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.

Drug resistance and genotypes of strains of Mycobacterium tuberculosis isolated from human immunodeficiency virus-infected and non-infected tuberculosis patients in Bauru, São Paulo, Brazil

Little is known about transmission and drug resistance of tuberculosis (TB) in Bauru, State of São Paulo. The objective of this study was to evaluate risk factors for transmission of Mycobacterium tuberculosis strains in this area. Strains were collected from patients attended at ambulatory services in the region and susceptibility towards the main first line antibiotics was determined and fingerprinting performed. A total of 57 strains were submitted to susceptibility testing: 23 (42.6%) were resistant to at least one drug while 3 (13%) were resistant against both rifampicin and isoniazide. Resistant strains had been isolated from patients that had not (n = 13) or had (n = 9) previously been submitted to anti-TB treatment, demonstrating a preoccupying high level of primary resistance in the context of the study. All strains were submitted to IS6110 restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element PCR (DRE-PCR). Using IS6110-RFLP, 26.3% of the strains were clustered and one cluster of 3 patients included 2 HIV-infected individuals that had been hospitalized together during 16 days; clustering of strains of patients from the hospital was however not higher than that of patients attended at health posts. According to DRE-PCR, 55.3% belonged to a cluster, confirming the larger discriminatory power of IS6110-RFLP when compared to DRE-PCR, that should therefore be used as a screening procedure only. No clinical, epidemiological or microbiological characteristics were associated with clustering so risk factors for transmission of TB could not be defined in the present study.

IS6110 restriction fragment length polymorphism of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in Campinas, Brazil: evidence of intercontinental distribution of strains

Tuberculosis (TB) is a major concern in developing countries. In Brazil, few genotyping studies have been conducted to verify the number of IS6110 copies present in local prevalent strains of Mycobacterium tuberculosis, the distribution and clustering of strains. IS6110 DNA fingerprinting was performed on a sample of M. tuberculosis isolates from patients with AFB smear-positive pulmonary TB, at a hospital in Brazil. The IS6110 profiles were analyzed and compared to a M. tuberculosis database of the Houston Tuberculosis Initiative, Houston, US. Seventy-six fingerprints were obtained from 98 patients. All M. tuberculosis strains had an IS6110 copy number between 5-21 allowing for differentiation of the isolates. Human immunodeficiency virus infection was confirmed in nearly half the patients of whom data was available. Fifty-eight strains had unique patterns, while 17 strains were grouped in 7 clusters (2 to 6 strains). When compared to the HTI database, 6 strains matched isolates from El Paso, Ciudad de Juarez, Houston, and New York. Recently acquired infections were documented in 19% of cases. The community transmission of infection is intense, since some clustered strains were recovered during the four-year study period. The intercontinental dissemination of M. tuberculosis strains is suspected by demonstration of identical fingerprints in a distant country.

Spatial analysis of the distribution of leprosy in the State of Ceará, Northeast Brazil

The aim of this study was to describe spatial patterns of the distribution of leprosy and to investigate spatial clustering of incidence rates in the state of Ceará, Northeast Brazil. The average incidence rate of leprosy for the period of 1991 to 1999 was calculated for each municipality of Ceará. Maps were used to describe the spatial distribution of the disease, and spatial statistics were applied to explore large- and small-scale variations of incidence rates. Three regions were identified in which the incidence of leprosy was particularly high. A spatial gradient in the incidence rates was identified, with a tendency of high rates to be concentrated on the North-South axis in the middle region of the state. Moran's I statistic indicated that a significant spatial autocorrelation also existed. The spatial distribution of leprosy in Ceará is heterogeneous. The reasons for spatial clustering of disease rates are not known, but might be related to an heterogeneous distribution of other factors such as crowding, social inequality, and environmental characteristics which by themselves determine the transmission of Mycobacterium leprae.

IS6110 restriction fragment length polymorphism typing of Mycobacterium tuberculosis isolates from East Azerbaijan Province of Iran

To investigate the genetic variation among Mycobacterium tuberculosis isolates in the East Azerbaijan Province of Iran and to evaluate the level of and risk factors for recent transmission of tuberculosis (TB), we performed IS6110-based restriction fragment length polymorphism analysis of strains, isolated from 105 patients during the period of September 2002 to March 2003 in TB centers and university hospitals of the province. Among 105 isolates, 81 different IS6110 patterns were found, of which 70 were observed only once and 11 were shared by two to eight isolates. Ninety-six isolates (91.4%) were found to have more than five copies of IS6110 and together with high patterns polymorphism, shows that IS6110-RFLP typing could be useful for studying the epidemiology of TB in Azerbaijan. The minimum estimated rate of recent transmission was 23%, suggesting that the degree of recent transmission in East Azerbaijan Province is relatively low. Clustering was not associated with age, sex or site of infection of TB but drug-resistant isolates were less likely to be clustered than sensitive isolates (p < 0.05).

The spatial distribution of Schistosoma mansoni infection before and after chemotherapy in the Jequitinhonha Valley in Brazil

Schistosomiasis prevalence and egg counts remained low one year after chemotherapy in most households in a hyperendemic rural area in northern Minas Gerais but several distinct spatial patterns could be observed in relation to IgE levels and to a lesser extent to exposure risk (TBM) and type of water supply. An inverse relationship between pre-treatment household prevalence and egg counts on the one hand and post-treatment IgE levels on the other were noted in two of the five communities. Low exposure risk was associated with the low pre-treatment infection rates in the central village but did not contribute to the decline of infection rates after chemotherapy in the study area, as indicated by the significant increase in water contact during the posttreatment period (p < 0.0001). Distance between households and the streams and socioeconomic factors were also unimportant in predicting the spatial distribution of infection. These results are consistent with the production and antiparasitic effect of high levels of IgE in Schistosoma mansoni infection.

Man biting rate seasonal variation of malaria vectors in Roraima, Brazil

Malaria control has been directed towards regional actions where more detailed knowledge of local determinants of transmission is of primary importance. This is a short report on range distribution and biting indices for Anopheles darlingi and An. albitarsis during the dry and rainy season that follows river level variation in a savanna/alluvial forest malaria system area in the Northern Amazon Basin. Distribution range and adult biting indices were at their highest during the rainy season for both An. darlingi and An. albitarsis. During the rainy season the neighboring alluvial forest was extensively flooded. This coincided with highest rates in malaria transmission with case clustering near the river. As the river receded, anopheline distribution range and density decreased. This decrease in distribution and density corresponded to a malaria decrease in the near area. An exponential regression function was derived to permit estimations of An. darlingi distribution over specified distances. Anopheline spatio-temporal variations lead to uneven malaria case distribution and are of important implications for control strategies.

Typing of Mycobacterium tuberculosis strains isolated in Community Health Centers of Rio de Janeiro City, Brazil

Fingerprinting of Mycobacterium tuberculosis strains from tuberculosis (TB) patients attended in Community Health Centers (CHCs) of Rio de Janeiro was performed to verify possible risk factors for TB transmission. A prospective community-based study was performed during the period of July 1996 to December 1996 by collecting sputum samples of 489 patients in 11 different CHCs in four different planning areas (APs) of the city. Bacteriological, clinical, and epidemiological information was collected and M. tuberculosis genotypes defined after restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element (DRE) fingerprinting of RFLP-clustered cases. Risk factors for TB transmission were looked for using three levels of cluster stringency. Among 349 (71%) positive cultures obtained, IS6110-RFLP typing could be performed on strains from 153 different patients. When using identity of RFLP patterns as cluster definition, 49 (32%) of the strains belonged to a cluster and none of the clinical or epidemiologic characteristics was associated with higher clustering levels. However, higher clustering level was observed in the AP including the central region of the city when compared to others. This strongly suggests that more recent transmission occurs in that area and this may be related with higher incidence of TB and HIV in this region.

A preliminary analysis of the population genetics and molecular phylogenetics of Onchocerca volvulus (Nematoda: Filarioidea) using nuclear ribosomal second internal transcribed spacer sequences

Nuclear internal transcribed spacer 2 (ITS2) rDNA sequences were used for a molecular phylogenetics analysis of five Onchocerca species. The sister species of the human parasite O. volvulus was found to be the cattle parasite O. ochengi and not O. gibsoni, contrary to chromosomal evidence. The genetic differentiation of two African populations (representing the two African strains) and a Brazilian population of O. volvulus was also studied. Phylogenetic and network reconstruction did not show any clustering of ITS2 alleles on geographic or strain grounds. Furthermore, population genetics tests showed no indication of population differentiation but suggested gene flow among the three populations.

Spatial distribution of Schistosoma mansoni infection before and after chemotherapy with two praziquantel doses in a community of Pernambuco, Brazil

Praziquantel chemotherapy has been the focus of the Schistosomiasis Control Program in Brazil for the past two decades. Nevertheless, information on the impact of selective chemotherapy against Schistosoma mansoni infection under the conditions confronted by the health teams in endemic municipalities remains scarce. This paper compares the spatial pattern of infection before and after treatment with either a 40 mg/kg or 60 mg/kg dose of praziquantel by determining the intensity of spatial cluster among patients at 180 and 360 days after treatment. The spatial-temporal distribution of egg-positive patients was analysed in a Geographic Information System using the kernel smoothing technique. While all patients became egg-negative after 21 days, 17.9% and 30.9% reverted to an egg-positive condition after 180 and 360 days, respectively. Both the prevalence and intensity of infection after treatment were significantly lower in the 60 mg/kg than in the 40 mg/kg treatment group. The higher intensity of the kernel in the 40 mg/kg group compared to the 60 mg/kg group, at both 180 and 360 days, reflects the higher number of reverted cases in the lower dose group. Auxiliary, preventive measures to control transmission should be integrated with chemotherapy to achieve a more enduring impact.

Classification of immature mosquito species according to characteristics of the larval habitat in the subtropical province of Chaco, Argentina

To classify mosquito species based on common features of their habitats, samples were obtained fortnightly between June 2001-October 2003 in the subtropical province of Chaco, Argentina. Data on the type of larval habitat, nature of the habitat (artificial or natural), size, depth, location related to sunlight, distance to the neighbouring houses, type of substrate, organic material, vegetation and algae type and their presence were collected. Data on the permanence, temperature, pH, turbidity, colour, odour and movement of the larval habitat's water were also collected. From the cluster analysis, three groups of species associated by their degree of habitat similarity were obtained and are listed below. Group 1 consisted of Aedes aegypti. Group 2 consisted of Culex imitator, Culex davisi, Wyeomyia muehlensi and Toxorhynchites haemorrhoidalis separatus. Within group 3, two subgroups are distinguished: A (Psorophora ferox, Psorophora cyanescens, Psorophora varinervis, Psorophora confinnis, Psorophora cingulata, Ochlerotatus hastatus-oligopistus, Ochlerotatus serratus, Ochlerotatus scapularis, Culex intrincatus, Culex quinquefasciatus, Culex pilosus, Ochlerotatus albifasciatus, Culex bidens) and B (Culex maxi, Culex eduardoi, Culex chidesteri, Uranotaenia lowii, Uranotaenia pulcherrima, Anopheles neomaculipalpus, Anopheles triannulatus, Anopheles albitarsis, Uranotaenia apicalis, Mansonia humeralis and Aedeomyia squamipennis). Principal component analysis indicates that the size of the larval habitats and the presence of aquatic vegetation are the main characteristics that explain the variation among different species. In contrast, water permanence is second in importance. Water temperature, pH and the type of larval habitat are less important in explaining the clustering of species.

Tuberculosis in a southern Brazilian prison

The occurrence of tuberculosis (TB) in prisons has been described as an alarming public health problem in many countries, especially in developing nations. The objective of this study was to conduct a survey among prisoners with TB respiratory symptoms in order to estimate the incidence of the disease, to analyze the drug susceptibility profile and genotype the isolates of Mycobacterium tuberculosis in the city of Charqueadas, southern of Brazil. The TB incidence was 55/1,900 inhabitants in the prison; this corresponds to an incidence of 3,789/100,000 inhabitants, with a prevalence of 72/1,900 (4,960/100,000 inhabitants). Drug susceptibility test was performed and, among the analyzed isolates, 85% were susceptible to all drugs tested and 15% were resistant to at least one drug, of which 89% were resistant only to isoniazid (INH) or in combination with another drug. The genotype classification of spoligotyping analysis showed that 40% of the isolates belong to LAM family, 22% to T family, 17.5% to Haarlem family, 12.5% to U family and 3% to X family. The shared international spoligotypes most frequently found were 729 (27%), 50 (9.5%), 42 (8%), 53 (8%) and 863 (8%). In conclusion, it was observed that TB in this specific population had been caused, mostly, by strains that have been transmitted in the last few years, as demonstrated by the large level of genotype clustering. In addition, it was found specific large clusters, which were not often found in the general population from the same period and in the same region.

Distinct population structure for co-occurring Anopheles goeldii and Anopheles triannulatus in Amazonian Brazil

To evaluate whether environmental heterogeneity contributes to the genetic heterogeneity in Anopheles triannulatus, larval habitat characteristics across the Brazilian states of Roraima and Pará and genetic sequences were examined. A comparison with Anopheles goeldii was utilised to determine whether high genetic diversity was unique to An. triannulatus. Student t test and analysis of variance found no differences in habitat characteristics between the species. Analysis of population structure of An. triannulatus and An. goeldii revealed distinct demographic histories in a largely overlapping geographic range. Cytochrome oxidase I sequence parsimony networks found geographic clustering for both species; however nuclear marker networks depicted An. triannulatus with a more complex history of fragmentation, secondary contact and recent divergence. Evidence of Pleistocene expansions suggests both species are more likely to be genetically structured by geographic and ecological barriers than demography. We hypothesise that niche partitioning is a driving force for diversity, particularly in An. triannulatus.

Ecology of cutaneous leishmaniasis in Sinai: linking parasites, vectors and hosts

Cutaneous leishmaniasis (CL) is a neglected clinical form of public health importance that is quite prevalent in the northern and eastern parts of Egypt. A comprehensive study over seven years (January 2005-December 2011) was conducted to track CL transmission with respect to both sandfly vectors and animal reservoirs. The study identified six sandfly species collected from different districts in North Sinai: Phlebotomus papatasi, Phlebotomus kazeruni, Phlebotomus sergenti, Phlebotomus alexandri, Sergentomyia antennata and Sergentomyia clydei. Leishmania (-)-like flagellates were identified in 15 P. papatasi individuals (0.5% of 3,008 dissected females). Rodent populations were sampled in the same districts where sandflies were collected and eight species were identified: Rattus norvegicus (n = 39), Rattus rattus frugivorous (n = 13), Rattus rattus alexandrinus (n = 4), Gerbillus pyramidum floweri (n = 38), Gerbillus andersoni (n = 28), Mus musculus (n = 5), Meriones sacramenti (n = 22) and Meriones crassus (n = 10). Thirty-two rodents were found to be positive for Leishmania infection (20.12% of 159 examined rodents). Only Leishmania major was isolated and identified in 100% of the parasite samples. The diversity of both the vector and rodent populations was examined using diversity indices and clustering approaches.

Antimicrobial susceptibility patterns, emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.