139 resultados para Raoul Ruiz
Resumo:
CA88 is the first long nuclear repetitive DNA sequence identified in the blood fluke, Schistosoma mansoni. The assembled S. mansoni sequence, which contains the CA88 repeat, has 8,887 nucleotides and at least three repeat units of approximately 360 bp. In addition, CA88 also possesses an internal CA microsatellite, identified as SmBr18. Both PCR and BLAST analysis have been used to analyse and confirm the CA88 sequence in other S. mansoni sequences in the public database. PCR-acquired nuclear repetitive DNA sequence profiles from nine Schistosoma species were used to classify this organism into four genotypes. Included among the nine species analysed were five sequences of both African and Asian lineages that are known to infect humans. Within these genotypes, three of them refer to recognised species groups. A panel of four microsatellite loci, including SmBr18 and three previously published loci, has been used to characterise the nine Schistosoma species. Each species has been identified and classified based on its CA88 DNA fingerprint profile. Furthermore, microsatellite sequences and intra-specific variation have also been observed within the nine Schistosoma species sequences. Taken together, these results support the use of these markers in studying the population dynamics of Schistosoma isolates from endemic areas and also provide new methods for investigating the relationships between different populations of parasites. In addition, these data also indicate that Schistosoma magrebowiei is not a sister taxon to Schistosoma mattheei, prompting a new designation to a basal clade.
Resumo:
Non-invasive markers of fibrosis have been used to diagnose liver fibrosis in a variety of diseases. Hyaluronic acid (HA) and collagen IV (C-IV) levels were measured in the sera of patients from an endemic area for schistosomiasis in Brazil to diagnose and to rank the intensity of liver fibrosis. Seventy-nine adult patients with schistosomiasis, in the age range of 21-82 years (49 ± 13.4) were submitted to clinical and ultrasonographic examinations. Ultrasound was employed to diagnose and categorise liver fibrosis according to World Health Organization patterns. Serum HA and C-IV levels were measured using commercial ELISA kits. Ultrasound revealed six patients with intense liver fibrosis, 21 with moderate, 23 with light and 29 without. Serum HA was able to separate individuals with fibrosis from those without (p < 0.001) and light from intense fibrosis (p = 0.029), but C-IV was not (p = 0.692). The HA diagnostic accuracy for fibrosis was 0.89. The 115.4 ng/mL cut-off level diagnosed patients with fibrosis (sensitivity 0.98, specificity 0.64). HA correlated positively with portal hypertension. Periportal fibrosis (subjective evaluation), age and collateral circulation predicted HA increase. In conclusion, we propose that serum HA can be used to identify patients with liver fibrosis in an endemic area for schistosomiasis mansoni in Brazil.
Resumo:
Mitochondrial DNA of Biomphalaria tenagophila, a mollusc intermediate host of Schistosoma mansoni in Brazil, was sequenced and characterised. The genome size found for B. tenagophila was 13,722 bp and contained 13 messenger RNAs, 22 transfer RNAs (tRNA) and two ribosomal RNAs (rRNA). In addition to sequencing, the mitochondrial DNA (mtDNA) genome organization of B. tenagophila was analysed based on its content and localization of both coding and non-coding regions, regions of gene overlap and tRNA nucleotide sequences. Sequences of protein, rRNA 12S and rRNA 16S nucleotides as well as gene organization were compared between B. tenagophila and Biomphalaria glabrata, as the latter is the most important S. mansoni intermediate host in Brazil. Differences between such species were observed regarding rRNA composition. The complete sequence of the B. tenagophila mitochondrial genome was deposited in GenBank (accession EF433576). Furthermore, phylogenetic relationships were estimated among 28 mollusc species, which had their complete mitochondrial genome deposited in GenBank, using the neighbour-joining method, maximum parsimony and maximum likelihood bootstrap. B. tenagophila was positioned at a branch close to B. glabrata and Pulmonata molluscs, collectively comprising a paraphyletic group, contrary to Opistobranchia, which was positioned at a single branch and constituted a monophyletic group.
Resumo:
The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.
Resumo:
The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.
Resumo:
We utilized two-dimensional gel electrophoresis and immunoblotting (2D-immunoblotting) with anti-Sporothrix schenckii antibodies to identify antigenic proteins in cell wall preparations obtained from the mycelial and yeast-like morphologies of the fungus. Results showed that a 70-kDa glycoprotein (Gp70) was the major antigen detected in the cell wall of both morphologies and that a 60-kDa glycoprotein was present only in yeast-like cells. In addition to the Gp70, the wall from filament cells showed four proteins with molecular weights of 48, 55, 66 and 67 kDa, some of which exhibited several isoforms. To our knowledge, this is the first 2D-immunoblotting analysis of the S. schenckii cell wall.
Resumo:
The sandfly Phlebotomus perniciosus is the most widespread vector of Leishmania infantum in Spain. Laboratory colonisation represents the most feasible source of information on the biology of these insects, but in conducting any study, the density of individuals in the colony may drop to such an extent that it is sometimes difficult to recover the initial population levels. A new technique was tested for the recovery of sandfly eggs in three different colonies; the recovery rate was studied by comparing the standard method of mass rearing with this new method of colony management. The results demonstrate a mean increase of 18.4% in adult production, a growth in colony productivity that justifies the inclusion of this process in the routine maintenance of any colony of sandflies.
Resumo:
Here we present a comprehensive review of the literature on the vectorial importance of the major Anopheles malaria vectors in Colombia. We provide basic information on the geographical distribution, altitudinal range, immature habitats, adult behaviour, feeding preferences and anthropophily, endophily and infectivity rates. We additionally review information on the life cycle, longevity and population fluctuation of Colombian Anopheles species. Emphasis was placed on the primary vectors that have been epidemiologically incriminated in malaria transmission: Anopheles darlingi, Anopheles albimanus and Anopheles nuneztovari. The role of a selection of local, regional or secondary vectors (e.g., Anopheles pseudopunctipennis and Anopheles neivai) is also discussed. We highlight the importance of combining biological, morphological and molecular data for the correct taxonomical determination of a given species, particularly for members of the species complexes. We likewise emphasise the importance of studying the bionomics of primary and secondary vectors along with an examination of the local conditions affecting the transmission of malaria. The presence and spread of the major vectors and the emergence of secondary species capable of transmitting human Plasmodia are of great interest. When selecting control measures, the anopheline diversity in the region must be considered. Variation in macroclimate conditions over a species' geographical range must be well understood and targeted to plan effective control measures based on the population dynamics of the local Anopheles species.
Resumo:
Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide.
Resumo:
A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.
Resumo:
Orally transmitted Chagas disease (ChD), which is a well-known entity in the Brazilian Amazon Region, was first documented in Venezuela in December 2007, when 103 people attending an urban public school in Caracas became infected by ingesting juice that was contaminated with Trypanosoma cruzi. The infection occurred 45-50 days prior to the initiation of the sampling performed in the current study. Parasitological methods were used to diagnose the first nine symptomatic patients; T. cruzi was found in all of them. However, because this outbreak was managed as a sudden emergency during Christmas time, we needed to rapidly evaluate 1,000 people at risk, so we decided to use conventional serology to detect specific IgM and IgG antibodies via ELISA as well as indirect haemagglutination, which produced positive test results for 9.1%, 11.9% and 9.9% of the individuals tested, respectively. In other more restricted patient groups, polymerase chain reaction (PCR) provided more sensitive results (80.4%) than blood cultures (16.2%) and animal inoculations (11.6%). Although the classical diagnosis of acute ChD is mainly based on parasitological findings, highly sensitive and specific serological techniques can provide rapid results during large and severe outbreaks, as described herein. The use of these serological techniques allows prompt treatment of all individuals suspected of being infected, resulting in reduced rates of morbidity and mortality.
Resumo:
New Mycobacterium leprae protein antigens can contribute to improved serologic tests for leprosy diagnosis/classification and multidrug therapy (MDT) monitoring. This study describes seroreactivity to M. leprae proteins among participants from three highly endemic leprosy areas in Brazil: central-western Goiânia/Goiás (GO) (n = 225), Rondonópolis/Mato Grosso (MT) (n = 764) and northern Prata Village/Pará (PA) (n = 93). ELISA was performed to detect IgG to proteins (92f, 46f, leprosy IDRI diagnostic-1, ML0405, ML1213) and IgM to phenolic glycolipid-I (PGL-I). Multibacillary (MB) leprosy had positive rates for PGL-I that were similar to those for proteins; however, some anti-PGL-I-negative subjects were positive for proteins, suggesting that adding protein antigen to PGL-I can enhance the sensitivity of MB leprosy detection. In MT, different degrees of seroreactivity were observed and ranked for MB, former patients after MDT, paucibacillary (PB) leprosy, household contact (HHC) and endemic control (EC) groups. The seroreactivity of PB patients was low in GO and MT. HHCs from different endemic sites had similar IgG antibody responses to proteins. 46f and 92f were not recognised by most tuberculosis patients, ECs or HHCs within GO, an area with high BCG vaccination coverage. Low positivity in EC and HHC was observed in PA and MT. Our results provide evidence for the development of an improved serologic test that could be widely applicable for MB leprosy testing in Brazil.
Resumo:
Chagas disease, caused by Trypanosoma cruzi, represents an endemic among Latin America countries. The participation of free radicals, especially nitric oxide (NO), has been demonstrated in the pathophysiology of seropositive individuals with T. cruzi. In Chagas disease, increased NO contributes to the development of cardiomyopathy and megacolon. Metallothioneins (MTs) are efficient free radicals scavengers of NO in vitro and in vivo. Here, we developed a murine model of the chronic phase of Chagas disease using endemic T. cruzi RyCH1 in BALB/c mice, which were divided into four groups: infected non-treated (Inf), infected N-monomethyl-L-arginine treated (Inf L-NAME), non-infected L-NAME treated and non-infected vehicle-treated. We determined blood parasitaemia and NO levels, the extent of parasite nests in tissues and liver MT-I expression levels. It was observed that NO levels were increasing in Inf mice in a time-dependent manner. Inf L-NAME mice had fewer T. cruzi nests in cardiac and skeletal muscle with decreased blood NO levels at day 135 post infection. This affect was negatively correlated with an increase of MT-I expression (r = -0.8462, p < 0.0001). In conclusion, we determined that in Chagas disease, an unknown inhibitory mechanism reduces MT-I expression, allowing augmented NO levels.
Resumo:
Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.
Resumo:
In 2004, Aedes (Stegomyia) albopictus (Skuse, 1894) was observed for the first time in Catalonia, northeastern Spain. A decade later, it has spread throughout the eastern Mediterranean region of the country and the Balearic Islands. Framed within a national surveillance project, we present the results of monitoring in 2013 in the autonomous communities of the mainland Levante. The current study reveals a remarkable increase in the spread of the invasive mosquito in relation to results from 2012; the species was present and well-established in 48 municipalities, most of which were along the Mediterranean coastline from the Valencian Community to the Region of Murcia.