pedro, Sónia; Canto e Castro, Luísa; Rebelo-de-Andrade, HelenaAbstract Nucleotide sequences of the HA1 subunit of influenza B viruses isolated in Portugal between 1994 and 2003 influenza winter seasons were analyzed by the Neighbor-Joining algorithm and rates of HA1 evolution estimated by linear regression. From 1994 to 2002, all influenza B viruses studied were of the Yamagata lineage. Strains isolated from 1994 to 1996, 1996 to 1999, and 1999 to 2002 revealed a high similarity with B/Beijing/184/93, B/Yamanashi/166/98, and B/Sichuan/379/99, respectively, and strains isolated during 1994-1995, 1996-1997, and 1998-1999 clustered in more than one branch of the phylogenetic tree. Victoria-related strains reappeared during 2002/2003 and formed only one branch in the phylogenetic tree revealing a closer relationship to B/Shandong/7/97. Evolutionary rates for strains from the Yamagata lineage were estimated as 3.82x10(-3) nucleotides/site/year and 2.62x10(-3) nucleotides/site/year for Victoria-related strains. In order to identify putative influenza B HA1 codons under selective pressure, a codon-substitution model for heterogeneous selective pressure at amino acid sites was used. A percentage of 97.3% of codons under negative selective pressure and 2.7% of codons under positive selective pressure (omega=dN/dS=2.65) were estimated, with posterior probability higher than 0.90. Amino acid sites 75, 197, and 199 were found more likely to be under positive selective pressure.
- Natural polymorphisms of HIV type 2 pol sequences from drug-naive individualsPublication . Parreira, R.; Monteiro, F.; Pádua, E.; Piedade, J.; Venenno, T.; Paixão, M.T.; Esteves, A.Until today, the susceptibility of human immunodeficiency virus type 2 (HIV-2) to protease and nucleosidic reverse-transcriptase inhibitors (PI and NRTI, respectively) has not been clearly documented. In this report we studied HIV-2 proviral sequences (n = 30) from drug-naive patients. Our results revealed that several amino acid positions in the protease and reverse transcriptase coding sequence harbored residues that have been associated with drug resistance in HIV-1-infected patients. In particular, the M46I substitution in the protease was detected in 90% of the sequences analyzed, which, together with the other substitutions identified, may indicate a reduced susceptibility of HIV-2-infected drug-naive patients to PI. Furthermore, interpretation of genotypic data with four available algorithms, developed for interpretation of HIV-1 sequence data, suggested nonoverlapping profiles of drug resistance.
- Francisella tularensis, PortugalPublication . Lopes de Carvalho, Isabel; Escudero, R.; Garcia-Amil, C.; Falcão, H.; Anda, P.; Núncio, M.S.
- Molecular Epidemiology of Hepatitis A Virus in a Group of Portuguese Citizens Living in Lisbon AreaPublication . Rodrigues, Luisa; Pista, Angela; Oliveira, Ana; Água-Doce, Ivone; Manita, Carla; Paixão, TeresaHepatitis A virus (HAV) is the most important cause of acute infectious hepatitis worldwide. In Portugal, due to improvements in sanitation epidemic outbreaks of HAV infection have become less frequent. This report is the first, to our knowledge that characterized HAV in Portugal. For the detection and molecular characterization of HAV cases in a group of Portuguese individuals in the Lisbon area, 31 serum samples were tested: 8 from symptomatic children from an acute hepatitis A outbreak in a Roma (Gipsies) community (2004–2005), and 22 from patients with acuteHAV from sporadic cases (2005–2006). A sample of CSF involved in a case of meningitis was also included. IgM anti-HAV detection and nested reverse transcription (RT-PCR), with primers located at the VP1-P2a region, was undertaken to detect HAV genome. In positive samples, molecular characterization was followed by phylogenetic analysis. All samples (n¼31) were positive for IgM anti-HAV. HAV RNA was found in 96.7% of cases. All isolates were classified as genotype I: 22 belonged to sub-genotype IA (73.3%), and 8 to sub-genotype IB (26.7%). All strains obtained from an acute HAV outbreak had sub-genotype IA, in which seven isolates (87.5%) had identical sequences. In HAV sporadic cases sub-genotypes IA and IB were identified, and this may reflect the co-circulation of these two subgenotypes in Portugal. Molecular epidemiology of HAV infection in this group of Portuguese appears to be similar to other European countries. HAV phylogenetic studies can provide important information for the design of appropriate public health measures.