Final Report Summary - CHAGASEPINET (Comparative epidemiology of genetic lineages of Trypanosoma cruzi)
Executive summary:
Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered to be the most important parasitic disease in Latin America. The epidemiology of Chagas disease is complex, with distinct genetic lineages within the single species T. cruzi. The focus of this ambitious multidisciplinary CHAGASEPINET project, involving 15 research partners, was to apply high-resolution technologies to elucidate the epidemiology of the genetic lineages of T. cruzi, for improved understanding and control of Chagas disease. Achievements include:
1. The development of techniques such as multilocus sequence typing (MLST) and high-resolution microsatellite analysis (MLMT) for T. cruzi and their deployment in endemic regions have made widely available the capacity to identify T. cruzi genetic lineages, to unravel local transmission cycles and to plan local control strategies accordingly. Standardised polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs), MLST and MLMT methods have all been produced, with supplementary methods of data analysis, and technologies transferred, with high-impact outputs. The detailed description within CHAGASEPINET of the ecologies and epidemiologies of the six distinct genetic lineages of T. cruzi (TcI - TcVI) has provided an essential and fundamental platform for all subsequent research on the epidemiology of Chagas disease.
2. The TcI genome has been sequenced and several unforeseen additional genome sequences also provided.
3. A special achievement has been the first development and deployment of peptide based lineage-specific serology.
4. Development of PCR-oligochromatography (PCR-OC) proceeded as planned but it was not deployed to the field pending resolution of inconsistent results from different sources.
5. A large collection of clinical samples has been assembled, and pilot comparisons of genotyping and lineage-specific serology performed.
6. There has been an astonishing combined effort by South American and European partners to unravel in great detail the complex molecular epidemiology of T. cruzi infection, particularly in Bolivia, Brazil, Colombia, Ecuador and Venezuela, including ecological associations of the discrete typing units (DTUs), domestic and sylvatic cycle interactions, congenital transmission and oral outbreaks, also revealing extensive inter-lineage mitochondrial introgression and intra-lineage genetic exchange.
7. Cell invasion, morphometry, pathogenesis, congenital transmission and drug susceptibilities of the DTUs have been compared.
8. Resolution and congruence of DTU identification and population genetics methods have been assessed, with phylogenetics analysis of relationships and evolution.
9. A greatly expanded international cryobank has been established.
CHAGASEPINET has on several objectives exceeded expectation, transforming research capacity and the understanding of Chagas disease. Aspects of the research warrant further funding, for example: continuity and expansion of partnerships on molecular epidemiology, further development of peptide based specific serology, and more sensitive, extensive analysis of infecting genotypes against clinical outcome.
Project context and objectives:
Chagas disease is considered to be the most important parasitic disease in Latin America. The disease is caused by the protozoan Trypanosoma cruzi, which is transmitted primarily by bloodsucking triatomine bugs, not by their bite but by contamination of mucous membranes or abraded skin with the T. cruzi infected bug faeces and urine, deposited on the host as the bugs feed. Other methods of transmission occur, including transfusion of T. cruzi infected blood, transplantation of infected organs, congenital transmission, and oral transmission by contamination of food with infected triatomine faeces.
Control campaigns against triatomine bugs, by spraying infested houses, together with screening of blood and organ donors, have reduced the transmission of T. cruzi and the prevalence of infection in the human population. Nevertheless, it is still considered that 8 to 10 million people in Latin America carry T. cruzi infection. Mortality in the acute phase of infection is around 5 to 10 %, with infants and young adults being the most vulnerable. With rare exceptions, once an individual is infected with T. cruzi they are infected to life, despite the immune response to infection. Approximately 30 % of those who survive the acute phase of infection will progress to severe chronic Chagas disease. The organism multiplies in cells of a wide range of tissues, most notably in terms of pathogenesis, in muscle of the heart and alimentary tract, where both muscle cells and neurones are destroyed. Thus, in chronic Chagas disease there is cardiomyopathy with electrocardiography (ECG) abnormalities, particularly right bundle branch block (RBB) associated with sudden death or progressive congestive heart failure. A proportion of patients with chronic Chagas disease may develop chagasic megaoesophagus and or megacolon, requiring surgery. T. cruzi is also opportunistic, such that carriers of infection who become immunocompromised by human immunodeficiency virus infection / acquired immunodeficiency syndrome (HIV / AIDS) may relapse into the acute phase. Both immunocompromised cases and congenital cases may be associated with meningoencephalitis. There is no vaccine against Chagas disease and no immunotherapy. There are only two drugs for chemotherapy, benznidazole and nifurtimox, which are not always curative, and both may have toxic side-effects, especially when administered to adults.
T. cruzi infection used to be considered to be almost entirely confined to Latin America. However, with increasing global migration from Latin America it is now estimated that 300,000 individuals carry T. cruzi infection in the United States of America (USA) and that tens of thousands of infections are present in several European countries. Cases of blood transfusion transmission and congenital transmission can arise far from Latin America. Thus the World Health Organisation (WHO) has launched a global awareness campaign to enhance recognition of Chagas disease outside the core endemic regions.
The epidemiology of Chagas disease is complex. In some endemic regions the triatomine bug vector species are confined to domestic and peridomestic sites, having arrived by dispersion or carriage from their original sylvatic locations. In other regions the same triatomine species may be present inside and outside the houses, with the threat of reinvasion from sylvatic habitats after spraying programmes. In the Amazon basin and the USA, although T. cruzi infection is present (enzootic) in a wide range of mammals the local bug species do not colonise houses and cases of vector borne Chagas disease are sporadic.
Oral outbreaks occasionally occur in the Amazon region and elsewhere, particularly associated with consumption of triatomine contaminated plant juices.
T. cruzi used to be considered to be a relatively homogeneous entity, and it is still a single species. However, the application of biochemical methods, particularly multilocus enzyme electrophoresis (MLEE) had a remarkable impact on perceptions of the agent of Chagas disease. In a landmark study, in a village in Bahia State Brazil, MLEE demonstrated that in that focus domestic and sylvatic strains were radically distinct, more so than recognized species of the agents of leishmaniasis (Leishmania) species. Follow-up research with MLEE revealed that there are at least six distinct genetic groups or lineages of T. cruzi.
Molecular methods have advanced greatly in the last two decades with the development of higher resolution technologies based on the amplification and analysis of deoxyribonucleic acid (DNA). These technologies have profound potential impact on the understanding of the complex epidemiology of Chagas disease and of other infectious diseases, in the context of strategies for disease prevention and control.
The focus of this ambitious multidisciplinary project, involving 15 research partners, was therefore to elucidate the epidemiology of the genetic lineages of T. cruzi, for improved understanding and control of Chagas disease. The project united skills in genotyping, genomics, genetics and pathogenesis in Europe with considerable compatible skills in South America, and with key research in endemic areas that have distinct characteristics. The project was intended to be high impact in terms of both research progress and the fostering of collaborative networks.
Aim
Elucidate the epidemiology of the genetic lineages of T. cruzi, for improved understanding and prevention of Chagas disease.
Objectives
Technology development:
1. Develop further and apply genotyping technologies including PCR-RFLP typing, MLST and multilocus microsatellite typing (MLMT) to the analysis of genetic populations of T. cruzi, making available improved standardised genotyping methods for routine use in endemic countries.
2. Sequence and annotate the unresolved genome of T. cruzi I.
3. Guided by comparative genomics of T. cruzi I and T. cruzi VI (CL Brener strain), assess lineage-specific diagnosis with the trypomastigote small surface antigen (TSSA) and with other candidate antigens.
4. Develop a rapid PCR-oligochromatography test for detection of T. cruzi infection.
Molecular epidemiology:
5. Pilot studies of association between genetic lineage, clinical outcome, and prevalence of congenital infection
6. Assemble new isolates and biological clones of T. cruzi to map the silvatic vector, silvatic mammal, human and ecological associations of the T. cruzi lineages II, IV, V, VI.
7. Compare lineage specific pathogenesis and transmissibility of congenital infection in a mouse model, and compare lineage susceptibility to drugs in vitro.
Population genetics and phylogenetics:
8. Based on an expanded set of molecular data re-evaluate the population genetics and evolution of T. cruzi lineages.
International cryobank and database:
9. Establish in South America an accessible, expanded, international cryobank for T. cruzi.
10. Establish a website and database for outputs of the project.
The project objectives encompassed the desirable characteristics prescribed by the relevant European Commission (EC) call, in that they included: genomics; effective, innovative relevance to disease, pathogenesis, drugs, interventions; an integrated multidisciplinarity, and capacity building, networking and training in endemic regions.
As this was a complex project with multiple objectives, for clarity the work performed since the beginning of the project and the main results achieved so far are summarised here sequentially for each of the 15 work packages (WPs) that comprised the project. The project ran for four years, with the fourth year being a no additional cost extension. Outputs of the research will emerge beyond the four-year life of the CHAGASEPINET project as data analyses and publications continue to be produced.
Project results:
1. Coordination
The CHAGASEPINET project was coordinated by Professor Michael A. Miles, of the London School of Hygiene and Tropical Medicine, ably assisted by Dr Martin Llewellyn and in the latter stages of the fourth year also by Dr Matthew Yeo. There were 14 other institutional partners, eight of these working in endemic regions in South America, in Bolivia, Venezuela, Argentina, Brazil (2 partners, Goiania and Rio de Janeiro), Colombia, Chile and Ecuador. European partners were in the UK (2 partners, London and Norwich), Belgium (2 partners, Antwerp and Brussels) France, Sweden and Spain. A consortium agreement united the partnership and was valid throughout the project, without amendment. Periodic meetings of the partners rotated between venues and were hosted in Colombia, Bolivia, Ecuador, Goiania, Rio de Janeiro, Antwerp and Paris. Each meeting was preceded by preparation of a detailed agenda and resulted in consensus action points for each partner and for each WP. In addition informal progress reports were provided by the partners between the more formal periodic meetings. Administrative, management and financial support was provided via these meetings to all partners, and when necessary by additional consultation. A mid-term review took place in Paris after the first 18 month period of the project, and progress was commended by external reviewers. A technology transfer workshop was run in Bogota. A second workshop, also planned for Bogota in early 2012 was moved to Goiania, Brazil in September 2012. The reason for this move was to combine a plenary scientific and financial meeting with three weeks of intensive research in the local laboratories, supervised by Gert Auwera and Alex Luquetti, on genotyping of the CHAGASEPINET library of T. cruzi isolates and DNA extracts from chronic Chagas disease. The coordinator organised a follow-up visit to Goiania by Tapan Bhattacharyya for comparative trials of lineage-specific genotyping. A particular task of the coordinator was to organise selection of a panel of reference T. cruzi strains and provision of DNA extracts to relevant partners. This was achieved during the first year of the project, with crucial support from the French partner (Michel Tibayrenc and Christian Barnabé).
A shared website and a shared database were established with support from partners 5 (Bjorn Andersson) and 12 (Alex Luquetti) (see WP15).
2. Polymerase chain reaction/restriction fragment length polymorphisms (PCR-RFLPs)
The second WP was dedicated to the further development of amplification of specific DNA targets for identification of the different T. cruzi lineages, or DTUs. In some cases the amplified DNA (amplicons) provided relevant information directly by the size of the amplicons, in others the products were cut into fragments of discriminatory sizes, when resolved by gel electrophoresis.
In the first phase of the WP, partner 1 and collaborators provided and published a provisional scheme for DTU identification using three targets. Extensive comparisons and cost analyses, principally by partner 2, then led to the selection of an improved panel of 4 PCRs for DTU assignment. These PCRs amplify parts of the gp72, 1f8, histone H3, and gp160 genomic loci. Specificity to T. cruzi was confirmed by testing amplification with human blood and with the related sympatric parasites Leishmania and Trypanosoma rangeli. DTU assignment by PCR-RFLPs was also validated by comparison with MLST (WP3) with a highly congruent a success rate of 99 %. A standard operating procedure (SOP) based on the 4 targets was disseminated to the project partners, and was updated on a regular basis. Trials were also run on DNA extracts of blood / guanidine samples from chronic patients. Results of PCR-RFLP genotyping were subsequently compared with lineage-specific serology (WP6). PCR-RFLP technology was transferred and applied widely for DTU identification by partners working in endemic regions of Bolivia, Venezuela, Argentina, Brazil, Colombia, Chile and Ecuador.
The PCR-RFLP approach provided specific, reliable and highly reproducible identification of the T. cruzi DTUs, transposable to laboratories in endemic regions. Sensitivity for the identification of DTUs directly on blood/guanidine clinical samples was not adequate for genotyping without recovery of live organisms. Alternative amplification methods with targets present as multiple copies in the genome were observed, in trials co-ordinated by partner 2, to lack user-friendly discriminatory power. DTU identification and genotyping methods applied to blood samples are also limited in that they may fail to reveal T. cruzi genotypes sequestered in the many other vulnerable tissues, particularly muscle of the heart and alimentary tract (see WP6).
3. MLST
MLST is essentially a derivative of MLEE. Instead of revealing and comparing the mobility of specific enzymes on electrophoretic gels, in MLST sections of housekeeping genes are amplified by PCR and the DNA is sequenced. The sequences are then aligned and compared. MLST data can provide DTU identification and with appropriate sampling and high-resolution targets can be applied to study population structure, presence of genetic exchange and phylogenetic relationships.
Partner 10 (Patricio Diosque) and partner 1 worked together to develop MLST for T. cruzi. After extensive development and trials of alternative targets a final MLST scheme has been optimised. Fifteen characterised gene fragments were applied to a panel of T. cruzi reference strains encompassing the known lineages. All possible combinations of loci were assessed. One combination of 7 genetic loci (Rb19, TcMPX, HMCOAR, RHO1, GPI, SODB and LAP) was the best according to proposed criteria. The 7 fragments discriminated all reference strains in the panel. A reduced scheme based on 4 gene fragments (MPX, CoAr, Gtp and GPX) also displayed high fidelity (bootstrap) values for all DTUs. The more diverse MLST targets are also applicable to population genetics, detection of genetic exchange and resolution of phylogenetic relationships. In an innovative extension of the project objectives, partner 1 (Louisa Messenger and Martin Llewellyn) devised a parallel mitochondrial MLST (MMLST) scheme with 10 targets. Both MLST and MMLST have been applied in endemic regions, as referred to again below and as described in resultant publications. In the absence of software able to deal conveniently with MLST data of diploid organisms, partner 10 developed MLSTest, a novel Windows based software for MLST data analysis in eukaryotes. The software is freely available at http://www.ipe.edu.ar
4. MLMT
In MLMT tandomly repeated DNA targets, usually dinucleotide or trinucleotide repeats, are amplified from the genome of interest, in this case T. cruzi, and the number of repeats determined by sizing the amplified fragments, usually on an automated DNA sequencer. The size of the fragments can be determined locally or DNA can be sent out for analysis commercially or to a collaborating laboratory. The advantage of MLMT is that microsatellites evolve rapidly and they are ideally suited for high-resolution population genetics analysis and for tracking the origins and spread of particular genotypes. Furthermore, they can be applied to estimate the time of divergence and emergence of genotypes. A large panel of MLMT targets was assessed for their capacity for application to T. cruzi DTUs. From this large panel a subset of targets was selected and simple protocols were optimised. Advanced training in MLMT analysis has been given to research staff from Venezuela, Brazil, Colombia, Mexico, Ecuador and Argentina, visiting partner 1 (London). The fourth year of CHAGASEPINET allowed the MLMT research to come to full fruition, with comparison of genotyping methods and deployment of MLMT to endemic regions. A remarkable finding has been the commonplace lack of congruence between MLMT nuclear genotypes of T. cruzi isolates and their corresponding mitochondrial, mMLST genotypes, as especially seen in research in Colombia (Juan-David Ramirez et al.) but also elsewhere. This phenomenon of inter-lineage mitochondrial introgression, in which the mitochondrial genome is exchanged between strains without apparent nuclear combination, implies that genetic exchange is not rare in natural populations of T. cruzi. In other research employing comparative MLMT and MLST analysis, the hybrid DTUs TcV and VI were studied in depth and compared with putative parental DTUs, TcII and TcIII. This analysis amply confirmed the hybrid nature of TcV and TcVI, indicating that they were derived from two recombination events. Furthermore, dating of the origin of TcV and TcVI and prediction of the origin of their parents, showed that these hybrid DTUs had emerged relatively recently and spread rapidly in the Southern Cone region of South America, possibly in the peridomestic habitat associated with human colonisation. This is consistent with the apparent extreme rarity of TcV and TcVI among silvatic mammals. Among other studies MLMT was applied to in-depth molecular epidemiological analysis of transmission dynamics in Colombia, definition of the origin of two recent oral outbreaks of disease in Venezuela, and the presence of recombination within sylvatic populations of T. cruzi in Bolivia. Some further details are provided in WP9, below, and in a substantial series of publications by the relevant partners.
5. Sequencing and annotation of the TcI genome
Prior to CHAGASEPINET only one T. cruzi genome had been sequenced thoroughly, the CL Brener strain, which is a TcVI hybrid, and therefore has unduly complicated problems of genome assembly but the advantage that aspects of both of the parental TcII and TcIII genomes are revealed within the CL Brener genome. Accordingly, an objective of CHAGASEPINET was to provide the first genome sequence of TcI, the predominant agent of Chagas disease in northen South America and Central America, and putatively associated with a different clinical presentation (absence of megasyndromes) compared to Chagas disease in the Southern Cone region of South America, which is predominantly due to TcII, TcV and TcVI.
The sequencing of the Sylvio X10/1 genome was completed and published ahead of schedule using shotgun 454 sequencing. The sequencing strategy was subsequently improved to include Illumina data. In view of the rapid progress with new high throughput low cost sequencing technology, four additional strains were selected for sequencing. T. cruzi marinkellei, a bat specific T. cruzi, has been sequenced, analysed and published; a TcIV strain from Venezuela and two additional TcI strains have been sequenced, assembled, annotated, and compared. An efficient pipeline for these analyses has been established. The resulting reference genome sequences will facilitate larger comparative genome studies and enhance understanding of the different ecologies and epidemiologies of the T. cruzi DTUs, including pathogenesis and the clinical prognosis of infection.
6. Lineage specific diagnostics
As mentioned above, a disadvantage of genotyping methods based on recovery of live organisms from blood or DNA from blood and the tissues is that they are dependent upon the presence of the required material in the small sample and do not reveal what genotypes may lie elsewhere sequestered at other sites within the host. The new concept of lineage-specific serology, in which serum antibodies to specific DTUs are detected, theoretically not only shows current exposure or infection with a DTU but also may reveal the historical DTU infection history of a patient.
Accordingly, comparative sequence analysis between strains by Partner 5 has been used to identify highly polymorphic genes that might be used as lineage-specific diagnostic tools. Comparative genomics of a small surface antigen (TSSA) allowed lineage-specific peptides to be designed and synthesised, and peptides antigens for TcII, TcIII, TcIV, TcV and TcVI were shown to be promising in experimental trials. In October 2012 Tapan Bhattacharyya worked with Alejandro Luquetti in Goiânia, Brazil, to perform ELISAs using the lineage- specific peptides and sera originating from Brazil, Ecuador, Venezuela, Argentina, and Bolivia. Results encouraged a new collaboration with the American Red cross to apply peptide-based lineage specific serology to their serum bank derived from candidate blood donors. A TcI specific diagnostic peptide has yet to be identified. Partner 13 demonstrated a high degree of TcI TSSA polymorphisms associated with peptide folding. In a separate unforeseen offshoot of this WP a promising pilot study was undertaken in Goiânia to explore biomarkers for distinction of cure and relapse following chemotherapy.
Partner 1 compared lysate antigens and commercial kits, observing that chagasic sera may give different titres with their presumed heterologous and homologous lysate antigens. Glycan profiling (partner 7) found no DTU associated differences in the relative amounts of each sugar nucleotide in the T. cruzi cytsolic pool. The monosaccharide galactose was shown to be present in larger amounts in DM28, ESM and X10 strains, compared to CL-Brener and Y strains.
Partner 13 assayed 13 anti-inflammatory and pro-inflammatory cytokines in sera from adult chagasic patients with infections genotyped as TcI, TcII or mixed TcI-TcII. Results suggested that regulation of anti-inflammatory and pro-inflammatory cytokines has a role in presentation of chagasic cardiomyopathy, and cytokine levels were significantly different in TcI and mixed TcI / TcII.
7. PCR-oligochromatography test
The PCR-oligochromatography test consists of PCR amplification of T. cruzi DNA, followed by detection of the product with a dipstick. Partner 2 dedicated considerable effort to evaluating three alternative tests and producing kits for use in the field. The intention was to have the advantage of high sensitivity combined with ease of use in the field. The alternative tests targetted satellite DNA or kinetoplast (kDNA) minicircles, in the latter case with two assay versions, V2, V3, of which V2 was selected for phase II trials.
Specificity was found to be high for non-endemic controls (100 %) and controls from Chile (> 90 %), but low for a Brazilian cohort of samples (< 50 %). High heterogeneity of sensitivities was observed between the different countries. Repeatability of the evaluated PCR-OC tests was moderate to low. The kDNA OligoC-TesT V2 showed a high sensitivity on the patient specimens from Chile but its sensitivity was lower on the Brazilian patients. No significant difference in sensitivity was observed with the patients from Spain and Argentina (p > 0.05). The overall results of the kDNA OligoC-TesT V2 look slightly better than those of the SatDNA OligoC-TesT, and the difference is statistically significant for the sensitivity (p = 0.038) but not significant for specificity (p > 0.05).
Reluctantly, further development of these tests was shelved pending explanation of inconsistent specificity and sensitivity results with samples from different sources. Additional funding would be required to reassess the performance and potential of these tests.
8. Pilot association studies
The intention of this WP was to undertake a pilot study of T. cruzi genotypes amongst patients with chronic disease from countries, applying both PCR-RFLPs and lineage-specific serology for determination of genotypes.
Goiânia was established as the primary site for assembly of the CHAGASEPINET international reference library for DNA analyses and serology. Partners 12, 3, 2 and others defined clinical groups and SOPs and an EpiInfo database was constructed. In 6 partner countries blood samples have been collected from 720 individuals, 552 with positive serology, 8 with doubtful serology and 160 with negative serology. A modified haemoculture procedure provided a dramatic improvement in the ability to isolate T. cruzi from chronic patients, attaining a tenfold increase in parasite isolation to > 30 %. More than one hundred T. cruzi stocks have been cryopreserved; a bank of more than 1 000 sera from different countries and their DNA counterparts is available. PCR-RFLP genotyping was highly effective with small numbers of organisms in primary cultures but lacked sensitivity for application directly to small volume blood samples. As mentioned above (WP2) alternative PCR-RFLPs based on multicopy targets were not readily and unequivocally interpreted. Lineage-specific serology with synthetic peptides worked remarkably well, with clear positive and negative results; sera from approximately 50% of patients in Goiânia reacted with peptides. Partner 8 processed 465 seropositive samples by RT-PCR, of which 129 were RT-PCR (+) and 334 were RT-PCR (-); 43 of the RT-PCR (+) samples were culture positive. T. cruzi was detected in the cord blood of 20 neonates. Partners 12 and 3 visited Venezuela and Ecuador, in particular to promote surveillance for congenital Chagas disease. As part of the BENEFIT study T. cruzi was genotyped from 240 chronic chagasic patients. The predominance of TcI in Colombia patients was corroborated, with a high prevalence of cardiac alterations. Comparative data from Venezuela on TcI and TcIV suggested an association between pathogenicity and T. cruzi DTU. Data analyses and preparation of further publications derived from this WP are still in progress.
9. Vectors and hosts of TcII, IV, V and VI
The intention of this WP was to provide a wealth of new information on the ecological and host associations of the T. cruzi DTUs. Work was not confined to the formal CHAGASEPINET partnership but included collaborations that arose during the project, most notably with Marta Teixeira in São Paulo, Brazil but also with Central America. In particular we wished to define the natural sylvatic hosts of TcII and TcIV, and whether there were natural sylvatic hosts of the hybrid DTUs TcV and TcVI.
Intensive programmes of field research were undertaken by the partners in endemic countries, in Bolivia, Venezuela, Argentina, Brazil, Colombia, Ecuador and Chile. Partner 8 conducted five major field campaigns along transects spanning the ecotopic diversity of Bolivia (Amazonian lowlands, Andean valleys and northern Chaco region) isolating and genotyping T. cruzi from sylvatic and domestic triatomines, sylvatic mammals, bats and dogs, while partner 4 focused on T. cruzi in sylvatic Triatoma infestans.
Partner 11 initially focused mainly on the Atlantic forest region of Brazil and was able to find TcII in sylvatic mammals and vectors. In 2012 alone, partner 11 undertook 6 field expeditions, including the Altlantic forest and Pantanal regions and enzootic sites with oral outbreaks. All except one species of carnivore in the Pantanal (Corumbá) and Savannah (Araguari) of Brazil were infected with T. cruzi and two procyonids species sustained high parasitaemia. Several genotypes were recovered from these carnivores, which were shown to act as concentration hosts, partially by predation. Domestic dogs in the Amazon region were shown to be useful sentinels to detect the presence of local T. cruzi genotypes. Hosts of TcIV were better understood as a result of collaboration with Marta Teixeira, and were shown to include Amazonian primates.
Partner 13 worked in several field sites in Colombia, with Casanare being of special-interest because of the presence of Rhodnius prolixus in palm trees as well as houses. In Colombia, T. cruzi was isolated and genotyped from cases of chronic Chagas disease and many mammal and triatomine species, revealing heterogeneity in TcI, a common domestic TcI genotype and allowing tracking of the origins of oral outbreaks. Partner 15 continued extensive field work in coastal central Ecuador. Occasional isolates of TcII and TcVI were found in Colombia and / or Ecuador, which may be migrants from further south. Convincing sylvatic hosts and vectors of TcV and TcVI could not be found, supporting work indicating that these hybrid genotypes have arisen recently in the peridomestic environment and spread rapidly through the Gran Chaco region. In Venezuela, TcIV was confirmed as the secondary agent of Chagas disease but local sylvatic hosts are not entirely clear. As in Colombia, the origins of oral outbreaks were tracked in Venezuela. A combination of PCR-RFLPs, MLST and MLMT were applied to analysis of the epidemiology in all these endemic areas, truly benefiting from the South American / European partnership.
It can be seen from the brief summary of this WP that unprecedented progress has been made via CHAGASEPINET in applying molecular epidemiology to understanding the complexities and implications of diverse DTU transmission cycles. It is not possible to do justice to this extensive work here and interested readers are referred to current and ongoing publications of the partners.
10. Comparative pathogenesis
The purpose of this WP was to compare invasion mechanisms of the different T. cruzi DTUs and to see if the susceptibility of different cell types reflected their hosts of origin, for example armadillos as opposed to marsupials, or indicated different issue tropisms. In addition, this was WP provided an opportunity to compare the morphometry of the different DTUs. As data analyses are still in progress details of results will not be given here.
Invasion and intracellular propagation assays and comparative morphometry analyses were completed for T. cruzi strains representing the DTUs, and for T. cruzi marinkellei, in bat, opossum, human and monkey cell lines. Not all T. cruzi strains could propagate in all cell lines, with evidence of DTU specificities. Partner 7 (Madrid) studied all six lineages in IFN-gamma-R1-/- animals, revealing differences between the strains in inflammation in liver and spleen, organ damage, and, with the aid of an improved quantitative PCR (qPCR) assay, in organ tropisms. Differences exist between the T. cruzi DTUs at both the immune response and organ damage levels.
11. Impact on gestation and congenital transmissibility of infection
Congenital transmission of T. cruzi infection is reported to occur in 2 to 5 % of premature births in Bolivia. Serological surveys have indicated that congenital transmission of T. cruzi is much less common in other endemic regions. It is not certain whether the high prevalence of congenital infection in Bolivia is due to the particularly high infestation rate of houses with Triatoma infestans, repeated T. cruzi infection during pregnancy, and high parasitaemias leading to more transfer across the placenta. An alternative explanation is that some T. cruzi DTUs have a greater capacity to cross the placenta than others. It is notable that the vast majority of congenital infections are due to TcV but this might be due to the fact that TcV is more common in Bolivia. Nevertheless, the congenital infection in Brazil appears to be highest in the extreme South, where TcV is the common DTU in human infections. The purpose of this WP was to establish mouse models of congenital disease, to compare the impact of maternal infection with different DTUs on gestation, and to determine whether TcV has a propensity to cross the placenta.
Briefly, partner 3 studied the impact of acute and chronic infection and re-infection on reproductive capacity and gestation, and congenital and breast feeding transmission. The main conclusions were that:
i) whatever the parasite genotype, acute infection after zygote implantation time in the uterus, or close to delivery, prevents or severely prejudices gestation outcome (inducing pup mortality and intra-uterine growth retardation);
ii) whatever the parasite genotype, gestation during chronic infection results in intra-uterine growth retardation, whereas re-inoculation of parasites (TcVI) during gestation in such mice, in addition, strongly increases pup mortality;
iii) in the mouse model congenital infection remains a rare consequence of infection;
iv) PCR is not a convenient method to detect congenital infection close to delivery, possibly because transfer of T. cruzi DNA across the placenta is not coincident with transfer of infection;
v) as in human infections transmission of parasites by breast milk is unlikely. Partner 10 focused on establishing a mouse model for TcV infection and demonstrated that this DTU was capable of transmission congenitally in this system, circumstantially supportive of the fact that TcV is the most common DTU found in association with congenital cases in humans.
12. Comparative susceptibility to drugs
As with other aspects of the biology of T. cruzi and epidemiology Chagas disease the occurrence of the distinct genetic lineages provides a framework for studying susceptibility to drugs. Only two drugs are available for treatment of Chagas disease, benznidazole and nifurtimox. Both of these drugs have significant side effects, particularly in adults, and neither drug guarantees cure. Treatment is recommended for all acute cases, all congenital cases, all immunocompromised cases, and all chronic cases in children and young adults under the age of 16 or 18, because they suffer fewer side effects. However, treatment of adults is more controversial and assessed on a case-by-case basis, for example dependent on likelihood of congenital transmission in women of childbearing age or the presence of symptoms and the ability to tolerate side-effects. The purpose of this WP was to investigate whether genetic lineage of T. cruzi provided a basis for understanding diverse susceptibility to drugs. Recent work has indicated that resistance mechanisms to benznidazole and nifurtimox are shared, and involved loss of a nitroreductase allele but there are also other mechanisms, which are not yet understood.
Partner 1 and collaborators tested susceptibility of DTU reference strains to benznidazole and nifurtimox and found that T. cruzi genetic lineage was not a reliable indicator in vitro of response to these drugs. Furthermore, a range of susceptibility to such drugs could occur within a single DTU. Partner 1 is now evaluating susceptibility of DTUs to posoconazole in vitro, with comparative analyses still in progress. However, partner 7 (Madrid) devised an improved new system for comparing drug susceptibilities and obtained results, now published, indicating that 15 T. cruzi strains representative of the known lineages displayed differential susceptibility to new drugs derived from Streptomyces sp. The same system has now been applied to amphotericin and benznidazole, with detailed findings to be reported. An algorithm that computes summation of drug effects at different concentrations, in conjunction with cluster analysis discriminates the strains into at least four groups resembling the lineage groups of T. cruzi.
Thus DTUs provide one logical framework for discovery of drugs effective against T. cruzi but are not necessarily predictive of response to drugs. The CHAGASEPINET coordinator participated in a DNDi meeting in Rio de Janeiro in October 2012 to advise on the selection of strains for high throughput screening of drugs and for follow-up of promising candidates.
13. Population genetics and phylogenetics
The new technologies developed during CHAGASEPINET and the extensive datasets from diverse endemic regions have enabled population genetics and phylogenetic analyses on an unprecedented size and scale, as published or in preparation. In addition to comparative MLST, mitochondrial MLST and MLMT analyses the application of discriminant analysis of principal components to T. cruzi population genetic data was pioneered. Bayesian estimates of phylogenetic uncertainty have also been incorporated and BEAST software has been applied to date emergence events. Results have supported the validity of the T. cruzi DTUs and fundamentally changed perceptions of relationships between and within DTUs. Thus inter-lineage mitochondrial introgression has been shown to be commonplace, indicating far greater genetic exchange between DTUs than hitherto expected, and extensive intra-lineage genetic change also appears to take place in natural populations of T. cruzi.
Epidemiologically this is profoundly important because it allows the continuous emergence of new genotypes that may differ in potential transmission pathways, virulence and susceptibility to drugs. High resolution analysis within DTUs, particularly by MLMT, has been able to unravel regional transmission pathways, revealing the widespread dispersion of a relatively homogeneous domestic genotype in Venezuela and Colombia, together with invasion of sylvatic strains into domestic cycles. MLMT has also allowed the tracking of sources of oral outbreaks in both Venezuela and Colombia. The relatively recent emergence of the hybrid DTUs TcV and TcVI has been dated and is consistent with their anthropogenic emergence and their lack of sylvatic reservoir hosts. Furthermore, robust dating based on mitochondrial DNA indicates that the emergence of a predominant domestic TcI clade in northern South America occurred 23 000 ± 12 000 years ago.
14. Expanded cryobank
The availability of an international cryobank of well-characterised and documented T. cruzi strains is fundamental to continuity of research effort. The CHAGASEPINET project has enabled expansion of an international T. cruzi cryobank established by partner 11 (Rio de Janeiro) linked to the COLTRYP FIOCRUZ collection. In 2012 alone, more than 100 sylvatic and human isolates were deposited, from carnivores in the Pantanal and Savannah regions of Brazil, Atlantic Forest and Pantanal, triatomines and human isolates from Brazil and Venezuela, with systematic deposition of biological and genotypic data on each T. cruzi strain or clone. An on-line catalogue home page is available at http://www.cria.org.br/~sidnei/fiocruz/coltryp/index. Import and export procedures for deposition/withdrawal of samples have been posted on the CHAGASEPINET website (see http://www.ki.se/CHAGASEPINET online). However, it has not been possible for some partners to transfer their strains for deposition in the cryobank and in this regard further progress is required.
Potential impact:
The socioeconomic and societal impact of CHAGASEPINET is abundantly apparent from the nature of the research undertaken and the outputs summarised above. Thus, technology has been developed specifically in the context of improving understanding of the epidemiology and the control of Chagas disease. Research by all the partners in the endemic regions has been conducted to define transmission pathways and guide better strategies for disease control, for example in showing whether reinvasion is likely after spraying campaigns or monitoring and tracking emergence of new risks. The partners in the endemic regions are either an integral part of or closely affiliated to the public health systems. Thus outputs of this research are and will impel modification of vector control strategies. Awareness and surveillance for congenital Chagas disease have been promoted, for example in Venezuela and Ecuador. Impact has encompassed technological innovation, capacity, understanding, awareness, process, performance, public health and policy. A very important impact has been the cementing of synergistic collaboration between the South American partners and the building of strong mutually supportive interactions between Europe and South America. New partnerships have arisen in South America as a result of the CHAGASEPINET initiative. A few examples of impact are reiterated below:
1. The detailed description within CHAGASEPINET of the ecologies and epidemiologies of the six distinct genetic lineages of T. cruzi (TcI -TcVI) has provided an essential and fundamental platform for all subsequent research on the epidemiology of Chagas disease.
2. The development of techniques such as MLST and MLMT for T. cruzi and their deployment in endemic regions has made widely available the capacity to identify T. cruzi genetic lineages, to unravel local transmission cycles and to plan local control strategies accordingly.
3. In Ecuador population genetics analysis indicated rapid dispersal of domestic / peridomestic T. cruzi and genetic exchange among the domestic/peridomestic T. cruzi strains. Mapping of endemic localities that are predominantly separated from the sylvatic cycle has facilitated vector control.
4. In Venezuela TcI was confirmed as the primary agent of Chagas disease. TcIV is a secondary, putatively more benign disease agent. A relatively homogeneous clone of T. cruzi was shown to be widely dispersed. Nevertheless, sporadic invasion of sylvatic Rhodnius prolixus and T. cruzi strains demanded modified spraying programmes incorporating longer term surveillance. Oral outbreaks were shown to be due to TcI from contaminating sylvatic triatomines. Recommendations were made for the limitation of oral outbreaks.
5. In Colombia complex patterns of Tc1 co-infection were revealed with genetic exchange among strains, congenital transmission and oral outbreaks that involved sylvatic strains.
6. T. cruzi lineage-specific serology with synthetic peptides has been deployed in Goiania, Brazil to identify patients infected with TcII / TcV / TcVI. With David Leiby of the USA, lineage-specific serological screening of Latin American migrants has been successfully trialled to identify high risk blood donors. A feasibility study to screen Latin American migrants to UK has also been conducted.
Dissemination has primarily been by the generation of a large number of publications in scientific journals, and this dissemination is evident from the list of publications provided separately in this final report. Further publications are in preparation. Wider scientific dissemination has been by representation of CHAGASEPINET at many international conferences through oral presentations and multiple poster presentations. In addition, there has been discussion and interaction with policy makers, either directly or indirectly, and media events such as the high profile and TV coverage in Ecuador, in conjunction with the plenary CHAGASEPINET meeting in Quito. Exploitation is essentially as described above; some technological developments might eventually yield products for (non-profit) wide exploitation.
List of websites: The website (see http://www.ki.se/CHAGASEPINET online) was established jointly by partners 5 (Sweden) and 11 (Rio de Janeiro). The website is updated periodically and linked to the cryobank catalogue (see http://www.cria.org.br/~sidnei/fiocruz/coltryp/index online) as well as the CHAGASEPINET sample database (see https://creator.zoho.com/panstrongylus/CHAGASEPINET-sample-database/ online).
Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered to be the most important parasitic disease in Latin America. The epidemiology of Chagas disease is complex, with distinct genetic lineages within the single species T. cruzi. The focus of this ambitious multidisciplinary CHAGASEPINET project, involving 15 research partners, was to apply high-resolution technologies to elucidate the epidemiology of the genetic lineages of T. cruzi, for improved understanding and control of Chagas disease. Achievements include:
1. The development of techniques such as multilocus sequence typing (MLST) and high-resolution microsatellite analysis (MLMT) for T. cruzi and their deployment in endemic regions have made widely available the capacity to identify T. cruzi genetic lineages, to unravel local transmission cycles and to plan local control strategies accordingly. Standardised polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs), MLST and MLMT methods have all been produced, with supplementary methods of data analysis, and technologies transferred, with high-impact outputs. The detailed description within CHAGASEPINET of the ecologies and epidemiologies of the six distinct genetic lineages of T. cruzi (TcI - TcVI) has provided an essential and fundamental platform for all subsequent research on the epidemiology of Chagas disease.
2. The TcI genome has been sequenced and several unforeseen additional genome sequences also provided.
3. A special achievement has been the first development and deployment of peptide based lineage-specific serology.
4. Development of PCR-oligochromatography (PCR-OC) proceeded as planned but it was not deployed to the field pending resolution of inconsistent results from different sources.
5. A large collection of clinical samples has been assembled, and pilot comparisons of genotyping and lineage-specific serology performed.
6. There has been an astonishing combined effort by South American and European partners to unravel in great detail the complex molecular epidemiology of T. cruzi infection, particularly in Bolivia, Brazil, Colombia, Ecuador and Venezuela, including ecological associations of the discrete typing units (DTUs), domestic and sylvatic cycle interactions, congenital transmission and oral outbreaks, also revealing extensive inter-lineage mitochondrial introgression and intra-lineage genetic exchange.
7. Cell invasion, morphometry, pathogenesis, congenital transmission and drug susceptibilities of the DTUs have been compared.
8. Resolution and congruence of DTU identification and population genetics methods have been assessed, with phylogenetics analysis of relationships and evolution.
9. A greatly expanded international cryobank has been established.
CHAGASEPINET has on several objectives exceeded expectation, transforming research capacity and the understanding of Chagas disease. Aspects of the research warrant further funding, for example: continuity and expansion of partnerships on molecular epidemiology, further development of peptide based specific serology, and more sensitive, extensive analysis of infecting genotypes against clinical outcome.
Project context and objectives:
Chagas disease is considered to be the most important parasitic disease in Latin America. The disease is caused by the protozoan Trypanosoma cruzi, which is transmitted primarily by bloodsucking triatomine bugs, not by their bite but by contamination of mucous membranes or abraded skin with the T. cruzi infected bug faeces and urine, deposited on the host as the bugs feed. Other methods of transmission occur, including transfusion of T. cruzi infected blood, transplantation of infected organs, congenital transmission, and oral transmission by contamination of food with infected triatomine faeces.
Control campaigns against triatomine bugs, by spraying infested houses, together with screening of blood and organ donors, have reduced the transmission of T. cruzi and the prevalence of infection in the human population. Nevertheless, it is still considered that 8 to 10 million people in Latin America carry T. cruzi infection. Mortality in the acute phase of infection is around 5 to 10 %, with infants and young adults being the most vulnerable. With rare exceptions, once an individual is infected with T. cruzi they are infected to life, despite the immune response to infection. Approximately 30 % of those who survive the acute phase of infection will progress to severe chronic Chagas disease. The organism multiplies in cells of a wide range of tissues, most notably in terms of pathogenesis, in muscle of the heart and alimentary tract, where both muscle cells and neurones are destroyed. Thus, in chronic Chagas disease there is cardiomyopathy with electrocardiography (ECG) abnormalities, particularly right bundle branch block (RBB) associated with sudden death or progressive congestive heart failure. A proportion of patients with chronic Chagas disease may develop chagasic megaoesophagus and or megacolon, requiring surgery. T. cruzi is also opportunistic, such that carriers of infection who become immunocompromised by human immunodeficiency virus infection / acquired immunodeficiency syndrome (HIV / AIDS) may relapse into the acute phase. Both immunocompromised cases and congenital cases may be associated with meningoencephalitis. There is no vaccine against Chagas disease and no immunotherapy. There are only two drugs for chemotherapy, benznidazole and nifurtimox, which are not always curative, and both may have toxic side-effects, especially when administered to adults.
T. cruzi infection used to be considered to be almost entirely confined to Latin America. However, with increasing global migration from Latin America it is now estimated that 300,000 individuals carry T. cruzi infection in the United States of America (USA) and that tens of thousands of infections are present in several European countries. Cases of blood transfusion transmission and congenital transmission can arise far from Latin America. Thus the World Health Organisation (WHO) has launched a global awareness campaign to enhance recognition of Chagas disease outside the core endemic regions.
The epidemiology of Chagas disease is complex. In some endemic regions the triatomine bug vector species are confined to domestic and peridomestic sites, having arrived by dispersion or carriage from their original sylvatic locations. In other regions the same triatomine species may be present inside and outside the houses, with the threat of reinvasion from sylvatic habitats after spraying programmes. In the Amazon basin and the USA, although T. cruzi infection is present (enzootic) in a wide range of mammals the local bug species do not colonise houses and cases of vector borne Chagas disease are sporadic.
Oral outbreaks occasionally occur in the Amazon region and elsewhere, particularly associated with consumption of triatomine contaminated plant juices.
T. cruzi used to be considered to be a relatively homogeneous entity, and it is still a single species. However, the application of biochemical methods, particularly multilocus enzyme electrophoresis (MLEE) had a remarkable impact on perceptions of the agent of Chagas disease. In a landmark study, in a village in Bahia State Brazil, MLEE demonstrated that in that focus domestic and sylvatic strains were radically distinct, more so than recognized species of the agents of leishmaniasis (Leishmania) species. Follow-up research with MLEE revealed that there are at least six distinct genetic groups or lineages of T. cruzi.
Molecular methods have advanced greatly in the last two decades with the development of higher resolution technologies based on the amplification and analysis of deoxyribonucleic acid (DNA). These technologies have profound potential impact on the understanding of the complex epidemiology of Chagas disease and of other infectious diseases, in the context of strategies for disease prevention and control.
The focus of this ambitious multidisciplinary project, involving 15 research partners, was therefore to elucidate the epidemiology of the genetic lineages of T. cruzi, for improved understanding and control of Chagas disease. The project united skills in genotyping, genomics, genetics and pathogenesis in Europe with considerable compatible skills in South America, and with key research in endemic areas that have distinct characteristics. The project was intended to be high impact in terms of both research progress and the fostering of collaborative networks.
Aim
Elucidate the epidemiology of the genetic lineages of T. cruzi, for improved understanding and prevention of Chagas disease.
Objectives
Technology development:
1. Develop further and apply genotyping technologies including PCR-RFLP typing, MLST and multilocus microsatellite typing (MLMT) to the analysis of genetic populations of T. cruzi, making available improved standardised genotyping methods for routine use in endemic countries.
2. Sequence and annotate the unresolved genome of T. cruzi I.
3. Guided by comparative genomics of T. cruzi I and T. cruzi VI (CL Brener strain), assess lineage-specific diagnosis with the trypomastigote small surface antigen (TSSA) and with other candidate antigens.
4. Develop a rapid PCR-oligochromatography test for detection of T. cruzi infection.
Molecular epidemiology:
5. Pilot studies of association between genetic lineage, clinical outcome, and prevalence of congenital infection
6. Assemble new isolates and biological clones of T. cruzi to map the silvatic vector, silvatic mammal, human and ecological associations of the T. cruzi lineages II, IV, V, VI.
7. Compare lineage specific pathogenesis and transmissibility of congenital infection in a mouse model, and compare lineage susceptibility to drugs in vitro.
Population genetics and phylogenetics:
8. Based on an expanded set of molecular data re-evaluate the population genetics and evolution of T. cruzi lineages.
International cryobank and database:
9. Establish in South America an accessible, expanded, international cryobank for T. cruzi.
10. Establish a website and database for outputs of the project.
The project objectives encompassed the desirable characteristics prescribed by the relevant European Commission (EC) call, in that they included: genomics; effective, innovative relevance to disease, pathogenesis, drugs, interventions; an integrated multidisciplinarity, and capacity building, networking and training in endemic regions.
As this was a complex project with multiple objectives, for clarity the work performed since the beginning of the project and the main results achieved so far are summarised here sequentially for each of the 15 work packages (WPs) that comprised the project. The project ran for four years, with the fourth year being a no additional cost extension. Outputs of the research will emerge beyond the four-year life of the CHAGASEPINET project as data analyses and publications continue to be produced.
Project results:
1. Coordination
The CHAGASEPINET project was coordinated by Professor Michael A. Miles, of the London School of Hygiene and Tropical Medicine, ably assisted by Dr Martin Llewellyn and in the latter stages of the fourth year also by Dr Matthew Yeo. There were 14 other institutional partners, eight of these working in endemic regions in South America, in Bolivia, Venezuela, Argentina, Brazil (2 partners, Goiania and Rio de Janeiro), Colombia, Chile and Ecuador. European partners were in the UK (2 partners, London and Norwich), Belgium (2 partners, Antwerp and Brussels) France, Sweden and Spain. A consortium agreement united the partnership and was valid throughout the project, without amendment. Periodic meetings of the partners rotated between venues and were hosted in Colombia, Bolivia, Ecuador, Goiania, Rio de Janeiro, Antwerp and Paris. Each meeting was preceded by preparation of a detailed agenda and resulted in consensus action points for each partner and for each WP. In addition informal progress reports were provided by the partners between the more formal periodic meetings. Administrative, management and financial support was provided via these meetings to all partners, and when necessary by additional consultation. A mid-term review took place in Paris after the first 18 month period of the project, and progress was commended by external reviewers. A technology transfer workshop was run in Bogota. A second workshop, also planned for Bogota in early 2012 was moved to Goiania, Brazil in September 2012. The reason for this move was to combine a plenary scientific and financial meeting with three weeks of intensive research in the local laboratories, supervised by Gert Auwera and Alex Luquetti, on genotyping of the CHAGASEPINET library of T. cruzi isolates and DNA extracts from chronic Chagas disease. The coordinator organised a follow-up visit to Goiania by Tapan Bhattacharyya for comparative trials of lineage-specific genotyping. A particular task of the coordinator was to organise selection of a panel of reference T. cruzi strains and provision of DNA extracts to relevant partners. This was achieved during the first year of the project, with crucial support from the French partner (Michel Tibayrenc and Christian Barnabé).
A shared website and a shared database were established with support from partners 5 (Bjorn Andersson) and 12 (Alex Luquetti) (see WP15).
2. Polymerase chain reaction/restriction fragment length polymorphisms (PCR-RFLPs)
The second WP was dedicated to the further development of amplification of specific DNA targets for identification of the different T. cruzi lineages, or DTUs. In some cases the amplified DNA (amplicons) provided relevant information directly by the size of the amplicons, in others the products were cut into fragments of discriminatory sizes, when resolved by gel electrophoresis.
In the first phase of the WP, partner 1 and collaborators provided and published a provisional scheme for DTU identification using three targets. Extensive comparisons and cost analyses, principally by partner 2, then led to the selection of an improved panel of 4 PCRs for DTU assignment. These PCRs amplify parts of the gp72, 1f8, histone H3, and gp160 genomic loci. Specificity to T. cruzi was confirmed by testing amplification with human blood and with the related sympatric parasites Leishmania and Trypanosoma rangeli. DTU assignment by PCR-RFLPs was also validated by comparison with MLST (WP3) with a highly congruent a success rate of 99 %. A standard operating procedure (SOP) based on the 4 targets was disseminated to the project partners, and was updated on a regular basis. Trials were also run on DNA extracts of blood / guanidine samples from chronic patients. Results of PCR-RFLP genotyping were subsequently compared with lineage-specific serology (WP6). PCR-RFLP technology was transferred and applied widely for DTU identification by partners working in endemic regions of Bolivia, Venezuela, Argentina, Brazil, Colombia, Chile and Ecuador.
The PCR-RFLP approach provided specific, reliable and highly reproducible identification of the T. cruzi DTUs, transposable to laboratories in endemic regions. Sensitivity for the identification of DTUs directly on blood/guanidine clinical samples was not adequate for genotyping without recovery of live organisms. Alternative amplification methods with targets present as multiple copies in the genome were observed, in trials co-ordinated by partner 2, to lack user-friendly discriminatory power. DTU identification and genotyping methods applied to blood samples are also limited in that they may fail to reveal T. cruzi genotypes sequestered in the many other vulnerable tissues, particularly muscle of the heart and alimentary tract (see WP6).
3. MLST
MLST is essentially a derivative of MLEE. Instead of revealing and comparing the mobility of specific enzymes on electrophoretic gels, in MLST sections of housekeeping genes are amplified by PCR and the DNA is sequenced. The sequences are then aligned and compared. MLST data can provide DTU identification and with appropriate sampling and high-resolution targets can be applied to study population structure, presence of genetic exchange and phylogenetic relationships.
Partner 10 (Patricio Diosque) and partner 1 worked together to develop MLST for T. cruzi. After extensive development and trials of alternative targets a final MLST scheme has been optimised. Fifteen characterised gene fragments were applied to a panel of T. cruzi reference strains encompassing the known lineages. All possible combinations of loci were assessed. One combination of 7 genetic loci (Rb19, TcMPX, HMCOAR, RHO1, GPI, SODB and LAP) was the best according to proposed criteria. The 7 fragments discriminated all reference strains in the panel. A reduced scheme based on 4 gene fragments (MPX, CoAr, Gtp and GPX) also displayed high fidelity (bootstrap) values for all DTUs. The more diverse MLST targets are also applicable to population genetics, detection of genetic exchange and resolution of phylogenetic relationships. In an innovative extension of the project objectives, partner 1 (Louisa Messenger and Martin Llewellyn) devised a parallel mitochondrial MLST (MMLST) scheme with 10 targets. Both MLST and MMLST have been applied in endemic regions, as referred to again below and as described in resultant publications. In the absence of software able to deal conveniently with MLST data of diploid organisms, partner 10 developed MLSTest, a novel Windows based software for MLST data analysis in eukaryotes. The software is freely available at http://www.ipe.edu.ar
4. MLMT
In MLMT tandomly repeated DNA targets, usually dinucleotide or trinucleotide repeats, are amplified from the genome of interest, in this case T. cruzi, and the number of repeats determined by sizing the amplified fragments, usually on an automated DNA sequencer. The size of the fragments can be determined locally or DNA can be sent out for analysis commercially or to a collaborating laboratory. The advantage of MLMT is that microsatellites evolve rapidly and they are ideally suited for high-resolution population genetics analysis and for tracking the origins and spread of particular genotypes. Furthermore, they can be applied to estimate the time of divergence and emergence of genotypes. A large panel of MLMT targets was assessed for their capacity for application to T. cruzi DTUs. From this large panel a subset of targets was selected and simple protocols were optimised. Advanced training in MLMT analysis has been given to research staff from Venezuela, Brazil, Colombia, Mexico, Ecuador and Argentina, visiting partner 1 (London). The fourth year of CHAGASEPINET allowed the MLMT research to come to full fruition, with comparison of genotyping methods and deployment of MLMT to endemic regions. A remarkable finding has been the commonplace lack of congruence between MLMT nuclear genotypes of T. cruzi isolates and their corresponding mitochondrial, mMLST genotypes, as especially seen in research in Colombia (Juan-David Ramirez et al.) but also elsewhere. This phenomenon of inter-lineage mitochondrial introgression, in which the mitochondrial genome is exchanged between strains without apparent nuclear combination, implies that genetic exchange is not rare in natural populations of T. cruzi. In other research employing comparative MLMT and MLST analysis, the hybrid DTUs TcV and VI were studied in depth and compared with putative parental DTUs, TcII and TcIII. This analysis amply confirmed the hybrid nature of TcV and TcVI, indicating that they were derived from two recombination events. Furthermore, dating of the origin of TcV and TcVI and prediction of the origin of their parents, showed that these hybrid DTUs had emerged relatively recently and spread rapidly in the Southern Cone region of South America, possibly in the peridomestic habitat associated with human colonisation. This is consistent with the apparent extreme rarity of TcV and TcVI among silvatic mammals. Among other studies MLMT was applied to in-depth molecular epidemiological analysis of transmission dynamics in Colombia, definition of the origin of two recent oral outbreaks of disease in Venezuela, and the presence of recombination within sylvatic populations of T. cruzi in Bolivia. Some further details are provided in WP9, below, and in a substantial series of publications by the relevant partners.
5. Sequencing and annotation of the TcI genome
Prior to CHAGASEPINET only one T. cruzi genome had been sequenced thoroughly, the CL Brener strain, which is a TcVI hybrid, and therefore has unduly complicated problems of genome assembly but the advantage that aspects of both of the parental TcII and TcIII genomes are revealed within the CL Brener genome. Accordingly, an objective of CHAGASEPINET was to provide the first genome sequence of TcI, the predominant agent of Chagas disease in northen South America and Central America, and putatively associated with a different clinical presentation (absence of megasyndromes) compared to Chagas disease in the Southern Cone region of South America, which is predominantly due to TcII, TcV and TcVI.
The sequencing of the Sylvio X10/1 genome was completed and published ahead of schedule using shotgun 454 sequencing. The sequencing strategy was subsequently improved to include Illumina data. In view of the rapid progress with new high throughput low cost sequencing technology, four additional strains were selected for sequencing. T. cruzi marinkellei, a bat specific T. cruzi, has been sequenced, analysed and published; a TcIV strain from Venezuela and two additional TcI strains have been sequenced, assembled, annotated, and compared. An efficient pipeline for these analyses has been established. The resulting reference genome sequences will facilitate larger comparative genome studies and enhance understanding of the different ecologies and epidemiologies of the T. cruzi DTUs, including pathogenesis and the clinical prognosis of infection.
6. Lineage specific diagnostics
As mentioned above, a disadvantage of genotyping methods based on recovery of live organisms from blood or DNA from blood and the tissues is that they are dependent upon the presence of the required material in the small sample and do not reveal what genotypes may lie elsewhere sequestered at other sites within the host. The new concept of lineage-specific serology, in which serum antibodies to specific DTUs are detected, theoretically not only shows current exposure or infection with a DTU but also may reveal the historical DTU infection history of a patient.
Accordingly, comparative sequence analysis between strains by Partner 5 has been used to identify highly polymorphic genes that might be used as lineage-specific diagnostic tools. Comparative genomics of a small surface antigen (TSSA) allowed lineage-specific peptides to be designed and synthesised, and peptides antigens for TcII, TcIII, TcIV, TcV and TcVI were shown to be promising in experimental trials. In October 2012 Tapan Bhattacharyya worked with Alejandro Luquetti in Goiânia, Brazil, to perform ELISAs using the lineage- specific peptides and sera originating from Brazil, Ecuador, Venezuela, Argentina, and Bolivia. Results encouraged a new collaboration with the American Red cross to apply peptide-based lineage specific serology to their serum bank derived from candidate blood donors. A TcI specific diagnostic peptide has yet to be identified. Partner 13 demonstrated a high degree of TcI TSSA polymorphisms associated with peptide folding. In a separate unforeseen offshoot of this WP a promising pilot study was undertaken in Goiânia to explore biomarkers for distinction of cure and relapse following chemotherapy.
Partner 1 compared lysate antigens and commercial kits, observing that chagasic sera may give different titres with their presumed heterologous and homologous lysate antigens. Glycan profiling (partner 7) found no DTU associated differences in the relative amounts of each sugar nucleotide in the T. cruzi cytsolic pool. The monosaccharide galactose was shown to be present in larger amounts in DM28, ESM and X10 strains, compared to CL-Brener and Y strains.
Partner 13 assayed 13 anti-inflammatory and pro-inflammatory cytokines in sera from adult chagasic patients with infections genotyped as TcI, TcII or mixed TcI-TcII. Results suggested that regulation of anti-inflammatory and pro-inflammatory cytokines has a role in presentation of chagasic cardiomyopathy, and cytokine levels were significantly different in TcI and mixed TcI / TcII.
7. PCR-oligochromatography test
The PCR-oligochromatography test consists of PCR amplification of T. cruzi DNA, followed by detection of the product with a dipstick. Partner 2 dedicated considerable effort to evaluating three alternative tests and producing kits for use in the field. The intention was to have the advantage of high sensitivity combined with ease of use in the field. The alternative tests targetted satellite DNA or kinetoplast (kDNA) minicircles, in the latter case with two assay versions, V2, V3, of which V2 was selected for phase II trials.
Specificity was found to be high for non-endemic controls (100 %) and controls from Chile (> 90 %), but low for a Brazilian cohort of samples (< 50 %). High heterogeneity of sensitivities was observed between the different countries. Repeatability of the evaluated PCR-OC tests was moderate to low. The kDNA OligoC-TesT V2 showed a high sensitivity on the patient specimens from Chile but its sensitivity was lower on the Brazilian patients. No significant difference in sensitivity was observed with the patients from Spain and Argentina (p > 0.05). The overall results of the kDNA OligoC-TesT V2 look slightly better than those of the SatDNA OligoC-TesT, and the difference is statistically significant for the sensitivity (p = 0.038) but not significant for specificity (p > 0.05).
Reluctantly, further development of these tests was shelved pending explanation of inconsistent specificity and sensitivity results with samples from different sources. Additional funding would be required to reassess the performance and potential of these tests.
8. Pilot association studies
The intention of this WP was to undertake a pilot study of T. cruzi genotypes amongst patients with chronic disease from countries, applying both PCR-RFLPs and lineage-specific serology for determination of genotypes.
Goiânia was established as the primary site for assembly of the CHAGASEPINET international reference library for DNA analyses and serology. Partners 12, 3, 2 and others defined clinical groups and SOPs and an EpiInfo database was constructed. In 6 partner countries blood samples have been collected from 720 individuals, 552 with positive serology, 8 with doubtful serology and 160 with negative serology. A modified haemoculture procedure provided a dramatic improvement in the ability to isolate T. cruzi from chronic patients, attaining a tenfold increase in parasite isolation to > 30 %. More than one hundred T. cruzi stocks have been cryopreserved; a bank of more than 1 000 sera from different countries and their DNA counterparts is available. PCR-RFLP genotyping was highly effective with small numbers of organisms in primary cultures but lacked sensitivity for application directly to small volume blood samples. As mentioned above (WP2) alternative PCR-RFLPs based on multicopy targets were not readily and unequivocally interpreted. Lineage-specific serology with synthetic peptides worked remarkably well, with clear positive and negative results; sera from approximately 50% of patients in Goiânia reacted with peptides. Partner 8 processed 465 seropositive samples by RT-PCR, of which 129 were RT-PCR (+) and 334 were RT-PCR (-); 43 of the RT-PCR (+) samples were culture positive. T. cruzi was detected in the cord blood of 20 neonates. Partners 12 and 3 visited Venezuela and Ecuador, in particular to promote surveillance for congenital Chagas disease. As part of the BENEFIT study T. cruzi was genotyped from 240 chronic chagasic patients. The predominance of TcI in Colombia patients was corroborated, with a high prevalence of cardiac alterations. Comparative data from Venezuela on TcI and TcIV suggested an association between pathogenicity and T. cruzi DTU. Data analyses and preparation of further publications derived from this WP are still in progress.
9. Vectors and hosts of TcII, IV, V and VI
The intention of this WP was to provide a wealth of new information on the ecological and host associations of the T. cruzi DTUs. Work was not confined to the formal CHAGASEPINET partnership but included collaborations that arose during the project, most notably with Marta Teixeira in São Paulo, Brazil but also with Central America. In particular we wished to define the natural sylvatic hosts of TcII and TcIV, and whether there were natural sylvatic hosts of the hybrid DTUs TcV and TcVI.
Intensive programmes of field research were undertaken by the partners in endemic countries, in Bolivia, Venezuela, Argentina, Brazil, Colombia, Ecuador and Chile. Partner 8 conducted five major field campaigns along transects spanning the ecotopic diversity of Bolivia (Amazonian lowlands, Andean valleys and northern Chaco region) isolating and genotyping T. cruzi from sylvatic and domestic triatomines, sylvatic mammals, bats and dogs, while partner 4 focused on T. cruzi in sylvatic Triatoma infestans.
Partner 11 initially focused mainly on the Atlantic forest region of Brazil and was able to find TcII in sylvatic mammals and vectors. In 2012 alone, partner 11 undertook 6 field expeditions, including the Altlantic forest and Pantanal regions and enzootic sites with oral outbreaks. All except one species of carnivore in the Pantanal (Corumbá) and Savannah (Araguari) of Brazil were infected with T. cruzi and two procyonids species sustained high parasitaemia. Several genotypes were recovered from these carnivores, which were shown to act as concentration hosts, partially by predation. Domestic dogs in the Amazon region were shown to be useful sentinels to detect the presence of local T. cruzi genotypes. Hosts of TcIV were better understood as a result of collaboration with Marta Teixeira, and were shown to include Amazonian primates.
Partner 13 worked in several field sites in Colombia, with Casanare being of special-interest because of the presence of Rhodnius prolixus in palm trees as well as houses. In Colombia, T. cruzi was isolated and genotyped from cases of chronic Chagas disease and many mammal and triatomine species, revealing heterogeneity in TcI, a common domestic TcI genotype and allowing tracking of the origins of oral outbreaks. Partner 15 continued extensive field work in coastal central Ecuador. Occasional isolates of TcII and TcVI were found in Colombia and / or Ecuador, which may be migrants from further south. Convincing sylvatic hosts and vectors of TcV and TcVI could not be found, supporting work indicating that these hybrid genotypes have arisen recently in the peridomestic environment and spread rapidly through the Gran Chaco region. In Venezuela, TcIV was confirmed as the secondary agent of Chagas disease but local sylvatic hosts are not entirely clear. As in Colombia, the origins of oral outbreaks were tracked in Venezuela. A combination of PCR-RFLPs, MLST and MLMT were applied to analysis of the epidemiology in all these endemic areas, truly benefiting from the South American / European partnership.
It can be seen from the brief summary of this WP that unprecedented progress has been made via CHAGASEPINET in applying molecular epidemiology to understanding the complexities and implications of diverse DTU transmission cycles. It is not possible to do justice to this extensive work here and interested readers are referred to current and ongoing publications of the partners.
10. Comparative pathogenesis
The purpose of this WP was to compare invasion mechanisms of the different T. cruzi DTUs and to see if the susceptibility of different cell types reflected their hosts of origin, for example armadillos as opposed to marsupials, or indicated different issue tropisms. In addition, this was WP provided an opportunity to compare the morphometry of the different DTUs. As data analyses are still in progress details of results will not be given here.
Invasion and intracellular propagation assays and comparative morphometry analyses were completed for T. cruzi strains representing the DTUs, and for T. cruzi marinkellei, in bat, opossum, human and monkey cell lines. Not all T. cruzi strains could propagate in all cell lines, with evidence of DTU specificities. Partner 7 (Madrid) studied all six lineages in IFN-gamma-R1-/- animals, revealing differences between the strains in inflammation in liver and spleen, organ damage, and, with the aid of an improved quantitative PCR (qPCR) assay, in organ tropisms. Differences exist between the T. cruzi DTUs at both the immune response and organ damage levels.
11. Impact on gestation and congenital transmissibility of infection
Congenital transmission of T. cruzi infection is reported to occur in 2 to 5 % of premature births in Bolivia. Serological surveys have indicated that congenital transmission of T. cruzi is much less common in other endemic regions. It is not certain whether the high prevalence of congenital infection in Bolivia is due to the particularly high infestation rate of houses with Triatoma infestans, repeated T. cruzi infection during pregnancy, and high parasitaemias leading to more transfer across the placenta. An alternative explanation is that some T. cruzi DTUs have a greater capacity to cross the placenta than others. It is notable that the vast majority of congenital infections are due to TcV but this might be due to the fact that TcV is more common in Bolivia. Nevertheless, the congenital infection in Brazil appears to be highest in the extreme South, where TcV is the common DTU in human infections. The purpose of this WP was to establish mouse models of congenital disease, to compare the impact of maternal infection with different DTUs on gestation, and to determine whether TcV has a propensity to cross the placenta.
Briefly, partner 3 studied the impact of acute and chronic infection and re-infection on reproductive capacity and gestation, and congenital and breast feeding transmission. The main conclusions were that:
i) whatever the parasite genotype, acute infection after zygote implantation time in the uterus, or close to delivery, prevents or severely prejudices gestation outcome (inducing pup mortality and intra-uterine growth retardation);
ii) whatever the parasite genotype, gestation during chronic infection results in intra-uterine growth retardation, whereas re-inoculation of parasites (TcVI) during gestation in such mice, in addition, strongly increases pup mortality;
iii) in the mouse model congenital infection remains a rare consequence of infection;
iv) PCR is not a convenient method to detect congenital infection close to delivery, possibly because transfer of T. cruzi DNA across the placenta is not coincident with transfer of infection;
v) as in human infections transmission of parasites by breast milk is unlikely. Partner 10 focused on establishing a mouse model for TcV infection and demonstrated that this DTU was capable of transmission congenitally in this system, circumstantially supportive of the fact that TcV is the most common DTU found in association with congenital cases in humans.
12. Comparative susceptibility to drugs
As with other aspects of the biology of T. cruzi and epidemiology Chagas disease the occurrence of the distinct genetic lineages provides a framework for studying susceptibility to drugs. Only two drugs are available for treatment of Chagas disease, benznidazole and nifurtimox. Both of these drugs have significant side effects, particularly in adults, and neither drug guarantees cure. Treatment is recommended for all acute cases, all congenital cases, all immunocompromised cases, and all chronic cases in children and young adults under the age of 16 or 18, because they suffer fewer side effects. However, treatment of adults is more controversial and assessed on a case-by-case basis, for example dependent on likelihood of congenital transmission in women of childbearing age or the presence of symptoms and the ability to tolerate side-effects. The purpose of this WP was to investigate whether genetic lineage of T. cruzi provided a basis for understanding diverse susceptibility to drugs. Recent work has indicated that resistance mechanisms to benznidazole and nifurtimox are shared, and involved loss of a nitroreductase allele but there are also other mechanisms, which are not yet understood.
Partner 1 and collaborators tested susceptibility of DTU reference strains to benznidazole and nifurtimox and found that T. cruzi genetic lineage was not a reliable indicator in vitro of response to these drugs. Furthermore, a range of susceptibility to such drugs could occur within a single DTU. Partner 1 is now evaluating susceptibility of DTUs to posoconazole in vitro, with comparative analyses still in progress. However, partner 7 (Madrid) devised an improved new system for comparing drug susceptibilities and obtained results, now published, indicating that 15 T. cruzi strains representative of the known lineages displayed differential susceptibility to new drugs derived from Streptomyces sp. The same system has now been applied to amphotericin and benznidazole, with detailed findings to be reported. An algorithm that computes summation of drug effects at different concentrations, in conjunction with cluster analysis discriminates the strains into at least four groups resembling the lineage groups of T. cruzi.
Thus DTUs provide one logical framework for discovery of drugs effective against T. cruzi but are not necessarily predictive of response to drugs. The CHAGASEPINET coordinator participated in a DNDi meeting in Rio de Janeiro in October 2012 to advise on the selection of strains for high throughput screening of drugs and for follow-up of promising candidates.
13. Population genetics and phylogenetics
The new technologies developed during CHAGASEPINET and the extensive datasets from diverse endemic regions have enabled population genetics and phylogenetic analyses on an unprecedented size and scale, as published or in preparation. In addition to comparative MLST, mitochondrial MLST and MLMT analyses the application of discriminant analysis of principal components to T. cruzi population genetic data was pioneered. Bayesian estimates of phylogenetic uncertainty have also been incorporated and BEAST software has been applied to date emergence events. Results have supported the validity of the T. cruzi DTUs and fundamentally changed perceptions of relationships between and within DTUs. Thus inter-lineage mitochondrial introgression has been shown to be commonplace, indicating far greater genetic exchange between DTUs than hitherto expected, and extensive intra-lineage genetic change also appears to take place in natural populations of T. cruzi.
Epidemiologically this is profoundly important because it allows the continuous emergence of new genotypes that may differ in potential transmission pathways, virulence and susceptibility to drugs. High resolution analysis within DTUs, particularly by MLMT, has been able to unravel regional transmission pathways, revealing the widespread dispersion of a relatively homogeneous domestic genotype in Venezuela and Colombia, together with invasion of sylvatic strains into domestic cycles. MLMT has also allowed the tracking of sources of oral outbreaks in both Venezuela and Colombia. The relatively recent emergence of the hybrid DTUs TcV and TcVI has been dated and is consistent with their anthropogenic emergence and their lack of sylvatic reservoir hosts. Furthermore, robust dating based on mitochondrial DNA indicates that the emergence of a predominant domestic TcI clade in northern South America occurred 23 000 ± 12 000 years ago.
14. Expanded cryobank
The availability of an international cryobank of well-characterised and documented T. cruzi strains is fundamental to continuity of research effort. The CHAGASEPINET project has enabled expansion of an international T. cruzi cryobank established by partner 11 (Rio de Janeiro) linked to the COLTRYP FIOCRUZ collection. In 2012 alone, more than 100 sylvatic and human isolates were deposited, from carnivores in the Pantanal and Savannah regions of Brazil, Atlantic Forest and Pantanal, triatomines and human isolates from Brazil and Venezuela, with systematic deposition of biological and genotypic data on each T. cruzi strain or clone. An on-line catalogue home page is available at http://www.cria.org.br/~sidnei/fiocruz/coltryp/index. Import and export procedures for deposition/withdrawal of samples have been posted on the CHAGASEPINET website (see http://www.ki.se/CHAGASEPINET online). However, it has not been possible for some partners to transfer their strains for deposition in the cryobank and in this regard further progress is required.
Potential impact:
The socioeconomic and societal impact of CHAGASEPINET is abundantly apparent from the nature of the research undertaken and the outputs summarised above. Thus, technology has been developed specifically in the context of improving understanding of the epidemiology and the control of Chagas disease. Research by all the partners in the endemic regions has been conducted to define transmission pathways and guide better strategies for disease control, for example in showing whether reinvasion is likely after spraying campaigns or monitoring and tracking emergence of new risks. The partners in the endemic regions are either an integral part of or closely affiliated to the public health systems. Thus outputs of this research are and will impel modification of vector control strategies. Awareness and surveillance for congenital Chagas disease have been promoted, for example in Venezuela and Ecuador. Impact has encompassed technological innovation, capacity, understanding, awareness, process, performance, public health and policy. A very important impact has been the cementing of synergistic collaboration between the South American partners and the building of strong mutually supportive interactions between Europe and South America. New partnerships have arisen in South America as a result of the CHAGASEPINET initiative. A few examples of impact are reiterated below:
1. The detailed description within CHAGASEPINET of the ecologies and epidemiologies of the six distinct genetic lineages of T. cruzi (TcI -TcVI) has provided an essential and fundamental platform for all subsequent research on the epidemiology of Chagas disease.
2. The development of techniques such as MLST and MLMT for T. cruzi and their deployment in endemic regions has made widely available the capacity to identify T. cruzi genetic lineages, to unravel local transmission cycles and to plan local control strategies accordingly.
3. In Ecuador population genetics analysis indicated rapid dispersal of domestic / peridomestic T. cruzi and genetic exchange among the domestic/peridomestic T. cruzi strains. Mapping of endemic localities that are predominantly separated from the sylvatic cycle has facilitated vector control.
4. In Venezuela TcI was confirmed as the primary agent of Chagas disease. TcIV is a secondary, putatively more benign disease agent. A relatively homogeneous clone of T. cruzi was shown to be widely dispersed. Nevertheless, sporadic invasion of sylvatic Rhodnius prolixus and T. cruzi strains demanded modified spraying programmes incorporating longer term surveillance. Oral outbreaks were shown to be due to TcI from contaminating sylvatic triatomines. Recommendations were made for the limitation of oral outbreaks.
5. In Colombia complex patterns of Tc1 co-infection were revealed with genetic exchange among strains, congenital transmission and oral outbreaks that involved sylvatic strains.
6. T. cruzi lineage-specific serology with synthetic peptides has been deployed in Goiania, Brazil to identify patients infected with TcII / TcV / TcVI. With David Leiby of the USA, lineage-specific serological screening of Latin American migrants has been successfully trialled to identify high risk blood donors. A feasibility study to screen Latin American migrants to UK has also been conducted.
Dissemination has primarily been by the generation of a large number of publications in scientific journals, and this dissemination is evident from the list of publications provided separately in this final report. Further publications are in preparation. Wider scientific dissemination has been by representation of CHAGASEPINET at many international conferences through oral presentations and multiple poster presentations. In addition, there has been discussion and interaction with policy makers, either directly or indirectly, and media events such as the high profile and TV coverage in Ecuador, in conjunction with the plenary CHAGASEPINET meeting in Quito. Exploitation is essentially as described above; some technological developments might eventually yield products for (non-profit) wide exploitation.
List of websites: The website (see http://www.ki.se/CHAGASEPINET online) was established jointly by partners 5 (Sweden) and 11 (Rio de Janeiro). The website is updated periodically and linked to the cryobank catalogue (see http://www.cria.org.br/~sidnei/fiocruz/coltryp/index online) as well as the CHAGASEPINET sample database (see https://creator.zoho.com/panstrongylus/CHAGASEPINET-sample-database/ online).
