Further studies on the molecular systematics of Biomphalaria snails from Brazil

The polymerase chain reaction and restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rRNA gene, using the enzyme DdeI were used for the molecular identification of ten species and one subspecies of Brazilian Biomphalaria. Emphasis is given to the analysis of B. oligoza, B. schrammi and B. amazonica. The RFLP profiles obtained using this enzyme were highly distinctive for the majority of the species and exhibited low levels of intraspecific polymorphism among specimens from different regions of Brazil. However, B. peregrina and B. oligoza presented very similar profiles that complicated their identification at the molecular level and suggested a very close genetic similarity between the two species. Others enzymes including HaeIII, HpaII, AluI and MnlI were tested for their ability to differentiate these species. For B. amazonica three variant profiles produced with DdeI were observed. The study demonstrated that the ITS contains useful genetic markers for the identification of these snails

Nuclear rDNA-based molecular clock of the evolution of triatominae (Hemiptera: Reduviidae), vectors of Chagas disease

The evolutionary history and times of divergence of triatomine bug lineages are estimated from molecular clocks inferred from nucleotide sequences of the small subunit SSU (18S) and the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA of these reduviids. The 18S rDNA molecular clock rate in Triatominae, and Prosorrhynchan Hemiptera in general, appears to be of 1.8% per 100 million years (my). The ITS-2 molecular clock rate in Triatominae is estimated to be around 0.4-1% per 1 my, indicating that ITS-2 evolves 23-55 times faster than 18S rDNA. Inferred chronological data about the evolution of Triatominae fit well with current hypotheses on their evolutionary histories, but suggest reconsideration of the current taxonomy of North American species complexes.

Molecular epidemiology of methicillin resistant Staphylococcus aureus isolated from newborns in a hospital in Rio de Janeiro, Brazil

Methicillin resistant Staphylococcus aureus (MRSA) is an organism that is frequently transmitted in hospitals and perinatal units. The MRSA is considered a public health problem in neonatology because of its strong potential for dissemination in the wards associated with high rates of morbidity and mortality. In this study we describe the bacteriological, epidemiological and molecular characteristics of MRSA isolated from anterior nares and blood cultures of newborns hospitalized in a public maternity hospital in the city of Rio de Janeiro, Brazil. The frequency of MRSA isolated from nasal swabs of newborns was 47.8% (43/90). The genetic analysis of MRSA strains from anterior nares, showed 8 different pulsed field gel electrophoresis patterns (PFGE). Upon analysis of PFGE patterns of the 12 MRSA strains isolated from blood cultures, 8 different patterns were observed, 9 (75%) strains were genetic related to nasal secretion isolates patterns. In conclusion, our data demonstrate the importance of screening of newborns for the presence of MRSA in Brazilian hospitals and the usefulness of genetic typing of these pathogen during epidemiologic studies. This should lead to a better knowledge on the significancy and spreading of MRSA in the hospitals.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Transmission of tuberculosis in Havana, Cuba: a molecular epidemiological study by IS6110 restriction fragment length polymorphism typing

The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55%) had unique IS6110 restriction fragment length polymorphism (RFLP) patterns, while isolates from 23 others (45%) had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.

Recent studies on Schistosoma intercalatum: taxonomic status, puzzling distribution and transmission foci revisited

Schistosoma intercalatum, which causes human rectal schistosomiasis in Africa, still presents a great interest for its imprecise taxonomic status and its puzzling distribution in Africa. Two geographically isolated strains of S. intercalatum are recognized, the Lower Guinea strain and the Congo strain, which differ from each other in a number of morphological, biological and biochemical characteristics. Recent molecular data using RAPD markers indicate high divergence between the two strains, with values of Nei and Li's similarity indice allowing recognition of two genetically distinct taxa: experiments on pre- and post-isolating mechanisms are in progress in order to re-evaluate the taxonomic status of this polytypic species. With regard to its geographical distribution, S. intercalatum is characterized by the existence of two stable endemic areas (localized in Lower Guinea and North East of Democratic Republic of Congo) which correspond to the historical areas of species discovery, and the emergence during the last 15 years of new foci of the Lower Guinea strain outside previously known endemic areas. The absence of local adaptation of the Lower Guinea strain to its intermediate host, supported by experimental studies, may help to facilitate the spread of this strain. Nevertheless, the present restricted distribution of this species remains puzzling, because its potential snail hosts (bulinids) are widely distributed throughout much of Africa. Recent experimental and epidemiological studies suggest that interspecific sexual interactions between human schistosomes could have a role in limiting the distribution of S. intercalatum: the competitive sexual processes acting among human schistosomes show that S. haematobium and S. mansoni are always competitively dominant over S. intercalatum. These epidemiological observations lead the authors to distinguish three kinds of transmission foci for S. intercalatum.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

Pseudomonas aeruginosa: study of Antibiotic Resistance and Molecular Typing in Hospital Infection Cases in a Neonatal Intensive Care Unit from Rio de Janeiro City, Brazil

This study had the objective of to analyze the demographic and bacteriologic data of 32 hospitalized newborns in an neonatal intensive care unit of a public maternity hospital in Rio de Janeiro city, Brazil, seized by Pseudomonas aeruginosa sepsis during a period ranged from July 1997 to July 1999, and to determine the antimicrobial resistance percentage, serotypes and pulsed field gel electrophoresis (PFGE) patterns of 32 strains isolated during this period. The study group presented mean age of 12.5 days, with higher prevalence of hospital infection in males (59.4%) and vaginal delivery (81.2%), than females (40.6%) and cesarean delivery (18.8%), respectively. In this group, 20 (62.5%) patients received antimicrobials before positive blood cultures presentation. A total of 87.5% of the patients were premature, 62.5% presented very low birth weight and 40.6% had asphyxia. We detected high antimicrobial resistance percentage to b-lactams, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline among the isolated strains. All isolated strains were classified as multi-drug resistant. Most strains presented serotype O11 while PFGE analysis revealed seven distinct clones with isolation predominance of a single clone (75%) isolated from July 1997 to June 1998.

Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania)

To establish the relationships of the lizard- and mammal-infecting Leishmania, we characterized the intergenic spacer region of ribosomal RNA genes from L. tarentolae and L. hoogstraali. The organization of these regions is similar to those of other eukaryotes. The intergenic spacer region was approximately 4 kb in L. tarentolae and 5.5 kb in L. hoogstraali. The size difference was due to a greater number of 63-bp repetitive elements in the latter species. This region also contained another element, repeated twice, that had an inverted octanucleotide with the potential to form a stem-loop structure that could be involved in transcription termination or processing events. The ribosomal RNA gene localization showed a distinct pattern with one chromosomal band (2.2 Mb) for L. tarentolae and two (1.5 and 1.3 Mb) for L. hoogstraali. The study also showed sequence differences in the external transcribed region that could be used to distinguish lizard Leishmania from the mammalian Leishmania. The intergenic spacer region structure features found among Leishmania species indicated that lizard and mammalian Leishmania are closely related and support the inclusion of lizard-infecting species into the subgenus Sauroleishmania proposed by Saf'janova in 1982.

Experimental evidence and ecological perspectives for the adaptation of Schistosoma mansoni Sambon, 1907 (Digenea: Schistosomatidae) to a wild host, the water-rat, Nectomys squamipes Brants, 1827 (Rodentia: Sigmodontinae)

Due to the semi aquatic habits and the overlap of the geographical distribution of the water-rat, Nectomys spp., with schistosomiasis endemic areas, these wild rodents are very likely to acquire Schistosoma mansoni infection in their daily activities. The role of the water-rat in the S. mansoni cycle would be substantiated if one could prove that these rodents acquire the parasite during their own activity time, a completely independent time schedule of human activities. To pursue this goal, we performed two field experiments in the municipality of Sumidouro, State of Rio de Janeiro, Brazil, a schistosomiasis endemic area where N. squamipes is found naturally infected. One experiment was devised as a series of observations of activity time of the water-rat. The other experiment was a test of the occurrence of late transmission of S. mansoni to the water-rat. The daily activity pattern showed that the water-rat is active chiefly just after sunset. At both diurnal and late exposition essays the water-rat sentinels got infected by S. mansoni. These findings clarify ecological and behavioral components necessary to the adaptation of S. mansoni to the water-rat as a non human definitive host and the existence of a transmission cycle involving this animals as a reservoir.

Cloning and Molecular Characterization of the Schistosoma mansoni Genes RbAp48 and Histone H4

The human nuclear protein RbAp48 is a member of the tryptophan/aspartate (WD) repeat family, which binds to the retinoblastoma (Rb) protein. It also corresponds to the smallest subunit of the chromatin assembly factor and is able to bind to the helix 1 of histone H4, taking it to the DNA in replication. A cDNA homologous to the human gene RbAp48 was isolated from a Schistosoma mansoni adult worm library and named SmRbAp48. The full length sequence of SmRbAp48 cDNA is 1036 bp long, encoding a protein of 308 amino acids. The transcript of SmRbAp48 was detected in egg, cercariae and schistosomulum stages. The protein shows 84% similarity with the human RbAp48, possessing four WD repeats on its C-terminus. A hypothetical tridimensional structure for the SmRbAp48 C-terminal domain was constructed by computational molecular modeling using the b-subunit of the G protein as a model. To further verify a possible interaction between SmRbAp48 and S. mansoni histone H4, the histone H4 gene was amplified from adult worm genomic DNA using degenerated primers. The gene fragment of SmH4 is 294 bp long, encoding a protein of 98 amino acids which is 100% identical to histone H4 from Drosophila melanogaster.

Partial molecular characterization of Sm8, a tegumental antigen of Schistosoma mansoni

Sm8 is a major tegumental antigen of Schistosoma mansoni. The partial cDNA was isolated and analyzed. Sequence analysis revealed transmembrane compatible hydrophobic domains and a putative leucine zipper pattern. The mRNA and the protein are predominantly expressed in adult worms.

Molecular epidemiology of human immunodeficiency virus-1 in the state of Ceará, Northeast, Brazil

We analyzed, by env and gag heteroduplex mobility assay, 149 human immunodeficiency virus (HIV-1) positive samples collected in Ceará during the year 2000. The prevalence of subtype B was 81.2% and the prevalence of subtype F and B/F recombinants were both 2.7%. Eight (5.4%) and 12 (8%) out of 149 samples showed indeterminate results in the env and gag analysis respectively. By FokI restriction fragment length polymorphism, 34% of the subtype B samples were identified as the typical Brazilian subtype B.In the present study, we identified HIV-1 subtype F and B/F in Ceará for the first time. Our results contribute to the understanding of HIV in Brazil, and may prove useful for the development of vaccine candidates.

Hepatitis B and C in the hemodialysis unit of Tocantins, Brazil: serological and molecular profiles

A survey was conducted in the hemodialysis population of the state of Tocantins, Brazil, aiming to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, to analyze associated risk factors, and also to investigate these viruses genotypes distribution. During January and March 2001, all patients (n = 100) were interviewed at the unique dialysis unit in Tocantins. Blood samples were collected and serum samples were screened for HBV serological markers. Hepatitis B surface antigen positive samples were tested for HBV DNA. All samples were also tested for anti-HCV antibodies and HCV RNA. An overall prevalence of 45% was found for HBV infection (4% were HBsAg/anti-HBc positive, 2% were anti-HBc only and 39% had anti-HBc/anti-HBs markers). Concerning HCV infection, anti-HCV and HCV RNA were detected in 13% and 14% of the subjects, respectively. Three patients were HCV RNA positive and anti-HCV negative, resulting in an overall HCV prevalence of 16%. Univariate analysis of risk factors showed that only shift and length of time on hemodialysis were associated with HBV and HCV positivity, respectively. Among the four HBsAg-positive samples, HBV DNA was detected in three of them, which were identified as genotype A by restriction fragment length polymorphism (RFLP) analysis. All 14 HCV RNA-positive samples were genotyped by INNO-LiPA. Genotypes 1a and 3a were found in 85% and 15%, respectively. The present data show low HBsAg and HCV prevalence rates. The risk factors associated with HBV and HCV positivity suggest that nosocomial transmission may influence in spreading these viruses in the dialysis unit studied.