Chagas disease control-surveillance in the Americas: the multinational initiatives and the practical impossibility of interrupting vector-borne Trypanosoma cruzi transmission.

Chagas disease (CD) still imposes a heavy burden on most Latin American countries. Vector-borne and mother-to-child transmission cause several thousand new infections per year, and at least 5 million people carry Trypanosoma cruzi. Access to diagnosis and medical care, however, is far from universal. Starting in the 1990s, CD-endemic countries and the Pan American Health Organization-World Health Organization (PAHO-WHO) launched a series of multinational initiatives for CD control-surveillance. An overview of the initiatives' aims, achievements, and challenges reveals some key common themes that we discuss here in the context of the WHO 2030 goals for CD. Transmission of T. cruzi via blood transfusion and organ transplantation is effectively under control. T. cruzi, however, is a zoonotic pathogen with 100+ vector species widely spread across the Americas; interrupting vector-borne transmission seems therefore unfeasible. Stronger surveillance systems are, and will continue to be, needed to monitor and control CD. Prevention of vertical transmission demands boosting current efforts to screen pregnant and childbearing-aged women. Finally, integral patient care is a critical unmet need in most countries. The decades-long experience of the initiatives, in sum, hints at the practical impossibility of interrupting vector-borne T. cruzi transmission in the Americas. The concept of disease control seems to provide a more realistic description of what can in effect be achieved by 2030.

Chagas disease in the context of the 2030 agenda: global warming and vectors.

The 2030 Agenda for Sustainable Development is a plan of action for people, planet and prosperity. Thousands of years and centuries of colonisation have passed the precarious housing conditions, food insecurity, lack of sanitation, the limitation of surveillance, health care programs and climate change. Chagas disease continues to be a public health problem. The control programs have been successful in many countries in reducing transmission by T. cruzi; but the results have been variable. WHO makes recommendations for prevention and control with the aim of eliminating Chagas disease as a public health problem. Climate change, deforestation, migration, urbanisation, sylvatic vectors and oral transmission require integrating the economic, social, and environmental dimensions of sustainable development, as well as the links within and between objectives and sectors. While the environment scenarios change around the world, native vector species pose a significant public health threat. The man-made atmosphere change is related to the increase of triatomines' dispersal range, or an increase of the mobility of the vectors from their sylvatic environment to man-made constructions, or humans getting into sylvatic scenarios, leading to an increase of Chagas disease infection. Innovations with the communities and collaborations among municipalities, International cooperation agencies, local governmental agencies, academic partners, developmental agencies, or environmental institutions may present promising solutions, but sustained partnerships, long-term commitment, and strong regional leadership are required. A new world has just opened up for the renewal of surveillance practices, but the lessons learned in the past should be the basis for solutions in the future.

Chagas disease in the context of the 2030 agenda: global warming and vectors

The 2030 Agenda for Sustainable Development is a plan of action for people, planet and prosperity. Thousands of years and centuries of colonisation have passed the precarious housing conditions, food insecurity, lack of sanitation, the limitation of surveillance, health care programs and climate change. Chagas disease continues to be a public health problem. The control programs have been successful in many countries in reducing transmission by T. cruzi; but the results have been variable. WHO makes recommendations for prevention and control with the aim of eliminating Chagas disease as a public health problem. Climate change, deforestation, migration, urbanisation, sylvatic vectors and oral transmission require integrating the economic, social, and environmental dimensions of sustainable development, as well as the links within and between objectives and sectors. While the environment scenarios change around the world, native vector species pose a significant public health threat. The man-made atmosphere change is related to the increase of triatomines' dispersal range, or an increase of the mobility of the vectors from their sylvatic environment to man-made constructions, or humans getting into sylvatic scenarios, leading to an increase of Chagas disease infection. Innovations with the communities and collaborations among municipalities, International cooperation agencies, local governmental agencies, academic partners, developmental agencies, or environmental institutions may present promising solutions, but sustained partnerships, long-term commitment, and strong regional leadership are required. A new world has just opened up for the renewal of surveillance practices, but the lessons learned in the past should be the basis for solutions in the future.

Chagas disease control-surveillance in the Americas: the multinational initiatives and the practical impossibility of interrupting vector-borne Trypanosoma cruzi transmission

Chagas disease (CD) still imposes a heavy burden on most Latin American countries. Vector-borne and mother-to-child transmission cause several thousand new infections per year, and at least 5 million people carry Trypanosoma cruzi. Access to diagnosis and medical care, however, is far from universal. Starting in the 1990s, CD-endemic countries and the Pan American Health Organization-World Health Organization (PAHO-WHO) launched a series of multinational initiatives for CD control-surveillance. An overview of the initiatives' aims, achievements, and challenges reveals some key common themes that we discuss here in the context of the WHO 2030 goals for CD. Transmission of T. cruzi via blood transfusion and organ transplantation is effectively under control. T. cruzi, however, is a zoonotic pathogen with 100+ vector species widely spread across the Americas; interrupting vector-borne transmission seems therefore unfeasible. Stronger surveillance systems are, and will continue to be, needed to monitor and control CD. Prevention of vertical transmission demands boosting current efforts to screen pregnant and childbearing-aged women. Finally, integral patient care is a critical unmet need in most countries. The decades-long experience of the initiatives, in sum, hints at the practical impossibility of interrupting vector-borne T. cruzi transmission in the Americas. The concept of disease control seems to provide a more realistic description of what can in effect be achieved by 2030.

Infestation dynamics of Triatoma dimidiata in highly deforested tropical dry forest regions of Guatemala.

BACKGROUND: Deforestation, driven by anthropogenic change in land use, influences the behaviour and abundance of vector-borne diseases. For various species of Chagas disease vectors, there is evidence that change in land use affects population density and abundance. Triatoma dimidiata is the most important Chagas vector in Guatemala, and at least one million people live in T. dimidiata endemic areas; however, infestation dynamics vary among regions, from high infestation with all life stages to low seasonal infestation by sylvatic adults. OBJECTIVES: The aim of this study was to evaluate how land-use, combined with domiciliary risk factors, influences the infestation dynamics of T. dimidiata for four villages in a dry forest region with a strong deforestation history. METHODS: Land use, measured with drone and satellite images, was classified into four categories (houses, monocultures and pastures, woodland and shrubland, and bare soil). Domiciliary risk factors and infestation were assessed through entomological surveys. Statistical analyses compared infestation indices and the ability of land use and domiciliary risk factors to explain infestation. FINDINGS: Two villages had significantly higher infestation (26 and 30% vs. 5 and 6%), yet all villages had high colonisation (71-100% of infested houses had immature insects), with no significant difference among them. Because of the high level of deforestation across the study area, land use was not related to infestation; however, domiciliary risk factors were. A model based on four weighted domiciliary risk factors (adobe or bajareque walls, intradomicile animals, intradomicile clutter, and dirt floors) explains the infestation risk. MAIN CONCLUSIONS: Because almost all infested houses have reproducing populations in this deforested dry forest region and statistical analysis identified the domiciliary risk factors for infestation, intermediate and long-term control of Chagas disease vectors in this region requires management of these risk factors.

Novel Evolutionary Algorithm Identifies Interactions Driving Infestation of Triatoma dimidiata, a Chagas Disease Vector.

Chagas disease is a lethal, neglected tropical disease. Unfortunately, aggressive insecticide-spraying campaigns have not been able to eliminate domestic infestation of Triatoma dimidiata, the native vector in Guatemala. To target interventions toward houses most at risk of infestation, comprehensive socioeconomic and entomologic surveys were conducted in two towns in Jutiapa, Guatemala. Given the exhaustively large search space associated with combinations of risk factors, traditional statistics are limited in their ability to discover risk factor interactions. Two recently developed statistical evolutionary algorithms, specifically designed to accommodate risk factor interactions and heterogeneity, were applied to this large combinatorial search space and used in tandem to identify sets of risk factor combinations associated with infestation. The optimal model includes 10 risk factors in what is known as a third-order disjunctive normal form (i.e., infested households have chicken coops AND deteriorated bedroom walls OR an accumulation of objects AND dirt floors AND total number of occupants ≥ 5 AND years of electricity ≥ 5 OR poor hygienic condition ratings AND adobe walls AND deteriorated walls AND dogs). Houses with dirt floors and deteriorated walls have been reported previously as risk factors and align well with factors currently targeted by Ecohealth interventions to minimize infestation. However, the tandem evolutionary algorithms also identified two new socioeconomic risk factors (i.e., households having many occupants and years of electricity ≥ 5). Identifying key risk factors may help with the development of new Ecohealth interventions and/or reduce the survey time needed to identify houses most at risk.

Common pattern of distribution for Mesoamerican Triatoma dimidiata suggest geological and ecological association.

The phylogeny of the Triatoma dimidiata complex has been widely assessed with different genetic and morphological data, which has allowed to reach the consensus that the complex consists of at least three taxonomic units. However, these taxonomic units seem to have a distribution related to geography throughout Mesoamerica, with different groupings depending on the source of information used. In the present study, we aimed to determine if there is a common biogeographical, genetic and phenetic distribution pattern among the T. dimidiata species in Mesoamerica and if this pattern is related to ecological and geological variability of the region. We found that panbiogeographical analysis showed three generalized tracks that coincide with genetic/phenetic data which showed a general pattern of distribution in two big clusters to the north and south of Mesoamerica. We also found that these clusters were significantly related to geological tectonic plates and ecotypes. We conclude that the geological history may be a plausible explanation for the greater differentiation observed in the T. dimidiata complex, but that the current ecological characteristics of the morphotectonic units or ecotypes may be responsible for the additional variation observed and therefore differential control strategies for each cluster considering geological history and ecotype should be used. Further, more detailed biogeographical and landscape genetic analyses are necessary with the goal to elucidate T. dimidiata differentiation related with ecological and geological variables in the region and the possible epidemiological and evolutionary consequences.

Infestation dynamics of Triatoma dimidiata in highly deforested tropical dry forest regions of Guatemala

BACKGROUND Deforestation, driven by anthropogenic change in land use, influences the behaviour and abundance of vector-borne diseases. For various species of Chagas disease vectors, there is evidence that change in land use affects population density and abundance. Triatoma dimidiata is the most important Chagas vector in Guatemala, and at least one million people live in T. dimidiata endemic areas; however, infestation dynamics vary among regions, from high infestation with all life stages to low seasonal infestation by sylvatic adults. OBJECTIVES The aim of this study was to evaluate how land-use, combined with domiciliary risk factors, influences the infestation dynamics of T. dimidiata for four villages in a dry forest region with a strong deforestation history. METHODS Land use, measured with drone and satellite images, was classified into four categories (houses, monocultures and pastures, woodland and shrubland, and bare soil). Domiciliary risk factors and infestation were assessed through entomological surveys. Statistical analyses compared infestation indices and the ability of land use and domiciliary risk factors to explain infestation. FINDINGS Two villages had significantly higher infestation (26 and 30% vs. 5 and 6%), yet all villages had high colonisation (71-100% of infested houses had immature insects), with no significant difference among them. Because of the high level of deforestation across the study area, land use was not related to infestation; however, domiciliary risk factors were. A model based on four weighted domiciliary risk factors (adobe or bajareque walls, intradomicile animals, intradomicile clutter, and dirt floors) explains the infestation risk. MAIN CONCLUSIONS Because almost all infested houses have reproducing populations in this deforested dry forest region and statistical analysis identified the domiciliary risk factors for infestation, intermediate and long-term control of Chagas disease vectors in this region requires management of these risk factors.

Residual survival and local dispersal drive reinfestation by Triatoma dimidiata following insecticide application in Guatemala.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and transmitted by triatomine insect vectors. In Guatemala, insecticide spraying is an integral part of management of the main vector, Triatoma dimidiata. Spraying typically has low efficacy, which may be due to incomplete elimination from infested houses, within-village dispersal, or influx from other villages or sylvan environments. To evaluate how these mechanisms contribute to reinfestation, we conducted a time-course analysis of T. dimidiata infestation, abundance and household genetic structure in two nearby villages in Jutiapa, Guatemala; houses in the first village were surveyed, treated with insecticide if infested and then re-surveyed at eight and 22 months following spraying, while the second village served as an untreated control to quantify changes associated with seasonal dispersal. Insects were genotyped at 2-3000 SNP loci for kinship and population genetic analyses. Insecticide application reduced overall infestation and abundance, while the untreated village was stable over time. Nevertheless, within two years 35.5% of treated houses were reinfested and genetic diversity had largely recovered. Insects collected from reinfested houses post-spraying were most closely related to pre-spray collections from the same house, suggesting that infestations had not been fully eliminated. Immigration by unrelated insects was also detected within a year of spraying; when it occurred, dispersal was primarily local from neighboring houses. Similar dispersal patterns were observed following the annual dispersal season in the untreated village, with high-infestation houses serving as sources for neighboring homes. Our findings suggest that the efficacy of pyrethroid application is rapidly diminished by both within-house breeding by survivors and annual cycles of among-house movement. Given these patterns, we conclude that house structural improvements, an integral part of the Ecohealth approach that makes houses refractory to vector colonization and persistence, are critical for long-term reduction of T. dimidiata infestation.

Uncovering vector, parasite, blood meal and microbiome patterns from mixed-DNA specimens of the Chagas disease vector Triatoma dimidiata.

Chagas disease, considered a neglected disease by the World Health Organization, is caused by the protozoan parasite Trypanosoma cruzi, and transmitted by >140 triatomine species across the Americas. In Central America, the main vector is Triatoma dimidiata, an opportunistic blood meal feeder inhabiting both domestic and sylvatic ecotopes. Given the diversity of interacting biological agents involved in the epidemiology of Chagas disease, having simultaneous information on the dynamics of the parasite, vector, the gut microbiome of the vector, and the blood meal source would facilitate identifying key biotic factors associated with the risk of T. cruzi transmission. In this study, we developed a RADseq-based analysis pipeline to study mixed-species DNA extracted from T. dimidiata abdomens. To evaluate the efficacy of the method across spatial scales, we used a nested spatial sampling design that spanned from individual villages within Guatemala to major biogeographic regions of Central America. Information from each biotic source was distinguished with bioinformatics tools and used to evaluate the prevalence of T. cruzi infection and predominant Discrete Typing Units (DTUs) in the region, the population genetic structure of T. dimidiata, gut microbial diversity, and the blood meal history. An average of 3.25 million reads per specimen were obtained, with approximately 1% assigned to the parasite, 20% to the vector, 11% to bacteria, and 4% to putative blood meals. Using a total of 6,405 T. cruzi SNPs, we detected nine infected vectors harboring two distinct DTUs: TcI and a second unidentified strain, possibly TcIV. Vector specimens were sufficiently variable for population genomic analyses, with a total of 25,710 T. dimidiata SNPs across all samples that were sufficient to detect geographic genetic structure at both local and regional scales. We observed a diverse microbiotic community, with significantly higher bacterial species richness in infected T. dimidiata abdomens than those that were not infected. Unifrac analysis suggests a common assemblage of bacteria associated with infection, which co-occurs with the typical gut microbial community derived from the local environment. We identified vertebrate blood meals from five T. dimidiata abdomens, including chicken, dog, duck and human; however, additional detection methods would be necessary to confidently identify blood meal sources from most specimens. Overall, our study shows this method is effective for simultaneously generating genetic data on vectors and their associated parasites, along with ecological information on feeding patterns and microbial interactions that may be followed up with complementary approaches such as PCR-based parasite detection, 18S eukaryotic and 16S bacterial barcoding.

Description of Triatoma mopan sp. n. from a cave in Belize (Hemiptera, Reduviidae, Triatominae).

In this paper, Triatoma mopansp. n. is described based on five males and six females collected in the Rio Frio cave, Cayo District, Belize. This species is similar to Triatoma dimidiata (Latreille), but can be distinguished by characters found on the pronotum, legs, and abdomen. Geometric morphometry and phylogenetic comparisons are also provided. Presently, the species is known only from the type locality and is a potential Chagas vector.

Vectors of diversity: Genome wide diversity across the geographic range of the Chagas disease vector Triatoma dimidiata sensu lato (Hemiptera: Reduviidae).

To date, the phylogeny of Triatoma dimidiata sensu lato (s. l.) (Hemiptera: Reduviidae: Triatominae), the epidemiologically most important Chagas disease vector in Central America and a secondary vector in Mexico and northern South America, has only been investigated by one multi-copy nuclear gene (Internal Transcribed Spacer - 2) and a few mitochondrial genes. We examined 450 specimens sampled across most of its native range from Mexico to Ecuador using reduced representation next-generation sequencing encompassing over 16,000 single nucleotide polymorphisms (SNPs). Using a combined phylogenetic and species delimitation approach we uncovered two distinct species, as well as a well-defined third group that may contain multiple species. The findings are discussed with respect to possible drivers of diversification and the epidemiological importance of the distinct species and groups.

High seroconversion rates in Trypanosoma cruzi chronic infection treated with benznidazole in people under 16 years in Guatemala.

INTRODUCTION:: Geographical, epidemiological, and environmental differences associated with therapeutic response to Chagas etiological treatment have been previously discussed. This study describes high seroconversion rates 72 months after benznidazole treatment in patients under 16 years from a project implemented by Doctors without Borders in Guatemala. METHODS:: An enzyme-linked immunosorbent assay was used to detect Trypanosoma cruzi IgG antibodies in capillary blood samples from patients 72 months after treatment. Fisher's exact test was used to establish association between characteristics, such as sex, age, and origin of patients, and final seroconversion. Kappa index determined concordance between laboratory tests. The level of significance was set to 5%. RESULTS:: Ninety-eight patients, aged 6 months to 16 years, were available for follow-up. Sex and origin were not associated with seroconversion. Individuals older than 13 were more prone to maintain a positive result 72 months after treatment, although results were not highly significant. Laboratory tests presented elevated Kappa concordance (95% CI) = 0.8290 (0.4955-1), as well as high (97%) seroconversion rates. CONCLUSIONS:: The high seroconversion rate found in this study emphasizes the importance of access to diagnosis, treatment, and follow-up of individuals affected by Chagas disease. Moreover, it contradicts the idea that it is not possible to achieve a cure with the currently available drugs. This study strongly supports expanding programs for patients infected with T. cruzi in endemic and non-endemic countries.

High seroconversion rates in Trypanosoma cruzi chronic infection treated with benznidazole in people under 16 years in Guatemala

Abstract INTRODUCTION: Geographical, epidemiological, and environmental differences associated with therapeutic response to Chagas etiological treatment have been previously discussed. This study describes high seroconversion rates 72 months after benznidazole treatment in patients under 16 years from a project implemented by Doctors without Borders in Guatemala. METHODS: An enzyme-linked immunosorbent assay was used to detect Trypanosoma cruzi IgG antibodies in capillary blood samples from patients 72 months after treatment. Fisher's exact test was used to establish association between characteristics, such as sex, age, and origin of patients, and final seroconversion. Kappa index determined concordance between laboratory tests. The level of significance was set to 5%. RESULTS: Ninety-eight patients, aged 6 months to 16 years, were available for follow-up. Sex and origin were not associated with seroconversion. Individuals older than 13 were more prone to maintain a positive result 72 months after treatment, although results were not highly significant. Laboratory tests presented elevated Kappa concordance (95% CI) = 0.8290 (0.4955-1), as well as high (97%) seroconversion rates. CONCLUSIONS: The high seroconversion rate found in this study emphasizes the importance of access to diagnosis, treatment, and follow-up of individuals affected by Chagas disease. Moreover, it contradicts the idea that it is not possible to achieve a cure with the currently available drugs. This study strongly supports expanding programs for patients infected with T. cruzi in endemic and non-endemic countries.

Hypothesis testing clarifies the systematics of the main Central American Chagas disease vector, Triatoma dimidiata (Latreille, 1811), across its geographic range.

The widespread and diverse Triatoma dimidiata is the kissing bug species most important for Chagas disease transmission in Central America and a secondary vector in Mexico and northern South America. Its diversity may contribute to different Chagas disease prevalence in different localities and has led to conflicting systematic hypotheses describing various populations as subspecies or cryptic species. To resolve these conflicting hypotheses, we sequenced a nuclear (internal transcribed spacer 2, ITS-2) and mitochondrial gene (cytochrome b) from an extensive sampling of T. dimidiata across its geographic range. We evaluated the congruence of ITS-2 and cyt b phylogenies and tested the support for the previously proposed subspecies (inferred from ITS-2) by: (1) overlaying the ITS-2 subspecies assignments on a cyt b tree and, (2) assessing the statistical support for a cyt b topology constrained by the subspecies hypothesis. Unconstrained phylogenies inferred from ITS-2 and cyt b are congruent and reveal three clades including two putative cryptic species in addition to T. dimidiata sensu stricto. Neither the cyt b phylogeny nor hypothesis testing support the proposed subspecies inferred from ITS-2. Additionally, the two cryptic species are supported by phylogenies inferred from mitochondrially-encoded genes cytochrome c oxidase I and NADH dehydrogenase 4. In summary, our results reveal two cryptic species. Phylogenetic relationships indicate T. dimidiata sensu stricto is not subdivided into monophyletic clades consistent with subspecies. Based on increased support by hypothesis testing, we propose an updated systematic hypothesis for T. dimidiata based on extensive taxon sampling and analysis of both mitochondrial and nuclear genes.

Reproductive isolation revealed in preliminary crossbreeding experiments using field collected Triatoma dimidiata (Hemiptera: Reduviidae) from three ITS-2 defined groups.

Triatoma dimidiata, a Chagas disease vector distributed in Mexico, Central America, Colombia, Venezuela, Peru and Ecuador, has been studied using genetic markers and four groups have been defined by ITS-2 sequences: 1A, 1B, 2 and 3. To gather evidence on the divergence and reproductive isolation among T. dimidiata ITS-2 groups, we carried out 15 crossbreeding experiments with field-collected sylvan and domestic T. dimidiata from Guatemala where three groups are found: 1A, 2 and 3. Reciprocal crosses between individuals from groups 1A and 2, and a cross between group 2 individuals from different habitats, produced an average 129.78±42.29 eggs with hatching success ranging from 31.6 to 90.1%. The offspring of these crosses reached the adult stage, and crosses between F1 insects produced eggs. These results suggest that there are no pre- or post-zygotic reproductive barriers between groups 1A and 2, or within group 2. Crosses between group 3 females and males from groups 1A or 2 produced on average 85.67±30.26 eggs and none of them hatched. These results support the existence of pre-zygotic barriers between T. dimidiata group 3 and groups 1A and 2. The group 3 individuals were collected in sylvatic environments in Yaxha, Peten, Guatemala. Previously, distinct chromosomal characteristics (cytotype 3) were described in individuals from this population. Based on this evidence we suggest that this population is divergent at the species level from other T. dimidiata populations.

Phylogeographic pattern and extensive mitochondrial DNA divergence disclose a species complex within the Chagas disease vector Triatoma dimidiata.

BACKGROUND: Triatoma dimidiata is among the main vectors of Chagas disease in Latin America. However, and despite important advances, there is no consensus about the taxonomic status of phenotypically divergent T. dimidiata populations, which in most recent papers are regarded as subspecies. METHODOLOGY AND FINDINGS: A total of 126 cyt b sequences (621 bp long) were produced for specimens from across the species range. Forty-seven selected specimens representing the main cyt b clades observed (after a preliminary phylogenetic analysis) were also sequenced for an ND4 fragment (554 bp long) and concatenated with their respective cyt b sequences to produce a combined data set totalling 1175 bp/individual. Bayesian and Maximum-Likelihood phylogenetic analyses of both data sets (cyt b, and cyt b+ND4) disclosed four strongly divergent (all pairwise Kimura 2-parameter distances >0.08), monophyletic groups: Group I occurs from Southern Mexico through Central America into Colombia, with Ecuadorian specimens resembling Nicaraguan material; Group II includes samples from Western-Southwestern Mexico; Group III comprises specimens from the Yucatán peninsula; and Group IV consists of sylvatic samples from Belize. The closely-related, yet formally recognized species T. hegneri from the island of Cozumel falls within the divergence range of the T. dimidiata populations studied. CONCLUSIONS: We propose that Groups I-IV, as well as T. hegneri, should be regarded as separate species. In the Petén of Guatemala, representatives of Groups I, II, and III occur in sympatry; the absence of haplotypes with intermediate genetic distances, as shown by multimodal mismatch distribution plots, clearly indicates that reproductive barriers actively promote within-group cohesion. Some sylvatic specimens from Belize belong to a different species - likely the basal lineage of the T. dimidiata complex, originated ~8.25 Mya. The evidence presented here strongly supports the proposition that T. dimidiata is a complex of five cryptic species (Groups I-IV plus T. hegneri) that play different roles as vectors of Chagas disease in the region.

Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector, Triatoma dimidiata (Latreille, 1811).

Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.

Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector, Triatoma dimidiata (Latreille, 1811)

Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.