Angela Cristina Verissimo Junqueira

Emerging Chagas disease in Amazonian Brazil

In the Amazon Basin, Trypanosoma cruzi infection is enzootic, involving a variety of wild mammals and at least 10 of the 16 reported silvatic triatomine bug species. Human cases of Chagas disease are increasing, indicating that the disease may be emerging as a wider public health problem in the region: 38 cases from 1969 to 1992, and 167 in the past eight years. This article reviews the status of Chagas disease in Amazonian Brazil, including known reservoirs and vectors, and the genetic diversity of T. cruzi. At least three subspecific groups of T. cruzi-T. cruzilZ1, T. cruziZ3 and T. cruziZ3/Z1 ASAT--are present. It appears that T. cruzil has an extant capacity for genetic exchange. Attention is also drawn to the risk of domestic endemicity, in addition to the tasks facing the disease control authorities.

A mini-exon multiplex polymerase chain reaction to distinguish the major groups of Trypanosoma cruzi and T. rangeli in the Brazilian Amazon

0. Fernandesi*3, S. S. Santos’, E. CupoUlo’, B. Mendonga’, R. Derre3, A. C. V. Junqueira’, L. C. Santos’, N. R. Sturm4, R. l3. NaifP, T. V. Barret’, Il. A. Campbell4 and J. R. Coura’ ‘Department of Tropical Medicine and 2Department of Imw~nunology, Instiwo OswaEdo Cruz, Fiocruz, Rio de Janeiro, Brazil; 3DepaPtment of Pathology, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil; 4Department of Microbiology, InwunoZogy and MolecuZar Genetics, Uv 51nstituto National de Pesquisas da AmazBnia, INPA, Manaus, Brazil Keywords:Americantrypanosomiasis, Chagasdisease,

A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA.

We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.

Chagas disease in the Amazon Region

The risk that Chagas disease becomes established as a major endemic threat in Amazonia (the worlds largest tropical biome, today inhabited by over 30 million people) relates to a complex set of interacting biological and social determinants. These include intense immigration from endemic areas (possibly introducing parasites and vectors), extensive landscape transformation with uncontrolled deforestation, and the great diversity of wild Trypanosoma cruzi reservoir hosts and vectors (25 species in nine genera), which maintain intense sylvatic transmission cycles. Invasion of houses by adventitious vectors (with infection rates > 60%) is common, and focal adaptation of native triatomines to artificial structures has been reported. Both acute (approximately 500) and chronic cases of autochthonous human Chagas disease have been documented beyond doubt in the region. Continuous, low-intensity transmission seems to occur throughout the Amazon, and generates a hypoendemic pattern with seropositivity rates of approximately 1-3%. Discrete foci also exist in which transmission is more intense (e.g., in localized outbreaks probably linked to oral transmission) and prevalence rates higher. Early detection-treatment of acute cases is crucial for avoiding further dispersion of endemic transmission of Chagas disease in Amazonia, and will require the involvement of malaria control and primary health care systems. Comprehensive eco-epidemiological research, including prevalence surveys or the characterization of transmission dynamics in different ecological settings, is still needed. The International Initiative for Chagas Disease Surveillance and Prevention in the Amazon provides the framework for building up the political and scientific cooperation networks required to confront the challenge of preventing Chagas disease in Amazonia.

Comparison of the polymerase chain reaction with two classical parasitological methods for the diagnosis of Chagas disease in an endemic region of north-eastern Brazil

The sensitivities for Chagas disease diagnosis of haemoculture, xenodiagnosis, and polymerase chain reaction (PCR) amplification of Trypanosoma cruzi kinetoplast deoxyribonucleic acid (DNA) were compared for 101 patients living in an endemic region who were serologically positive for T. cruzi. PCR gave 60 positive results (59.4%), while a haemoculture was positive in 26 cases (25.7%) and xenodiagnosis in 36 (35.6%). Four xenodiagnosis-positive but PCR-negative patients were examined in detail. The discrepancies were not due to inhibition of the PCR reactions, as the samples were used successfully to amplify a human sequence. Nor were they due to a variation in kinetoplast DNA sequences, as the kinetoplast DNA of the parasite strains isolated from these patients after xenodiagnosis gave rise to the expected product when amplified by the PCR. We concluded that no parasite was present in the 5 mL of blood used for PCR, while probably a single T. cruzi cell was present in the blood volume ingested by the insects during xenodiagnosis (about 3 mL). This suggests that the total blood quantity collected for the PCR may be important with patients with low parasitaemia.

Trypanosoma cruzi in Brazilian Amazonia: Lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission ☆

In this study, we provide phylogenetic and biogeographic evidence that the Trypanosoma cruzi lineages T. cruzi I (TCI) and T. cruzi IIa (TCIIa) circulate amongst non-human primates in Brazilian Amazonia, and are transmitted by Rhodnius species in overlapping arboreal transmission cycles, sporadically infecting humans. TCI presented higher prevalence rates, and no lineages other than TCI and TCIIa were found in this study in wild monkeys and Rhodnius from the Amazonian region. We characterised TCI and TCIIa from wild primates (16 TCI and five TCIIa), Rhodnius spp. (13 TCI and nine TCIIa), and humans with Chagas disease associated with oral transmission (14 TCI and five TCIIa) in Brazilian Amazonia. To our knowledge, TCIIa had not been associated with wild monkeys until now. Polymorphisms of ssrDNA, cytochrome b gene sequences and randomly amplified polymorphic DNA (RAPD) patterns clearly separated TCIIa from TCIIb-e and TCI lineages, and disclosed small intra-lineage polymorphisms amongst isolates from Amazonia. These data are important in understanding the complexity of the transmission cycles, genetic structure, and evolutionary history of T. cruzi populations circulating in Amazonia, and they contribute to both the unravelling of human infection routes and the pathological peculiarities of Chagas disease in this region.

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.

Comparative phylogeography of Trypanosoma rangeli and Rhodnius (Hemiptera: Reduviidae) supports a long coexistence of parasite lineages and their sympatric vectors

To make reliable interpretations about evolutionary relationships between Trypanosoma rangeli lineages and their insect vectors (triatomine bugs of the genus Rhodnius) and, thus, about the determinant factors of lineage segregation within T. rangeli, we compared phylogenies of parasite isolates and vector species. Sixty‐one T. rangeli isolates from invertebrate and vertebrate hosts were initially evaluated in terms of polymorphism of the spliced‐leader gene (SL). Further analysis based on SL and SSUrRNA sequences from 33 selected isolates, representative of the overall phylogenetic diversity and geographical range of T. rangeli, supported four phylogenetic lineages within this species. By comparing the phylogeny of Rhodnius species with that inferred for T. rangeli isolates and through analysis of the geographical range of the isolates, we showed that there is a very significant overlap in the distribution of Rhodnius species and T. rangeli lineages. Congruence between phylogeographical analysis of both T. rangeli lineages and complexes of Rhodnius species are consistent with the hypothesis of a long coexistence of parasites and their vectors, with lineage divergence associated with sympatric species of Rhodnius apparently without association with particular vertebrate hosts. Separation of T. rangeli isolates from vectors of distinct complexes living in sympatry favours the absence of gene flow between the lineages and suggests evolution of T. rangeli lineages in independent transmission cycles, probably associated to specific Rhodnius spp. ecotopes. A polymerase chain reaction assay based on SL intergenic sequences was developed for simultaneous identification and lineage genotyping of T. rangeli in epidemiological surveys.

Chagas disease: from bush to huts and houses. Is it the case of the Brazilian amazon?

Two of the major problems facing the Amazon - human migration from the other areas and uncontrolled deforestation - constitute the greatest risk for the establishment of endemic Chagas disease in this part of Brazil. At least 18 species of triatomines had been found in the Brazilian Amazon, 10 of them infected with Trypanosoma cruzi, associated with numerous wild reservoirs. With wide-range deforestation, wild animals will perforce be driven into other areas, with tendency for triatomines to become adapted to alternative food sources in peri and intradomicilies. Serological surveys and cross-sectional studies for Chagas disease, carried out in rural areas of the Rio Negro, in the Brazilian Amazon, showed a high level of seropositivity for T. cruzi antibodies. A strong correlation of seroreactivity with the contact of gatherers of piaçava fibers with wild triatomines could be evidenced.

Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron

Trypanosoma cruzi is currently classified into 2 major phylogenetic lineages, T. cruzi I and II, that correlate with the formerly described zymodeme 1 and 2, respectively. Another isoenzymic group (zymodeme 3-Z3) was also described. In this study, we analysed the genetic diversity among Z3 isolates of the Brazilian Amazon by restriction fragment length polymorphism of the intergenic transcribed spacers (ITSs) of the ribosomal RNA cistron and the size of the divergent domain D7 of the 24Salpha rRNA gene. DNAs from 12 T. cruzi Z3 isolates obtained from humans (2), Panstrongylus geniculatus (1), and Rhodnius brethesi (9) were submitted to PCR amplification of the ITSs plus the 5.8S rDNA. The PCR products were digested with 4 distinct endonucleases and the profiles analysed by a numerical methodology. The phenetic dendrogram revealed a clear dichotomy in the Z3 group, defining 2 groups that were named Z3-A and Z3-B. Dimorphism was also found in the band sizes of the amplified D7 divergent domain of the 24Salpha rDNA, which showed a perfect correlation with the ITSs clustering. The organization of the ribosomal cistron was investigated by Southern blotting and shown to be conserved in the genome of the 2 Z3 groups. This study shows that the rDNA cistron allows the definition of 2 distinct subclusters in Z3 isolates.