The population structure of Trypanosoma cruzi: expanded analysis of 54 strains using eight polymorphic CA-repeat microsatellites

Recently we cloned and sequenced the first eight Trypanosoma cruzi polymorphic microsatellite loci and studied 31 clones and strains to obtain valuable information about the population structure of the parasite. We have now studied 23 further strains, increasing from 11 to 31 the number of strains obtained from patients with chronic Chagas disease. This expanded set of 54 strains and clones analyzed with the eight microsatellites markers confirmed the previously observed diploidy, clonal population organization and very high polymorphism of T. cruzi. Moreover, this new study disclosed two new features of the population genetic structure of T. cruzi. The first was the discovery that, similarly to what we had previously shown for strains isolated from insect vectors, mammals and humans with acute disease, isolates from patients in the chronic phase of Chagas disease could also be multiclonal, albeit at a reduced proportion. Second, when we used parsimony to display the genetic relationship among the clonal lineages in an unrooted Wagner network we observed, like before, a good correlation of the tree topography with the classification in three clusters on the basis of single locus analysis of the ribosomal RNA genes. However, a significant new finding was that now the strains belonging to cluster 2 split in two distant sub-clusters. This observation suggests that the evolutionary history of T. cruzi may be more complex than we previously thought.

The noble enigma: Chagas' nominations for the Nobel Prize

Carlos Chagas, a Brazilian physician, discovered the American trypanosomiasis in 1909. Like other remarkable discoveries of those days, his work helped to articulate the insect-vector theory and other theoretical guidelines in tropical medicine. Unlike all other discoveries, all the stages of this work were accomplished in a few months and by a single man. Chagas' discovery was widely recognized at home and abroad. He was twice nominated for the Nobel Prize - in 1913 and in 1921-, but never received the award. Evidence suggests that the reasons for this failure are related to the violent opposition that Chagas faced in Brazil. The contentions towards Chagas were related to a rejection of the meritocratic procedures that gave him prominence, as well as to local petty politics.

Chagas infection transmission control: situation of transfusional transmission in Brazil and other countries of Latin America

The transmission of the transfusion-associated Chagas disease is an important mechanism of its dissemination in several Latin American countries. The transmission risk depends on five factors: prevalence of infection in blood donors, degree of serological coverage, sensibility of used tests, safety of obtained results and infection risk. The Southern Cone Iniciative set off by the Pan-American Health Organization, in 1991, is contributing to the implementation of blood law in each endemic country, and to reduce the risk of transfusional transmission of this horrible disease. Despite the clear improvement of Brasilian hemotherapy after 1980 (with the creation of the Blood National Program - Pró-Sangue) and the significant reduction of the chagasic infection among its blood donors; socio-economic, politic and cultural unlevels, prevent it from reaching the necessary universality and security. In order to assure both, the Brazilian Ministry of Health decided to restructure its blood system. In May, 1998, a great program was launched, to reach a specific goal: Blood - 100% with quality safety in all its process until 2003. It was divided in 12 projects, intends to guarantee the quality and self sufficiency in blood and hemoderivates.

alpha-glycerophosphate dehydrogenase activity in flight muscles of triatomine bugs Panstrongylus megistus and Triatoma sordida

The alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity in flight muscles of Panstrongylus megistus and Triatoma sordida, vectors of Chagas disease in Brazil, was studied. Both species showed higher enzymatic activities in fliers than in non-fliers insects. T. sordida exhibited a higher proportion of flier insects than P. megistus. A possible role of alpha-GPDH on triatomines flight is discussed.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Serodiagnosis of chronic Chagas infection by using EIE-Recombinant-Chagas-Biomanguinhos kit

A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the refered kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.

The Triatomines of Northern Peru, with Emphasis on the Ecology and Infection by Trypanosomes of Rhodnius ecuadoriensis (Triatominae)

Information on the distribution and synanthropic behaviour of triatomines is essential for Chagas disease vector control. This work summarises such information from northern Peru, and presents new data on Rhodnius ecuadoriensis - an important local vector infesting 10-35% of dwellings in some zones. Three species are strongly synanthropic and may be suitable targets for chemical control of domestic/peridomestic bug populations. Panstrongylus herreri, the main domestic vector in the area, is probably present in sylvatic ecotopes in the Marañón river system. R. ecuadoriensis and Triatoma dimidiata seem exclusively domestic; biogeographical and ecological data suggest they might have spread in association with humans in northern Peru. Confirmation of this hypothesis would result in a local eradication strategy being recommended. Presence of trypanosome natural infection was assessed in 257 R. ecuadoriensis; Trypanosoma rangeli was detected in 4% of bugs. Six further triatomine species are potential disease vectors in the region (T. carrioni, P. chinai, P. rufotuberculatus, P. geniculatus, R. pictipes, and R. robustus), whilst Eratyrus mucronatus, E. cuspidatus, Cavernicola pilosa, Hermanlentia matsunoi, and Belminus peruvianus have little or no epidemiological significance. A strong community-based entomological surveillance system and collaboration with Ecuadorian public health authorities and researchers are recommended.

Changes in nuclear phenotype frequencies following sequential cold shocks in Triatoma infestans (Hemiptera, Reduviidae)

The nuclear phenotypes of Malpighian tubule cells in fifth instar nymphs of Triatoma infestans, one of the most important vectors of Chagas disease, were studied following sequential shocks at 0ºC, separated by intervals of 8 h and 24 h at 30ºC, under conditions of moderate fasting and full nourishment. The insects pertained to colonies reared in the laboratory and originated from domestic specimens collected in the Brazilian states of São Paulo (north) and Minas Gerais (south). Since nuclear phenotypes in this species are affected by single cold shocks, it was expected that these phenotypes could also be changed by sequential shocks. Nuclear phenotypes indicative of mechanisms of cell survival (nuclear fusion and heterochromatin decondensation) and cell death (apoptosis and necrosis) were observed concomitantly in all the conditions tested. Nuclear fusion and heterochromatin decondensation were not found relevant for the presumed acquisition of the cold-hardening response in T. infestans. The decreased frequency of apoptosis and necrosis following sequential cold shocks including under fasting conditions, indicated that tolerance to sequential cold shocks occurred in T. infestans of the mentioned origin.

Feeding and defaecation behaviour of Triatoma patagonica (Del Ponte, 1929) (Hemiptera: Reduviidae)

Among the vectors of Chagas disease, Triatoma patagonica is a species in the process of adaptation to the human environment being recently registered in urban and suburban zones. However, its importance as a vector of Chagas disease is unknown. The aim of this work was to evaluate two aspects of vectorial competence: the feeding behaviour and the defaecation pattern. These processes were studied in females of T. patagonica fed ad libitum on a restrained pigeon. The results showed that the blood meal size was negatively correlated with the time of first defaecation (r = -0.42). The first defaecation was emitted before the first 10 min and defaecations during feeding were frequent. A total of 73% of females, defaecated during the first 30 min post-feeding. These results suggest that if this species subsequently colonizes the domicile, it would be capable of transmitting Trypanosoma cruzi.

Impact of residual spraying on Rhodnius prolixus and Triatoma dimidiata in the department of Zacapa in Guatemala

As a vector control program to control Chagas disease in Guatemala, residual spraying of Rhodnius prolixus and Triatoma dimidiata was performed, and its impact was measured in the department of Zacapa. In order to identify infested villages and determine the degree of infestation, a baseline entomological survey to identify municipalities infested with vectors followed by an additional vector survey in areas known to be infested was conducted. Residual spraying using pyrethroid insecticides was performed at all the villages identified as being infested with the vectors. The residual spraying was shown to be highly effective against both vectors by the decrease in infestation indices after spraying. Analysis of the cost-effectiveness of the spraying showed that the average cost of insecticides per house is high when compared with that in Southern Cone countries.

Foco de doença de Chagas em Arcádia, estado do Rio de Janeiro, Brasil

Following the report of triatomine nymphs in a house in Arcadia, Miguel Pereira, state of Rio de Janeiro, Brazil, the infested dwelling was checked. Several eggs and 46 specimens of Triatoma vitticeps (Stal, 1859) were collected. Among them, adults and nymphal instars accounted for 43.5% and 56.5%, respectively. Analysis of blood meals showed the ecletism of this species; 24 (52.2%) were single feeds, 18 insects (39.1%) fed on two hosts and 4 (8.7%) on three hosts. Trypanosoma cruzi infection rate of examined specimens was 13%. Finally one of the residents of the house was positive for anti-T. cruzi antibodies using indirect immunofluorescence.

Morphometric analysis of Triatoma dimidiata populations (Reduviidae:Triatominae) from Mexico and Northern Guatemala

Triatoma dimidiata is one of the major vectors of Chagas disease in Latin America. Its range includes Mexico, all countries of Central America, Colombia, and Ecuador. In light of recent genetic analysis suggesting that the possible origin of this species is the Yucatan peninsula, we have analyzed populations from the state of Yucatan, San Luis Potosi, and Veracruz in Mexico, and a population from the southern region of the Yucatan peninsula located in Northern Guatemala, the region of El Peten. Classical morphometry including principal component, discriminant, sexual dimorphism, and wing asymmetry was analyzed. San Luis Potosi and Veracruz populations were indistinguishable while clearly separate from Yucatan and Peten populations. Despite important genetic differences, Yucatan and Peten populations were highly similar. Yucatan specimens were the smallest in size, while females were larger than males in all populations. Only head characters were necessary to distinguish population level differences, although wing fluctuating asymmetry was present in all populations. These results are discussed in light of recent findings suggesting genetic polymorphism in most populations of Triatoma dimidiata south of Chiapas to Ecuador.

Evaluation of cultures of Saccharomyces cerevisae as baits for Triatoma dimidiata and Triatoma pallidipennis

We tested the attraction of Triatoma dimidiata and T. pallidipennis to traps baited with yeast volatiles. Two traps were simultaneously presented in opposite sides of an experimental arena. One trap presented a yeast culture in sucrose solution, while the other contained sucrose solution as control. A first experimental series was done without offering a central refuge for bugs. In a second series, one shelter where the insects could hide was offered and the traps were presented afterwards. In the first series, yeast baited traps attracted significantly more insects than control ones for both species. In the second series, T. pallidipennis was significantly attracted to yeast, whereas T. dimidiata was not attracted. The potential use of yeast baited traps for capturing these vectors of Chagas disease is discussed.

Triatoma mexicana (Hemiptera: Reduviidae) in Guanajuato, Mexico: house infestation and seasonal variation

Triatoma mexicana was described by Herrich-Schaeffer in 1848. In 1940, a male specimen was found in Hidalgo. In 1970, this species was recorded in the state of Queretaro. Later, it was registered in Guanajuato and San Luis Potosi. In the present paper we performed an investigation in 545 dwellings from three counties in the state of Guanajuato, Mexico, from March 2003 to May 2004. The search and capture of triatomines were seasonally performed indoors and outdoors. Entomological indexes were calculated. The risk and no risk relations between triatomine presence and housing construction materials were analyzed. Fourteen triatomines were collected indoors and 151 outdoors. The vectors were collected in houses built with either risky and non-risky materials. Adults go indoors but do not settle there, hence, no relationship was found between the building materials and infestation of houses. Conventional interventions like house improvement or insecticide spraying are not efficient for the control of T. mexicana, because its developmental cycle is accomplished outdoors in the area surrounding the houses.