Evaluation of the Effectiveness of Chemical Control for Chagas Disease Vectors in Loja Province, Ecuador.

The objective of this study was to evaluate the effectiveness of selective and community-wide house insecticide spraying in controlling triatomines in the subtropical areas of Loja Province, Ecuador. We designed a quasi-experimental pre-post-test without a control group to compare entomological levels before and after spraying. The baseline study was conducted in 2008. Second, third, and fourth visits were conducted in 2010, 2011, and 2012 in three rural communities. Out of the 130 domestic units (DU) visited, 41 domestic units were examined in each of the four visits. Selective and community-wide insecticide interventions included spraying with 5% deltamethrin at 25 mg/m2 active ingredient. At each visit, a questionnaire was administered to identify the characteristics of households, and DUs were searched for triatomine bugs. In addition, parasitological analysis was carried out in life triatomines. One and two rounds of selective insecticide spraying decreased the probability of infestation by 62% (pairwise odds ratios [POR] 0.38, 95% confidence interval [CI] 0.17-0.89, p = 0.024) and 51% (POR 0.49, 95% CI 0.23-1.01, p = 0.054), respectively. A similar effect was observed after one round of community-wide insecticide application in Chaquizhca and Guara (POR 0.55, CI 0.24-1.25, p = 0.155) and Bellamaria (POR 0.62, CI 0.22-1.79, p = 0.379); however, it was not statistically significant. Trypanosoma cruzi infection in triatomines (n = 483) increased overtime, from 2008 (42.9% and 8.5% for Rhodnius ecuadoriensis and Panstrongylus chinai, respectively) to 2012 (79.5% and 100%). Neither of the two spraying methodologies was effective for triatomine control in this area and our results point to a high likelihood of reinfestation after insecticide application. This underscores the importance of the implementation of physical barriers that prevent invasion and colonization of triatomines in households, such as home improvement initiatives, accompanied by a concerted effort to address the underlying socioeconomic issues that keep this population at risk of developing Chagas disease.

Evaluation of Selective Deltamethrin Application with Household and Community Awareness for the Control of Chagas Disease in Southern Ecuador.

Chagas disease is endemic in ~70% of Ecuador. Rhodnius ecuadoriensis and Triatoma carrioni (Hemiptera: Reduviidae) are the primary vectors of Chagas disease in Southern Ecuador. This study tested the effectiveness of selective deltamethrin application of Domiciliary Units (DUs) infested with triatomines, coupled with community education activities and a community-based surveillance system. Ten communities were selected in Loja Province, 466 DUs were examined, of these, 5.6% were infested with R. ecuadoriensis (Density [D] = 4 triatomines/DUs searched, Crowding [CR] = 71 triatomines/infested house, Colonization Index [CI] = 77% infested DUs with nymphs) and 8% with T. carrioni (D = 0.6, CR = 7, CI = 64%). Infested DUs were sprayed with deltamethrin. Subsequent visits were conducted at 6 and 12 mo after spraying. At each time point, new entomological searches were carried out in all DUs. All entomological indexes dropped significantly for the primary vector species one year after the initial intervention (R. ecuadoriensis: I = 2%, D = 0.1, CR = 7, CI = 100%; T. carrioni: I = 1.6%, D = 0.1, CR = 5.5, CI = 50%). Fifteen min educational talks were conducted in every DUs and workshops for schoolchildren were organized. Community-based surveillance system was established. However, there is a high risk of DUs reinfestation, possibly from sylvatic habitats (especially of R. ecuadoriensis) and reinforcing educational and surveillance activities are necessary.

Population genomics and geographic dispersal in Chagas disease vectors: Landscape drivers and evidence of possible adaptation to the domestic setting.

Accurate prediction of vectors dispersal, as well as identification of adaptations that allow blood-feeding vectors to thrive in built environments, are a basis for effective disease control. Here we adopted a landscape genomics approach to assay gene flow, possible local adaptation, and drivers of population structure in Rhodnius ecuadoriensis, an important vector of Chagas disease. We used a reduced-representation sequencing technique (2b-RADseq) to obtain 2,552 SNP markers across 272 R. ecuadoriensis samples from 25 collection sites in southern Ecuador. Evidence of high and directional gene flow between seven wild and domestic population pairs across our study site indicates insecticide-based control will be hindered by repeated re-infestation of houses from the forest. Preliminary genome scans across multiple population pairs revealed shared outlier loci potentially consistent with local adaptation to the domestic setting, which we mapped to genes involved with embryogenesis and saliva production. Landscape genomic models showed elevation is a key barrier to R. ecuadoriensis dispersal. Together our results shed early light on the genomic adaptation in triatomine vectors and facilitate vector control by predicting that spatially-targeted, proactive interventions would be more efficacious than current, reactive approaches.

Human Blood Meals in Sylvatic Triatomines Challenges Domestic-Centered Strategies for Prevention of Trypanosoma cruzi Transmission in Ecuador.

Transmission risk of Chagas disease has been associated with human-vector contacts and triatomines colonizing dwellings, but alternative scenarios, independent of domestic colonization, are poorly documented. In the present work, we estimated the frequency of human blood meals in triatomines from domicile, peridomicile, and sylvatic environments in two endemic regions in Ecuador. Blood meal origins were identified by sequencing a cytb gene fragment. Human blood meals were detected in 42% of the triatomines among 416 analyzed, including 48% of sylvatic triatomines (both adults and nymphs). In triatomines from domicile and peridomicile, Trypanosoma cruzi infection rate was > 20%, and reached 48% in sylvatic triatomines. Human is a common source of blood for triatomines whether they live in or near dwellings in both regions, and the high rate of T. cruzi infection represents an important risk of transmission of Chagas disease. Consequently, control strategies should also take into account possible nondomestic transmission.

Molecular data supports monophyly of Triatoma dispar complex within genus Triatoma.

The genus Triatoma contains numerous species, principal or secondary vectors of Chagas disease, which have been included in the three main lineages of Triatomini tribe based on morphological and biogeographical characteristics: North American, South American, and T. dispar complex. The three members of the T. dispar complex are distributed in Ecuador. This complex has been scarcely studied through molecular approaches, and the taxonomic position of this complex is not confirmed. In this study, we explored the phylogenetic relationships within the genus Triatoma, including five species from North and Central America, six from South America, and the three species belonging to the T. dispar complex. Partial sequences of four mitochondrial genes (Cyt b, COII, 16S-rRNA, 12S-rRNA) and two nuclear genes (18S-rRNA, ITS2) were obtained from 74 specimens. Phylogenetic trees were built with concatenated and single sequences through maximum likelihood (ML), maximum parsimony (MP), and Bayesian methods. The trees built using concatenated sequences showed three main branches (clusters) highly supported by significant bootstrap values; the T. dispar complex appeared as a monophyletic group separate from species of North and Central American origin and South American origin. On the contrary, for each gene tree, the three main clusters were not always significantly supported, mostly because genetic information is dramatically reduced when a single gene is considered. Consequently, concatenation of genes gives relevant results and is highly recommended for further in-depth examination of the relationships of several species and complexes of triatomines that remain unresolved. Moreover, our current molecular data fully revealed the division of genus Triatoma into at least three main genetic groups.

Would tropical climatic variations impact the genetic variability of triatomines: Rhodnius ecuadoriensis, principal vector of Chagas disease in Ecuador?

Rhodnius ecuadoriensis is one of the most important vector species of Chagas disease in Ecuador. This species is distributed in the Central coast region and in the south Andean region, and an incipient speciation process between these geographical populations was previously proposed. The current population genetics study only focused on the Central coast region and analyzed 96 sylvatic specimens of R. ecuadoriensis associated with Phytelephas aequatorialis palm trees. We used Cytb and 16S-rRNA sequences and a Cytb-16S-rRNA concatenated set to explore (i) the genetic variability, spatial structuring, and demographic history of R. ecuadoriensis, and to determine (ii) the relationship between the genetic and climatic variabilities. A particularly high genetic variability was observed without detectable general genetic structure; only some terminal genetic clusters were observed. We did not observe isolation by geographical distance (IBD), and it is likely that ancient expansion occurred, according to Fs index and mismatch distribution for Cytb-16S-rRNA concatenated sequences. Hierarchical clustering showed that the current locality origins of the bugs were grouped into four bioclimatic clusters. Genetic and bioclimatic distances were not correlated, but some genetic clusters were associated with bioclimatic ones. The results showed an ancient evolution of the species in the region with a possible old expansion. The absence of spatial genetic structure could be due to climatic conditions (possible selection of singular genotypes) and to passive transportation of palms tree materials where R. ecuadoriensis are living.

Chagas vectors Panstrongylus chinai (Del Ponte, 1929) and Panstrongylus howardi (Neiva, 1911): chromatic forms or true species?

BACKGROUND: Chagas disease is a parasitic infection transmitted by "kissing bugs" (Hemiptera: Reduviidae: Triatominae) that has a huge economic impact in Latin American countries. The vector species with the upmost epidemiological importance in Ecuador are Rhodnius ecuadoriensis (Lent & Leon, 1958) and Triatoma dimidiata (Latreille, 1811). However, other species such as Panstrongylus howardi (Neiva, 1911) and Panstrongylus chinai (Del Ponte, 1929) act as secondary vectors due to their growing adaptation to domestic structures and their ability to transmit the parasite to humans. The latter two taxa are distributed in two different regions, they are allopatric and differ mainly by their general color. Their relative morphological similarity led some authors to suspect that P. chinai is a melanic form of P. howardi. METHODS: The present study explored this question using different approaches: antennal phenotype; geometric morphometrics of heads, wings and eggs; cytogenetics; molecular genetics; experimental crosses; and ecological niche modeling. RESULTS: The antennal morphology, geometric morphometrics of head and wing shape and cytogenetic analysis were unable to show distinct differences between the two taxa. However, geometric morphometrics of the eggs, molecular genetics, ecological niche modeling and experimental crosses including chromosomal analyses of the F1 hybrids, in addition to their coloration and current distribution support the hypothesis that P. chinai and P. howardi are separate species. CONCLUSIONS: Based on the evidence provided here, P. howardi and P. chinai should not be synonymized. They represent two valid, closely related species.

Genetic data support speciation between Panstrongylus howardi and Panstrongylus chinai, vectors of Chagas disease in Ecuador.

Limited genetic data are currently available for three vectors of Chagas disease in Ecuador, Panstrongylus howardi, P. chinai, and P. rufotuberculatus. Previously regarded as mainly sylvatic, these species have been poorly studied. Recently, they have been more frequently reported in domiciles and peridomiciles and are now considered true secondary vectors of Chagas disease in a country where an estimated 200,000 people are infected by Trypanosoma cruzi, a causative agent of this disease. In order to fill this gap, we obtained DNA for sequencing from 53 insects belonging to these three species and mainly sampled from the two Ecuadorian provinces of Loja and Manabí. We used six mitochondrial loci (COI, COII, ND4, CytB, 16S, and 12S) and two nuclear ones (ITS2 and 18S). We interpreted the phylogenetic trees built with single and concatenated data through maximum likelihood, Bayesian Markov chain Monte Carlo, and maximum parsimony methods. We provide evidence that P. chinai and P. howardi are indeed two supported species closely related and derived from a common ancestor. Additionally, the phylogenetic position of P. rufotuberculatus was confirmed as being distant from P. chinai and P. howardi and clustered with Triatoma dimidiata, a species belonging to the Northern American Triatoma clade.

Distribution of triatomine species in domestic and peridomestic environments in central coastal Ecuador.

BACKGROUND: Although the central coast of the Ecuador is considered endemic for Chagas disease, few studies have focused on determining the risk of transmission in this region. In this study we describe the triatomine household infestation in Manabí province (Central Coast region), determine the rate of Trypanosoma cruzi infection and study the risk factors associated with infestation by Rhodnius ecuadoriensis. METHODOLOGY/PRINCIPAL FINDINGS: An entomological survey found three triatomine species (Rhodnius ecuadoriensis, Panstrongylus rufotuberculatus and P. howardi) infesting domiciles in 47.4% of the 78 communities visited (total infestation rate of 4.5%). Four percent of domiciles were infested, and nymphs were observed in 77% of those domiciles. The three species were found in altitudes below 500 masl and in all ecological zones except cloud forest. Within the domicile, we found the three species mostly in bedrooms. Rhodnius ecuadoriensis and P. rufotuberculatus were abundant in bird nests, including chicken coops and P. howardi associated with rats in piles of bricks, in the peridomicile. Triatomine infestation was characterized by high rates of colonization, especially in peridomicile. Flagelates infection was detected in only 12% of the samples by microscopy and Trypanosoma cruzi infection in 42% of the examined triatomines by PCR (n = 372). The most important risk factors for house infestation by R. ecuadoriensis were ecological zone (w = 0.99) and presence of chickens (w = 0.96). Determinants of secondary importance were reporting no insecticide applications over the last twelve months (w = 0.86) and dirt floor (w = 0.70). On the other hand, wood as wall material was a protective factor (w = 0.85). CONCLUSION/SIGNIFICANCE: According the results, approximately 571,000 people would be at high risk for T. cruzi infection in Manabí province. A multidisciplinary approximation and the adhesion to a periodic integrated vector management (IVM) program are essential to guarantee sustainable preventive and control strategies for Chagas disease in this region.

2b-RAD genotyping for population genomic studies of Chagas disease vectors: Rhodnius ecuadoriensis in Ecuador.

BACKGROUND: Rhodnius ecuadoriensis is the main triatomine vector of Chagas disease, American trypanosomiasis, in Southern Ecuador and Northern Peru. Genomic approaches and next generation sequencing technologies have become powerful tools for investigating population diversity and structure which is a key consideration for vector control. Here we assess the effectiveness of three different 2b restriction site-associated DNA (2b-RAD) genotyping strategies in R. ecuadoriensis to provide sufficient genomic resolution to tease apart microevolutionary processes and undertake some pilot population genomic analyses. METHODOLOGY/PRINCIPAL FINDINGS: The 2b-RAD protocol was carried out in-house at a non-specialized laboratory using 20 R. ecuadoriensis adults collected from the central coast and southern Andean region of Ecuador, from June 2006 to July 2013. 2b-RAD sequencing data was performed on an Illumina MiSeq instrument and analyzed with the STACKS de novo pipeline for loci assembly and Single Nucleotide Polymorphism (SNP) discovery. Preliminary population genomic analyses (global AMOVA and Bayesian clustering) were implemented. Our results showed that the 2b-RAD genotyping protocol is effective for R. ecuadoriensis and likely for other triatomine species. However, only BcgI and CspCI restriction enzymes provided a number of markers suitable for population genomic analysis at the read depth we generated. Our preliminary genomic analyses detected a signal of genetic structuring across the study area. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that 2b-RAD genotyping is both a cost effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales. As such, 2b-RAD represents a powerful tool in the hands of medical entomologists with limited access to specialized molecular biological equipment.

Pioneer study of population genetics of Rhodnius ecuadoriensis (Hemiptera: Reduviidae) from the central coastand southern Andean regions of Ecuador.

Effective control of Chagas disease vector populations requires a good understanding of the epidemiological components, including a reliable analysis of the genetic structure of vector populations. Rhodnius ecuadoriensis is the most widespread vector of Chagas disease in Ecuador, occupying domestic, peridomestic and sylvatic habitats. It is widely distributed in the central coast and southern highlands regions of Ecuador, two very different regions in terms of bio-geographical characteristics. To evaluate the genetic relationship among R. ecuadoriensis populations in these two regions, we analyzed genetic variability at two microsatellite loci for 326 specimens (n=122 in Manabí and n=204 in Loja) and the mitochondrial cytochrome b gene (Cyt b) sequences for 174 individuals collected in the two provinces (n=73 and=101 in Manabí and Loja respectively). The individual samples were grouped in populations according to their community of origin. A few populations presented positive FIS, possible due to Wahlund effect. Significant pairwise differentiation was detected between populations within each province for both genetic markers, and the isolation by distance model was significant for these populations. Microsatellite markers showed significant genetic differentiation between the populations of the two provinces. The partial sequences of the Cyt b gene (578bp) identified a total of 34 haplotypes among 174 specimens sequenced, which translated into high haplotype diversity (Hd=0.929). The haplotype distribution differed among provinces (significant Fisher's exact test). Overall, the genetic differentiation of R. ecuadoriensis between provinces detected in this study is consistent with the biological and phenotypic differences previously observed between Manabí and Loja populations. The current phylogenetic analysis evidenced the monophyly of the populations of R. ecuadoriensis within the R. pallescens species complex; R. pallescens and R. colombiensis were more closely related than they were to R. ecuadoriensis.

Comprehensive Survey of Domiciliary Triatomine Species Capable of Transmitting Chagas Disease in Southern Ecuador.

BACKGROUND: Chagas disease is endemic to the southern Andean region of Ecuador, an area with one of the highest poverty rates in the country. However, few studies have looked into the epidemiology, vectors and transmission risks in this region. In this study we describe the triatomine household infestation in Loja province, determine the rate of Trypanosoma cruzi infection in triatomines and study the risk factors associated with infestation. METHODOLOGY/PRINCIPAL FINDINGS: An entomological survey found four triatomine species (Rhodnius ecuadoriensis, Triatoma carrioni, Panstrongylus chinai, and P. rufotuberculatus) infesting domiciles in 68% of the 92 rural communities examined. Nine percent of domiciles were infested, and nymphs were observed in 80% of the infested domiciles. Triatomines were found in all ecological regions below 2,200 masl. We found R. ecuadoriensis (275 to 1948 masl) and T. carrioni (831 to 2242 masl) mostly in bedrooms within the domicile, and they were abundant in chicken coops near the domicile. Established colonies of P. chinai (175 to 2003 masl) and P. rufotuberculatus (404 to 1613 masl) also were found in the domicile. Triatomine infestation was associated with surrogate poverty indicators, such as poor sanitary infrastructure (lack of latrine/toilet [w = 0.95], sewage to environment [w = 1.0]). Vegetation type was a determinant of infestation [w = 1.0] and vector control program insecticide spraying was a protective factor [w = 1.0]. Of the 754 triatomines analyzed, 11% were infected with Trypanosoma cruzi and 2% were infected with T. rangeli. CONCLUSIONS/SIGNIFICANCE: To date, only limited vector control efforts have been implemented. Together with recent reports of widespread sylvatic triatomine infestation and frequent post-intervention reinfestation, these results show that an estimated 100,000 people living in rural areas of southern Ecuador are at high risk for T. cruzi infection. Therefore, there is a need for a systematic, sustained, and monitored vector control intervention that is coupled with improvement of socio-economic conditions.

A national survey to determine prevalence of Trypanosoma cruzi infection among pregnant women in Ecuador.

A nationwide survey was conducted to obtain an estimate of Chagas disease prevalence among pregnant women in Ecuador. As part of a national probability sample, 5,420 women seeking care for delivery or miscarriage at 15 healthcare facilities were recruited into the study. A small minority of participants reported knowing about Chagas disease or recognized the vector. A national seroprevalence of 0.1% (95% confidence interval [95% CI] = 0.0-0.2%) was found; cases were concentrated in the coastal region (seroprevalence = 0.2%; 95% CI = 0.0-0.4%). No cases of transmission to neonates were identified in the sample. Seropositive participants were referred to the National Chagas Program for evaluation and treatment. Additional studies are necessary to determine if areas of higher prevalence exist in well-known endemic provinces and guide the development of a national strategy for elimination of mother-to-child transmission of Chagas disease in Ecuador.

Abundance, natural infection with trypanosomes, and food source of an endemic species of triatomine, Panstrongylus howardi (Neiva 1911), on the Ecuadorian Central Coast.

The elimination of domestic triatomines is the foundation of Chagas disease control. Regional initiatives are eliminating introduced triatomine species. In this scenario, endemic triatomines can occupy the ecological niches left open and become a threat to long-term Chagas disease control efforts. This study determined the abundance, colonization, and Trypanosoma cruzi infection rate of the endemic Panstrongylus howardi in 10 rural communities located in Ecuador's Manabí Province. In total, 518 individuals of P. howardi were collected. Infestation indices of 1.4% and 6.6% were found in the domestic and peridomestic environments, respectively. We determined a T. cruzi infection rate of 53.2% (N = 47) in this species. P. howardi has a high capacity to adapt to different habitats, especially in the peridomicile. This implies a considerable risk of transmission because of the frequency of intradomicile invasion. Therefore, this species needs to be taken into account in Chagas control and surveillance efforts in the region.

HIV and syphilis infection in pregnant women in Ecuador: prevalence and characteristics of antenatal care.

OBJECTIVES: This study aimed to obtain nationally representative estimates of HIV and syphilis prevalence and coverage of preventive antenatal services in pregnant women in Ecuador, in order to develop a national strategy for the elimination of mother-to-child transmission of HIV and syphilis. METHODS: A national probability sample of 5988 women presenting for delivery or miscarriage services was selected from 15 healthcare facilities during 2011-2012, using a two-stage cluster sample technique. Biological specimens were collected and an interview and review of medical records were performed. Agreement between these last two sources was measured. Estimates were adjusted for the sampling design. RESULTS: Estimated national HIV prevalence (0.60%) was higher than confirmed syphilis infection prevalence (0.25%). In the coastal region, HIV prevalence (1.13%) exceeded the threshold that defines a generalised epidemic and syphilis prevalence reached 0.37%. An estimated 5.9% of women did not use antenatal care services while 73.0% completed at least four consultations. HIV testing coverage (89.9%) was higher than for syphilis (71.6%). Agreement between medical records and interviews was mostly moderate (0.40-0.75). Important variables were frequently not recorded, such as timing of syphilis testing, which was not recorded in 49.6%. CONCLUSIONS: The concentration of HIV and syphilis infections in the coastal region of Ecuador highlights the need for intensified prevention and a response tailored to local epidemic conditions. Major challenges for the elimination initiative include achieving universal, early access to antenatal care, improving coverage of HIV and syphilis testing, and improving the quality of medical records to support progress monitoring.

Limitations of selective deltamethrin application for triatomine control in central coastal Ecuador.

BACKGROUND: This year-long study evaluated the effectiveness of a strategy involving selective deltamethrin spraying and community education for control of Chagas disease vectors in domestic units located in rural communities of coastal Ecuador. RESULTS: Surveys for triatomines revealed peridomestic infestation with Rhodnius ecuadoriensis and Panstrongylus howardi, with infestation indices remaining high during the study (13%, 17%, and 10%, at initial, 6-month, and 12-month visits, respectively), which indicates a limitation of this strategy for triatomine population control. Infestation was found 6 and 12 months after spraying with deltamethrin. In addition, a large number of previously vector-free domestic units also were found infested at the 6- and 12-month surveys, which indicates new infestations by sylvatic triatomines. The predominance of young nymphs and adults suggests new infestation events, likely from sylvatic foci. In addition, infection with Trypanosoma cruzi was found in 65%, 21% and 29% at initial, 6-month and 12-month visits, respectively. All parasites isolated (n = 20) were identified as TcI. CONCLUSION: New vector control strategies need to be devised and evaluated for reduction of T. cruzi transmission in this region.