Hydroxymethylnitrofurazone treatment in indeterminate form of chronic Chagas disease: Reduced intensity of tissue parasitism and inflammation-A histopathological study.

Hydroxymethylnitrofurazone (NFOH) is a nitrofurazone prodrug effective in vivo during acute infections, and it has less hepatotoxicity effect than the standard drug benznidazole (BZN) which has been used during short- and long-term treatment. In the present study, we induced the indeterminate form of Chagas disease in mice with a Y strain of Trypanosoma cruzi and analysed the histopathological data about the effects of NFOH and BZN on different tissues, including the heart, skeletal muscle, liver, kidney, colon, spleen and brain. After infection, BALB/c mice were treated with NFOH (150 mg/kg) and BZN (60 mg/kg) for 60 days and then submitted to immunosuppression using dexamethasone (5 mg/kg) for 14 days. Two trained analysts, as part of a blind evaluation, examined the results using serial sections of 3 mm diameter in two different moments. The results showed reactivation of the disease only in the infected nontreated group (POS). After treatment, amastigote nests were found in the heart, colon, liver and skeletal muscle in the POS group and in the heart and liver of the BZN group. Interestingly, amastigote nests were not found in the NFOH and NEG groups. The histopathological analysis showed fewer tissue lesions and parasite infiltrates in the NFOH group when compared with the BZN and POS groups. We have not observed any increase in the levels of hepatocellular injury biomarkers (AST/ALT) in the NFOH group. These in vivo studies show the potential for NFOH as an effective and safe compound useful as an anti-T. cruzi agent.

Combined phylogenetic and morphometric information to delimit and unify the Triatoma brasiliensis species complex and the Brasiliensis subcomplex.

"Triatoma brasiliensis species complex" was defined as a monophyletic group of the species: T. brasiliensis, T. juazeirensis, T. melanica, and T. sherlocki. An alternative grouping scheme proposed the concept of "Brasiliensis subcomplex" which included the former species together with T. melanocephala, T. petrocchiae, T. lenti, T. tibiamaculata, and T. vitticeps. To evaluate the relationship among these taxa we combined the results obtained with four mitochondrial genes (12S, 16S, COI and Cytb, adding to 1811bp) and geometric morphometric analysis of wings and heads. Panstrongylus megistus was included in the analysis as it was previously found related to T. tibiamaculata, T. melanocephala and T. vitticeps. The results of both molecular and morphometric approaches clearly grouped the species analyzed into two monophyletic units, supported by both genetic and wing variability. The first one (G1) comprises the four species originally included in the T. brasiliensis species complex plus T. lenti and T. petrocchiae. The second group (G2) was composed by T. melanocephala, T. tibiamaculata and T. vitticeps, and remarkably, P. megistus if considering wing variability and phylogenetic results. Nevertheless, geometric morphometrics of heads provided a quantitative measurement that discriminates Panstrongylus from the Triatoma species based on the position of the antennal insertion relative to eyes, as it is used as the generic distinctive character. The discrepancy among approaches questions the validity of this character to define Panstrongylus genus. Independently of the chosen group definition -"T. brasiliensis species complex" or "Brasiliensis subcomplex"-we propose to delimit it to species of G1 that are all associated with the Caatinga biome in the Brazilian Northeast. G2 are the ones associated with the Atlantic Forest biome.

Multiple mitochondrial genes of some sylvatic Brazilian Triatoma: non-monophyly of the T. brasiliensis subcomplex and the need for a generic revision in the Triatomini.

Multiple fragments of mitochondrial DNA genes (cytochrome b, cytochrome oxidase I, and 16S rDNA) were used to evaluate the phylogenetic relationships among Triatoma melanocephala, Triatoma tibiamaculata, Triatoma vitticeps, and other members of Triatoma brasiliensis subcomplex under a Bayesian framework and maximum parsimony criterion. With the addition of new sequences of T. tibiamaculata and T. vitticeps, Triatoma juazeirensis, Triatoma melanica and the newly sequenced T. melanocephala, the three first sylvatic species, T. melanocephala, T. tibiamaculata and T. vitticeps, were strongly recovered into a clade separate from the other with the remaining Triatoma species from South America, such as the members of T. brasiliensis subcomplex. Panstrongylus megistus was recovered as a sister to T. tibiamaculata, whereas T. vitticeps was a sister to T. melanocephala. This study revealed the non-monophyly of the T. brasiliensis subcomplex, and the polyphyly of Triatoma was reinforced by the placement of these three sylvatic species with Dipetalogaster, Meccus, Mepraia, and Panstrongylus. The results herein shown highlight the need of generic revision in Triatomini.

A novel association between Rhodnius neglectus and the Livistona australis palm tree in an urban center foreshadowing the risk of Chagas disease transmission by vectorial invasions in Monte Alto City, São Paulo, Brazil.

After several public notifications of domiciliary invasions, palm trees were investigated in downtown Monte Alto City, São Paulo State, Brazil, in proximity to the city hall building, the main church, condominiums and marketing establishments. One hundred seventy four palm trees of 10 species were investigated, in which 72 specimens of Rhodnius neglectus, a potential Chagas disease vector, were captured via manual methods. All insects were collected from dead leaves, organic debris and bird nests in the only three Livistona australis palm trees in the central park square. This was the first record of R. neglectus colonizing this palm species. Although no Trypanosoma cruzi was found by abdominal compression followed by light microscopy, the poor nutritional status of the bugs hampered the examination of gut contents for parasite detection. Furthermore, the central crowns of the trees, which shelter bats (Chiroptera: Mammalia), could not be carefully searched for insects due to difficult access. This new finding highlights the sudden alteration in insect behavior, probably as a result of man's interference. This report aims to warn those involved in the health system about this new threat, justifying detailed research of the area to evaluate the magnitude of this emerging public health issue.

Biological and molecular characterization of a Trypanosoma cruzi isolate obtained from Panstrongylus megistus captured in Sao Paulo State, Brazil.

An isolate of Trypanosoma cruzi obtained from P. megistus captured in the peridomicile area of a home in Santo Antonio do Jardim city in the State of Sao Paulo, denominated T. cruzi Mogi, was characterized biologically and molecularly. The RFLP analysis of the D7 divergent domain in the 24Sα rDNA and of the mini-exon positioned the T. cruzi isolate within the TcI group. Phylogenetic analysis performed with the trypanosomatid barcode confirmed that the isolate belongs to the TcI group, with high homology to the 3014 c1 T.cruzi strain. The biological characterization of the isolate in rats showed a prepatent period of about 8 days, low parasitemia and tropism for cardiac, skeletal and colonic muscles. In Swiss mice the T. cruzi Mogi isolate showed a prepatent period of about 22 days, intermittent parasitemia in some animals, and tropism for cardiac and colonic muscles. Despite the inherent difficulty of identifying correlations amongst the molecular and biological characteristics of different T. cruzi groups, the tropism for colonic muscle demonstrated by T. cruzi Mogi represented a peculiarity of this isolate within the TcI group.

Evolutionary relationships of the Triatoma matogrossensis subcomplex, the endemic triatoma in Central-Western Brazil, based on mitochondrial DNA sequences.

The phylogenetic relationships among species of Triatoma matogrossensis subcomplex (T. baratai, T. guazu, T. matogrossensis, T. sordida, T. vandae, and T. williami) was addressed by using fragments of cytochrome oxidase I (COI), 16S rDNA (16S), and cytochrome b (Cytb) through Bayesian and parsimony analyses. We did not recover a monophyletic T. matogrossensis subcomplex, and their members were found clustered in three strongly supported clades, as follows: i) T. jurbergi + T. matogrossensis + T. vandae + T. garciabesi + T. sordida; ii) with T. guasayana as the sister group of clade (i); and iii) T. williami + T. guazu, however not closely related to the clade formed by the previously mentioned species. The other two endemic species from Central-Western Brazil, T. baratai and T. costalimai, were not recovered with strong clade support as related to other members of this subcomplex. Results call for a further revision in the classification of the subcomplexes within the genus Triatoma.

Cross-mating experiments detect reproductive compatibility between Triatoma sherlocki and other members of the Triatoma brasiliensis species complex.

Phylogenetic approaches based on mitochondrial DNA variation (fragments of Cyt B and 16S ribosomal RNA) have revealed Triatoma sherlocki as the most recent species addition to the Triatoma brasiliensis species complex; a monophyletic group which includes T. brasiliensis, Triatoma melanica, and Triatoma juazeirensis. T. sherlocki is the most differentiated among all species of this complex: it is unable to fly, possesses longer legs than the other members, and has reddish tonality in some parts of its exochorion. We question whether these species are reproductively compatible because of this pronounced morphological differentiation, and therefore, we present a series of cross breeding experiments that test compatibility between T. sherlocki and other members of the T. brasiliensis complex. We extended our analyses to include crosses between T. sherlocki and Triatoma lenti, because the latter has been suggested as a possible member of this complex. T. sherlocki male×T. lenti female pairs failed to produce hybrids. All other crosses of T. sherlocki and members of T. brasiliensis species complex, as well as backcrosses, produced viable offspring through the third generation. This study stresses the importance of searching for the features that may isolate members of the T. brasiliensis species complex.

Phylogeny of Triatoma sherlocki (Hemiptera: Reduviidae: Triatominae) inferred from two mitochondrial genes suggests its location within the Triatoma brasiliensis complex.

The phylogenetic position of Triatoma sherlocki within triatomines group was inferred by analyzing mtDNA fragments of Cyt B and 16S ribosomal RNA by using maximum parsimony and Bayesian analysis. Despite being differentiated from members of the T. brasiliensis complex on morphologic grounds, molecular phylogenetic analysis suggests T. sherlocki is a member of this complex; moreover, it was placed as a sister species of T. melanica. These suggestions were supported by robust credibility rates. Hence, we show evidence for the paraphyletic group of the "Triatoma brasiliensis complex," which should be composed of T. brasiliensis brasiliensis, T. brasiliensis macromelasoma, T. juazeirensis, T. melanica, and T. sherlocki.

Comparative phylogeography of Trypanosoma cruzi TCIIc: new hosts, association with terrestrial ecotopes, and spatial clustering.

We characterized 28 new isolates of Trypanosoma cruzi IIc (TCIIc) of mammals and triatomines from Northern to Southern Brazil, confirming the widespread distribution of this lineage. Phylogenetic analyses using cytochrome b and SSU rDNA sequences clearly separated TCIIc from TCIIa according to terrestrial and arboreal ecotopes of their preferential mammalian hosts and vectors. TCIIc was more closely related to TCIId/e, followed by TCIIa, and separated by large distances from TCIIb and TCI. Despite being indistinguishable by traditional genotyping and generally being assigned to Z3, we provide evidence that TCIIa from South America and TCIIa from North America correspond to independent lineages that circulate in distinct hosts and ecological niches. Armadillos, terrestrial didelphids and rodents, and domestic dogs were found infected by TCIIc in Brazil. We believe that, in Brazil, this is the first description of TCIIc from rodents and domestic dogs. Terrestrial triatomines of genera Panstrongylus and Triatoma were confirmed as vectors of TCIIc. Together, habitat, mammalian host and vector association corroborated the link between TCIIc and terrestrial transmission cycles/ecological niches. Analysis of ITS1 rDNA sequences disclosed clusters of TCIIc isolates in accordance with their geographic origin, independent of their host species.

Rural Triatoma rubrovaria from southern Brazil harbors Trypanosoma cruzi of lineage IIc.

Triatoma infestans, the main vector of Chagas disease, has nearly been eliminated from Brazil. Nevertheless, other triatominae species are involved in the domiciliation process, including Triatoma rubrovaria in Rio Grande do Sul State (RS). Previous studies showed that 1.6% of the T. rubrovaria specimens collected at the rural district of Quaraí, RS, were naturally infected by Trypanosoma cruzi. In this study, five T. cruzi isolates obtained from infected triatomines were characterized molecularly and biologically. Genotyping of the T. cruzi isolates showed that they belong to lineage IIc of T. cruzi (TCIIc). Biological characterization showed miotropism and myositis during acute and chronic phases of infection, respectively. Virulence and mortality rates were variable among isolates. To our knowledge, this study corresponds to the first characterization of T. cruzi isolates from T. rubrovaria and the first description of TCIIc in the sylvatic cycle of T. cruzi from the southern region of Brazil.

[Morphological and histological study of eggs of six species of the Triatoma genus (Hemiptera: Reduviidae)]. / Estudo morfológico e histológico dos ovos de seis espécies do gênero Triatoma (Hemiptera: Reduviidae).

The objective of this study was to analyze the morphological and histological characteristics of eggs from six triatomine species in order to obtain a generic and specific characterization of this group of Trypanosoma cruzi vectors and better understand their phylogenetic and taxonomic aspects. The eggs of the respective species came from the collection of the Laboratório de Triatomíneos e Culicídeos da Faculdade de Saúde Pública/USP, from the Insetário do Serviço Especial de Saúde de Araraquara - SESA and from the Triatomine insectary of the Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos (FIOCRUZ), in Rio de Janeiro. Morphological studies were done with optical microscopy and scanning electron microscopy. Histological analyses used only optical microscopy. All of the species analyzed showed a predominance of hexagonal cells. The exchorion of Triatoma breyeri Del Ponte, T. costalimai Verano Galvão, and T. tibiamaculata (Pinto) consists of unornamented cells, overlapping or slightly separated, with a smooth, padded appearance. Meanwhile, in T. matogrossensis Leite Barbosa, T. sherlocki Papa, Juberg, Carcavallo, Cerqueira Barata, and T. williami Galvão, Souza Lima, the exchorion cells are ornamented on their entire surface with perforations and fissures. Egg histology showed undulations, pores, and orifices. The study helps to expand the generic and specific knowledge of the Triatominae subfamily. The characteristics identified in the exchorion of the eggshells can help separate close species. These new parameters will back the elaboration of future dichotomous keys, help to determine each vector species role and expand knowledge of the various species.

[Differences in the size of eggshells among three Pangstrongylus megistus colonies]. / Diferença no tamanho de cascas de ovos de colônias de Panstrongylus megistus.

The objective of the study was to evaluate the measurements of eggshells from three colonies of P. megistus, two from the lab and one from a sylvatic habitat. Fifty eggshells from each colony were measured with the use of a stereoscopic microscope and the Qwin image analysis system,. The statistical analysis was performed using unpaired t-test in INSTAT program. The length, width and diameter of the opercular opening of the eggshells from the sylvatic colony were larger than the two laboratorial colonies of P. megistus.