Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies

Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi, is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy.

Genetic variability of Brazilian phytoplasma and spiroplasma isolated from maize plants

The objective of this work was to characterize the genetic variability of phytoplasma and Spiroplasma kunkelii isolated from maize plants showing symptoms of stunt collected from different Brazilian geographic regions. A DNA fragment of 500 base pairs (bp) was amplified from the spiralin gene in S. kunkelii and one fragment of 1,200 bp was generated from 16S rDNA gene in phytoplasma. The partial sequences of the spiralin gene showed similarity of 98% among the isolates of S. kunkelii analyzed. These sequences were compared with the sequence of the spiralin gene from other Spiroplasma species deposited in the GenBank, resulting in a similarity varying from 76.9% to 88.1%. The 16S rDNA sequence from the phytoplasma were completely similar within the Brazilian isolates and showed up to 98% of the similarity with sequences already found from other phytoplasmas. A very narrow genetic variability was detected by these gene fragments within phytoplasma and Spiroplasma analyzed. However, other genomic regions with higher polymorphic levels shall be identified in order to better evaluate the genetic diversity within these microorganisms population.

Evaluation of Xylella fastidiosa genetic diversity by fAFPL markers

The first phytopathogenic bacterium with its DNA entirely sequenced is being detected and isolated from different host plants in several geographic regions. Although it causes diseases in cultures of economic importance, such as citrus, coffee, and grapevine little is known about the genetic relationships among different strains. Actually, all strains are grouped as a single species, Xylella fastidiosa, despite colonizing different hosts, developing symptoms, and different physiological and microbiological observed conditions. The existence of genetic diversity among X. fastidiosa strains was detected by different methodological techniques, since cultural to molecular methods. However, little is know about the phylogenetic relationships developed by Brazilian strains obtained from coffee and citrus plants. In order to evaluate it, fAFLP markers were used to verify genetic diversity and phylogenetic relationships developed by Brazilian and strange strains. fAFLP is an efficient technique, with high reproducibility that is currently used for bacterial typing and classification. The obtained results showed that Brazilian strains present genetic diversity and that the strains from this study were grouped distinctly according host and geographical origin like citrus-coffee, temecula-grapevine-mulberry and plum-elm.

Constituintes voláteis de própolis piauiense

The essential oils of five samples obtained in different regions of the state of Piaui were analyzed by GC-MS. Ninety-one volatile constituents were tentatively identified and eight were positively identified. The principal components obtained from each sample were: 1,8-cineole, exo-fenchol, terpin-4-ol and fenchone (Teresina), a-pinene, caryophyllene oxide, b-pinene and a-copaene (Pio IX), (E)-caryophyllene, a-copaene, a-pinene, caryophyllene oxide and d-cadinene (Campo Maior), (E)-caryophyllene, a-gurjunene and b-selinene (Pedro II) and (E)-caryophyllene, a-gurjunene, d-cadinene and a-copaene (Lagoa de Sao Francisco). The five samples presented differences in the chemical composition of volatile fractions. The studies pointed out the need of characterizing propolis from Piaui by geographic regions and by seasons (drought and rainy periods).

Sequences of the coat protein gene from brazilian isolates of Papaya ringspot virus

Papaya ringspot virus (PRSV) is the causal agent of the main papaya (Carica papaya) disease in the world. Brazil is currently the world's main papaya grower, responsible for about 40% of the worldwide production. Resistance to PRSV on transgenic plants expressing the PRSV coat protein (cp) gene was shown to be dependent on the sequence homology between the cp transgene expressed in the plant genome and the cp gene from the incoming virus, in an isolate-specific fashion. Therefore, knowledge of the degree of homology among the cp genes from distinct PRSV isolates which are present in a given area is important to guide the development of transgenic papaya for the control of PRSV in that area. The objective of the present study was to assess the degree of homology among the PRSV cp genes of several Brazilian isolates of this virus. Papaya and PRSV are present in many different ecosystems within Brazil. Twelve PRSV isolates, collected in eight different states from four different geographic regions, were used in this study. The sequences of the cp gene from these isolates were compared among themselves and to the gene used to generate transgenic papaya for Brazil. An average degree of homology of 97.3% at the nucleotide sequence was found among the Brazilian isolates. When compared to 27 isolates from outside Brazil in a homology tree, the Brazilian isolates were clustered with Australian, Hawaiian, and Central and North American isolates, with an average degree of homology of 90.7% among them.

Pathogenicity, molecular characterization, and cercosporin content of Brazilian isolates of Cercospora kikuchii

Cercospora kikuchii, involved with the defoliation of soybean (Glycine max) plants, is normally associated with Septoria glycines in late season. Seventy-two isolates from different regions in Brazil, obtained mainly from purple stained seeds, showed phenotypic variation. Cercosporin content and rate of colony growth was higly variable among isolates. A strong correlation between cercosporin content and virulence was identified. Genetic variation among and within populations was evaluated based on 86 RAPD loci. The RAPD analysis clustered all isolates into seven groups. No relationship was observed between RAPD groups and geographic origin or cercosporin content. The sequence of the internal spacer regions (ITS1-5.8S-ITS2) from 13 isolates chosen according to the previous RAPD and clustering analysis showed high similarity (97%-100%) to the GenBank sequences of C. kikuchii (AY266160, AY266161, AY152577 and AF291708). It is clear from this work that Brazilian isolates of C. kikuchii from different geographic regions, are variable in relation to virulence, RAPD profiles and cercosporin content. Cercosporin content could be a good parameter for choosing an adequate isolate for screening resistant or tolerant cultivars. Considering that this pathogen is easily seed-borne, findings are expected to show the same haplotypes in different regions. Migration could be favoured by infected seeds as demonstrated by RAPD analysis.

Serum neutralization with different types and subtypes of bovine herpesvirus 1 and 5

The serum neutralization (SN) test is the gold standard method to measure neutralizing antibodies to bovine herpesviruses. However, in view of the further subdivisions of bovine herpesviruses in types/subtypes, defining which virus to use at challenge in SN tests may be difficult. In view of that, this study was carried out to re-evaluate (SN) sensitivity with different types/subtypes of bovine herpesviruses types 1 (BoHV-1) and 5 (BoHV-5) as challenge viruses. Bovine sera (n=810) were collected from two distinct geographic regions and tested by SN with three type 1 viruses (BoHV-1.1 strains "Los Angeles" and "EVI123/98"; BoHV-1.2a strain "SV265/96") and three type 5 viruses (BoHV-5a strain "EVI88/95"; BoHV-5b strain "A663" and BoHV-5c "ISO97/95"). SN tests were performed with a 1 hour incubation of the serum-virus mixtures at 37ºC against 100 TCID50 of each of the viruses. SN sensitivity varied greatly depending on the challenge virus used in the test. The highest sensitivity (327 positive/810 total sera tested; 40.37%) was attained when the positive results to the six viruses were added together. No association could be found between any particular type or subtype of virus and the sensitivity of the test. When positive results to each single strain were considered, SN sensitivity varied from 41.7% to 81.7%, depending on the virus and the geographic region of origin of the sera. Variation was detected even when challenge viruses belonged to the same subtype, where disagreement between positive results reached 41%. These results indicate that one hour incubation SN tests against single viruses, as performed here, may display a significantly low sensitivity (p=0.05); performing SN tests against a number of different viruses may increase considerably SN sensitivity. Furthermore, the choice of virus used for challenge is critical in SN tests. In addition, sera from different geographic regions may give rise to disagreeing results with different strains of BoHV-1 and BoHV-5. This might be particularly relevant for control programs and in international trade, were maximum sensitivity should be targeted.

Profile and geographic distribution of reported cases of visceral leishmaniasis in Campo Grande, State of Mato Grosso do Sul, Brazil, from 2002 to 2009

INTRODUCTION: This study sought to describe the profile and geographic distribution of reported cases of visceral leishmaniasis (VL) in the City of Campo Grande, State of Mato Grosso do Sul (MS), Brazil, from 2002 to 2009. METHODS: Human data were collected from the Brazilian National Information System for Notifiable Diseases. Canine cases and entomological data were obtained from the Information Service for Canine Visceral Leishmaniasis Control/Campo Grande, MS. RESULTS: A total of 951 records from 2002 to 2009 were investigated. The number of reported cases of VL in males was significantly higher (p < 0.0001) than that in females. The higher frequency observed among males was associated with age (p < 0.0001), which increased in individuals aged 40 years and older. The overall fatality rate was 7.4%. Entomological surveys conducted in 2006, 2007, and 2009 showed the insect vector Lutzomyia longipalpis to be present in all urban regions of the county. CONCLUSIONS: VL cases in humans and dogs, as well as in vectors, occurs in all urban regions of Campo Grande. Despite not observing tendencies of increase or reduction in the incidence of the disease due to aging, the major incidence in men is higher in those aged 40 years or above.

Geographic weighted regression: applicability to epidemiological studies of leprosy

Abstract: INTRODUCTION: Geographic information systems (GIS) enable public health data to be analyzed in terms of geographical variability and the relationship between risk factors and diseases. This study discusses the application of the geographic weighted regression (GWR) model to health data to improve the understanding of spatially varying social and clinical factors that potentially impact leprosy prevalence. METHODS: This ecological study used data from leprosy case records from 1998-2006, aggregated by neighborhood in the Duque de Caxias municipality in the State of Rio de Janeiro, Brazil. In the GWR model, the associations between the log of the leprosy detection rate and social and clinical factors were analyzed. RESULTS: Maps of the estimated coefficients by neighborhood confirmed the heterogeneous spatial relationships between the leprosy detection rates and the predictors. The proportion of households with piped water was associated with higher detection rates, mainly in the northeast of the municipality. Indeterminate forms were strongly associated with higher detections rates in the south, where access to health services was more established. CONCLUSIONS: GWR proved a useful tool for epidemiological analysis of leprosy in a local area, such as Duque de Caxias. Epidemiological analysis using the maps of the GWR model offered the advantage of visualizing the problem in sub-regions and identifying any spatial dependence in the local study area.

Tocopherol profile of Brazil nut oil from different geographic areas of the Amazon region

The tocopherol content of Brazil nut oil from different Amazon regions (Manicoré-AM, Rio Preto da Eva-AM, São João da Baliza-RR, Caroebe-RR, Belém-PA, and Xapurí-AC) was investigated by normal-phase high-performance liquid chromatography. For all authentic oils, two isomers: α- and γ-tocopherols were observed (37.92-74.48 µg g-1, 106.88-171.80 µg g-1, respectively), and their levels were relatively constant among the oils having these geographic origins, which would enable to distinguish Brazil nut oil from other plant oils for authentication purposes. Commercial Brazil nut oils were also evaluated, and some of these oils demonstrated a tocopherol content that was very different from that of the authentic oils. Therefore, we suggest that the tocopherol profile of Brazil nut oil can be useful chemical marker for quality control and authentication.

Geographic distribution of HBsAg subtypes in Brazil

HBsAg positive serum samples (896) from five brazilian regions were analysed for HBsAg subtypes. The presence of five different subtypes (ayw2, ayw3, ayw4, adw2 and adw4) was detected. In Northern region subtypes adw4 (41.2%) and adw2 (37.2%) were predominant. In the North East only subtype adw2 was encountered. In Central West, South-East and South, subtypes ayw2, ayw3, adw2 and adw4 were present, with predominance of adw2 in Central West and South East (84.3% and 69.4% respectively) whereas in the South the predominant subtype was ayw3 (41.9%) followed by ayw2 (36.4%). Subtypes ayw1, ayr and adr were not found among the samples studied. These results show the difference in the incidence of HBsAg subtypes in the different regions of Brazil and their significance in relation to the colonization and migrations in this country.

Genetic polymorphism in Taenia solium metacestodes from different Brazilian geographic areas

The aim of the present study is to investigate genetic polymorphisms in Taenia solium metacestodes from different Brazilian geographical areas and to relate them to antibody recognition in serum samples of neurocysticercosis (NC) patients. Metacestodes were obtained from the Distrito Federal (DF), Bahia, Minas Gerais (MG) and São Paulo (SP) regions of Brazil. Samples of human sera from 49 individuals with NC, 68 individuals with other helminthiasis and 40 healthy volunteers were analysed (157 individuals in total). Antigens were prepared and used in enzyme-linked immunosorbent assay and western blotting assays to detect specific immunoglobulin G antibodies. Genetic distances between metacestode populations were analysed using random amplified polymorphic DNA (RAPD) analysis. Our results show that there was a higher frequency of reactivity in the DF region in the sera from NC patients (p < 0.05), while discrimination between active and inactive NC was seen only in extracts from the MG and SP regions (p < 0.05). Using RAPD, the sample from the DF region presented a greater increase compared to the other regions. A relationship between genetic polymorphisms among T. solium metacestodes from different areas in Brazil and the differences in antibody detection in patients with NC were established.

Geographic distribution of phlebotomine sandfly species (Diptera: Psychodidae) in Central-West Brazil

This study updates the geographic distributions of phlebotomine species in Central-West Brazil and analyses the climatic factors associated with their occurrence. The data were obtained from the entomology services of the state departments of health in Central-West Brazil, scientific collections and a literature review of articles from 1962-2014. Ecological niche models were produced for sandfly species with more than 20 occurrences using the Maxent algorithm and eight climate variables. In all, 2,803 phlebotomine records for 127 species were analysed. Nyssomyia whitmani,Evandromyia lenti and Lutzomyia longipalpiswere the species with the greatest number of records and were present in all the biomes in Central-West Brazil. The models, which were produced for 34 species, indicated that the Cerrado areas in the central and western regions of Central-West Brazil were climatically more suitable to sandflies. The variables with the greatest influence on the models were the temperature in the coldest months and the temperature seasonality. The results show that phlebotomine species in Central-West Brazil have different geographical distribution patterns and that climate conditions in essentially the entire region favour the occurrence of at least one Leishmania vector species, highlighting the need to maintain or intensify vector control and surveillance strategies.

Distribution and agroclimatic characterization of potential cultivation regions of physic nut in Mexico

The purposes of this study were to determine the distribution and climatic patterns of current and future physic nut (Jatropha curcas) cultivation regions in Mexico, and to identify possible locations for in vivo germplasm banks establishment, using geographic information systems. Current climatic data were processed by Floramap software to obtain distribution maps and climatic patterns of regions where wild physic nuts could be found. DIVA-GIS software analyzed current climatic data (Worldclim model) and climatic data generated by CCM3 model to identify current and future physic nut cultivation regions, respectively. The distribution map showed that physic nut was present in most of the tropical and subtropical areas of Mexico, which corresponded to three agroclimatic regions. Climate types were Aw2, Aw1, and Bs1, for regions 1, 2 and 3, respectively. Nontoxic genotypes were associated with region 2, and toxic genotypes were associated with regions 1 and 3. According to the current and future cultivation regions identified, the best suitable ones to establish in vivo germplasm collections were the coast of Michoacán and the Isthmus of Tehuantepec, located among the states of Veracruz, Oaxaca and Chiapas.

Occurrence of Akodon cursor (Rodentia, Cricetidae) with 14, 15 and 16 chromosome cytotypes in the same geographic area in Southern Brazil

The karyotype of Akodon cursor (initially identified as A. arviculoides) had been reported with chromosomal numbers 14 and 15 in the South and Southeast and 16 in Northeastern Brazil. We found the three cytotypes in a region of Southern Brazil. The G-band patterns of these specimens were the same as those from southeastern and northeastern regions. Seventeen different combinations of chromosomes due to a complex rearrangement in pair 1 and pericentric inversions in pairs 2 and 3 were identified. Seven of these combinations are new to in the literature.