BackgroundAmong Chagas disease triatomine vectors, the largest genus, Triatoma, includes species of high public health interest. Triatoma dimidiata, the main vector throughout Central America and up to Ecuador, presents extensive phenotypic, genotypic, and behavioral diversity in sylvatic, peridomestic and domestic habitats, and non-domiciliated populations acting as reinfestation sources. DNA sequence analyses, phylogenetic reconstruction methods, and genetic variation approaches are combined to investigate the haplotype profiling, genetic polymorphism, phylogeography, and evolutionary trends of T. dimidiata and its closest relatives within Triatoma. This is the largest interpopulational analysis performed on a triatomine species so far.Methodology and FindingsTriatomines from Mexico, Guatemala, Honduras, Nicaragua, Panama, Cuba, Colombia, Ecuador, and Brazil were used. Triatoma dimidiata populations follow different evolutionary divergences in which geographical isolation appears to have had an important influence. A southern Mexican–northern Guatemalan ancestral form gave rise to two main clades. One clade remained confined to the Yucatan peninsula and northern parts of Chiapas State, Guatemala, and Honduras, with extant descendants deserving specific status. Within the second clade, extant subspecies diversity was shaped by adaptive radiation derived from Guatemalan ancestral populations. Central American populations correspond to subspecies T. d. dimidiata. A southern spread into Panama and Colombia gave the T. d. capitata forms, and a northwestern spread rising from Guatemala into Mexico gave the T. d. maculipennis forms. Triatoma hegneri appears as a subspecific insular form.ConclusionsThe comparison with very numerous Triatoma species allows us to reach highly supported conclusions not only about T. dimidiata, but also on different, important Triatoma species groupings and their evolution. The very large intraspecific genetic variability found in T. dimidiata sensu lato has never been detected in a triatomine species before. The distinction between the five different taxa furnishes a new frame for future analyses of the different vector transmission capacities and epidemiological characteristics of Chagas disease. Results indicate that T. dimidiata will offer problems for control, although dwelling insecticide spraying might be successful against introduced populations in Ecuador.

Five commercially available enzyme-linked immunosorbent assays (ELISAs), one in-house ELISA, and two hemagglutination assays were evaluated to determine their diagnostic accuracy for Chagas' disease in two studies. In study 1, ELISA kits showed 100% sensitivity, but specificities ranged from 82.84% to 100% when leishmaniasis cases were included and from 95.57% to 100% when leishmaniasis cases were excluded. Kits using recombinant antigens or synthetic peptides are more specific than those using crude extracts from Trypanosoma cruzi epimastigote forms. Kits evaluated in Panama, in study 2, showed 75% to 100% sensitivity and 97.12% to 100% specificity. These data were obtained by using a Western blot assay with T. cruzi trypomastigote excreted-secreted antigens as a reference test to confirm T. cruzi infection.

The commercially available diagnostic tests for Chagas’ disease employ whole extracts or semipurified fractions ofTrypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas’ disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzirecombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas’ disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas’ disease.

An enzyme-linked immunosorbent assay to diagnose Chagas' disease by a serological test was performed with Trypanosoma cruzi recombinant antigens (JL8, MAP, and TcPo). High sensitivity (99.4%) and specificity (99.3%) were obtained when JL8 was combined with MAP (JM) and tested with 150 serum samples from chagasic and 142 nonchagasic individuals. Moreover, JM also diagnosed 84.2% of patients in the acute phase of T. cruzi infection.

At least two types of Wolbachia bacteria were detected in wild and insectarium-raised Rhodnius pallescens, a natural vector of Trypanosoma cruzi and Trypanosoma rangeli. Wolbachia was detected in all the organs and tissues studied and in the feces, and this provided a methodological advantage for determining the presence of this endosymbiont in this host, obviating the need to kill the specimens. The occurrence of trypanosomatids in wild individuals was also studied.

The occurrence of subgingival Aclinobacillus actinomycetemcomitans and Capnocytophaga in 12 localized juvenile periodontitis and 10 gingivitis patients from Panama was determined using selective culture techniques. A actinomycetemcomitans was present in all localized juvenile periodontitis lesions studied and was, on average, recovered in hundredfold‐higher numbers from localized juvenile periodontitis lesions than from gingivitis lesions. Capnocytophaga was only recovered in approximately threefold‐higher numbers from localized juvenile periodontitis than from gingivitis. The study confirms and extends previous data indicating a close relationship between A actinomycetemcomitans and localized juvenile periodontitis. It is proposed that identification of A actinomycetemcomitans may be a valuable adjunct in the diagnosis of localized juvenile periodontitis.

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Trypanosoma rangeli epimastigote components, able to elicit an immune response, were defined and compared with those found in Trypanosoma cruzi epimastigotes. Using polyclonal antibodies against these parasites and immunoblotting analysis, an antigenic similarity index of 0.48 was found when anti-T. cruzi antibodies were used and 0.60 with anti-T. rangeli antibodies. Additionally, immunoblotting analyses using specific antibodies against a T. rangeli polypeptide eluted from polyacrylamide gels confirm an antigen of 43 kDa as a specific marker for T. rangeli.