Evidence of likely autochthonous Chagas disease in the southwestern United States: A case series of Trypanosoma cruzi seropositive blood donors.

BACKGROUND: Chagas disease is a parasitic infection that can insidiously cause non-ischemic cardiomyopathy. Given the largely silent nature of this progressive disease, asymptomatic blood donors pose potential blood transfusion risk. Blood donation screening has become an unintentional form of Chagas disease surveillance, with thousands of new cases identified since national surveillance was initiated in 2007. STUDY DESIGN AND METHODS: We recruited T. cruzi-positive blood donors identified from California and Arizona blood centers for confirmatory blood screening and assessment of lifetime infection risk. RESULTS: Among eight suspected cases, we identified four confirmed US autochthonous infections. The current manuscript details the transmission sources, healthcare-seeking behaviors post-blood donation resulting, and clinical course of disease among persons without any history of travel to endemic Latin American countries. DISCUSSION: This manuscript presents four additional US-acquired Chagas disease cases and identifies an opportunity for blood centers to assist in confronting barriers surrounding Chagas disease in the US.

Rapid detection of human blood in triatomines (kissing bugs) utilizing a lateral flow immunochromatographic assay - A pilot study.

OBJECTIVES: We tested a rapid and specific immunochromatographic assay (that detects human blood in forensic samples) to determine if human blood was present in triatomines and their fecal excreta. METHODS: We fed Triatoma rubida human blood (positive control) or mouse blood (negative control) and performed the assay on the abdominal contents and fecal excreta. Triatomine field specimens collected in and around human habitations and excreta were also tested. FINDINGS: The assay was positive in triatomines fed human blood (N = 5/5) and fecal excreta from bugs known to have ingested human blood (N = 5/5). Bugs feeding on mice (N = 15/15) and their fecal excreta (N = 8/8) were negative for human blood. Human blood was detected in 47% (N = 23/49) triatomines, representing six different species, collected in the field. MAIN CONCLUSIONS: The pilot study shows that this rapid and specific test may have applications in triatomine research. Further study is needed to determine the sensitivity of this assay compared to other well-established techniques, such as DNA- and proteomics-based methodologies and the assay's application in the field.

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.

Migration and Gene Flow Among Domestic Populations of the Chagas Insect Vector Triatoma dimidiata (Hemiptera: Reduviidae) Detected by Microsatellite Loci.

Triatoma dimidiata (Latreille, 1811) is the most abundant and significant insect vector of the parasite Trypanosoma cruzi in Central America, and particularly in Guatemala. Tr. cruzi is the causative agent of Chagas disease, and successful disease control requires understanding the geographic distribution and degree of migration of vectors such as T. dimidiata that frequently re-infest houses within months following insecticide application. The population genetic structure of T. dimidiata collected from six villages in southern Guatemala was studied to gain insight into the migration patterns of the insects in this region where populations are largely domestic. This study provided insight into the likelihood of eliminating T. dimidiata by pesticide application as has been observed in some areas for other domestic triatomines such as Triatoma infestans. Genotypes of microsatellite loci for 178 insects from six villages were found to represent five genetic clusters using a Bayesian Markov Chain Monte Carlo method. Individual clusters were found in multiple villages, with multiple clusters in the same house. Although migration occurred, there was statistically significant genetic differentiation among villages (FR T = 0.05) and high genetic differentiation among houses within villages (FSR = 0.11). Relatedness of insects within houses varied from 0 to 0.25, i.e., from unrelated to half-sibs. The results suggest that T. dimidiata in southern Guatemala moves between houses and villages often enough that recolonization is likely, implying the use of insecticides alone is not sufficient for effective control of Chagas disease in this region and more sustainable solutions are required.

Triatoma sanguisuga blood meals and potential for Chagas disease, Louisiana, USA.

To evaluate human risk for Chagas disease, we molecularly identified blood meal sources and prevalence of Trypanosoma cruzi infection among 49 Triatoma sanguisuga kissing bugs in Louisiana, USA. Humans accounted for the second most frequent blood source. Of the bugs that fed on humans, ≈40% were infected with T. cruzi, revealing transmission potential.

Factors associated with peridomestic Triatoma sanguisuga (Hemiptera: Reduviidae) presence in southeastern Louisiana.

Although rare, there have been isolated reports of autochthonous transmission of Trypanosoma cruzi Chagas in the United States. In June 2006, a human case of domestically transmitted T. cruzi was identified in southern Louisiana. To examine the localized risk of human T. cruzi infection in the area surrounding the initial human case, environmental surveys of households in the area and a serological survey of the residents were performed between September 2008 and November 2009. Human T. cruzi infection was determined using a rapid antigen field test, followed by confirmatory enzyme-linked immunosorbent assay testing in the laboratory. A perimeter search of each participating residence for Triatoma sanguisuga (LeConte), the predominant local triatomine species, was also performed. No participating individuals were positive for antibodies against T. cruzi; however, high levels of T. cruzi infection (62.4%) were detected in collected T. sanguisuga. Households with T. sanguisuga presence were less likely to use air conditioning, and more likely to have either chickens or cats on the property. While the human risk for T cruzi infection in southeastern Louisiana is low, a high prevalence of infected T. sanguisuga does indicate a substantial latent risk for T. cruzi peridomestic transmission. Further examination of the behavior and ecology of T. sanguisuga in the region will assist in refining local T. cruzi risk associations.

Genetic diversity of the Chagas vector Triatoma dimidiata s.l. (Hemiptera: Reduviidae) across geographic scales in a top-priority area for control.

Population genetic structure of arthropod disease vectors provides important information on vector movement and climate or other environmental variables that influence their distribution. This information is critical for data-driven vector control. In the first comprehensive study of the genetic structure of T. dimidiata s.l. (Latreille, 1811) we focus on an area of active transmission designated as a top priority for control. We examined a high number of specimens across a broad geographic area along the border of Guatemala and El Salvador including multiple spatial scales using a high number of genome-wide markers. Measuring admixture, pairwise genetic differentiation, and relatedness, we estimated the specimens represented three genetic clusters. We found evidence of movement (migration/gene flow) across all spatial scales with more admixture among locations in El Salvador than in Guatemala. Although there was significant isolation by distance, the 2 close villages in Guatemala showed either the most or least genetic variation indicating an additional role of environmental variables. Further, we found that social factors may be influencing the genetic structure. We demonstrated the power of genomic studies with a large number of specimens across a broad geographic area. The results suggest that for effective vector control movement must be considered on multiple spatial scales along with its contributing factors.

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.

Vector blood meals and Chagas disease transmission potential, United States.

A high proportion of triatomine insects, vectors for Trypanosoma cruzi trypanosomes, collected in Arizona and California and examined using a novel assay had fed on humans. Other triatomine insects were positive for T. cruzi parasite infection, which indicates that the potential exists for vector transmission of Chagas disease in the United States.

The long-term impact of an Ecohealth intervention: Entomological data suggest the interruption of Chagas disease transmission in southeastern Guatemala.

Triatoma dimidiata is the main vector of Chagas disease in southern Mexico, Central America and northern South America. As a native vector, it moves readily among domestic, peri­domestic and sylvatic environments, making it difficult to control only using insecticide as this requires regular application, and re-infestation frequently occurs. Other social innovation alternatives such as those based on Ecohealth principles can be used to tackle the dynamics of the disease in an integral way. We asked whether an Ecohealth intervention, implemented beginning in 2001 in a highly infested village, 41.8%, in southeastern Guatemala, was sustainable in the long term. This intervention included initial insecticide treatments, followed by making low-cost house improvements to eliminate transmission risk factors such as repairing cracked walls, covering dirt floors with a cement-like substance and moving domestic animals outside. We assessed the long-term sustainability through entomological and house condition surveys, as well as an analysis of community satisfaction. We found over a 19-year period, infestation with T. dimidiata was reduced to 2.2% and maintained at a level below the level (8%) where vector transmission is unlikely. This long-term maintenance of low infestation coincided with a large proportion of villagers (88.6%) improving their houses and completing other aspects of the Ecohealth approach to maintain the village at low risk for Chagas transmission. There was unanimous satisfaction among the villagers with their houses, following improvements using the Ecohealth method, which likely played a role in the long-term persistence of the modifications. Although the infestation has remained low, 11 years following the last intervention and as the population grew there has been an increase in the proportion of "at-risk" houses, to 33%, pointing out the necessity of maintaining vigilance. The Ecohealth approach is a low-cost, sustainable approach for the long-term control of vector-borne Chagas disease. We recommend this approach including ongoing community monitoring and institutional response for the long-term, integrated control of Chagas disease.

Insights from a comprehensive study of Trypanosoma cruzi: A new mitochondrial clade restricted to North and Central America and genetic structure of TcI in the region.

More than 100 years since the first description of Chagas Disease and with over 29,000 new cases annually due to vector transmission (in 2010), American Trypanosomiasis remains a Neglected Tropical Disease (NTD). This study presents the most comprehensive Trypanosoma cruzi sampling in terms of geographic locations and triatomine species analyzed to date and includes both nuclear and mitochondrial genomes. This addresses the gap of information from North and Central America. We incorporate new and previously published DNA sequence data from two mitochondrial genes, Cytochrome oxidase II (COII) and NADH dehydrogenase subunit 1 (ND1). These T. cruzi samples were collected over a broad geographic range including 111 parasite DNA samples extracted from triatomines newly collected across North and Central America, all of which were infected with T. cruzi in their natural environment. In addition, we present parasite reduced representation (Restriction site Associated DNA markers, RAD-tag) genomic nuclear data combined with the mitochondrial gene sequences for a subset of the triatomines (27 specimens) collected from Guatemala and El Salvador. Our mitochondrial phylogenetic reconstruction revealed two of the major mitochondrial lineages circulating across North and Central America, as well as the first ever mitochondrial data for TcBat from a triatomine collected in Central America. Our data also show that within mtTcIII, North and Central America represent an independent, distinct clade from South America, named here as mtTcIIINA-CA, geographically restricted to North and Central America. Lastly, the most frequent lineage detected across North and Central America, mtTcI, was also an independent, distinct clade from South America, noted as mtTcINA-CA. Furthermore, nuclear genome data based on Single Nucleotide Polymorphism (SNP) showed genetic structure of lineage TcI from specimens collected in Guatemala and El Salvador supporting the hypothesis that genetic diversity at a local scale has a geographical component. Our multiscale analysis contributes to the understanding of the independent and distinct evolution of T. cruzi lineages in North and Central America regions.

From e-voucher to genomic data: Preserving archive specimens as demonstrated with medically important mosquitoes (Diptera: Culicidae) and kissing bugs (Hemiptera: Reduviidae).

Scientific collections such as the U.S. National Museum (USNM) are critical to filling knowledge gaps in molecular systematics studies. The global taxonomic impediment has resulted in a reduction of expert taxonomists generating new collections of rare or understudied taxa and these large historic collections may be the only reliable source of material for some taxa. Integrated systematics studies using both morphological examinations and DNA sequencing are often required for resolving many taxonomic issues but as DNA methods often require partial or complete destruction of a sample, there are many factors to consider before implementing destructive sampling of specimens within scientific collections. We present a methodology for the use of archive specimens that includes two crucial phases: 1) thoroughly documenting specimens destined for destructive sampling-a process called electronic vouchering, and 2) the pipeline used for whole genome sequencing of archived specimens, from extraction of genomic DNA to assembly of putative genomes with basic annotation. The process is presented for eleven specimens from two different insect subfamilies of medical importance to humans: Anophelinae (Diptera: Culicidae)-mosquitoes and Triatominae (Hemiptera: Reduviidae)-kissing bugs. Assembly of whole mitochondrial genome sequences of all 11 specimens along with the results of an ortholog search and BLAST against the NCBI nucleotide database are also presented.

Catch me if you can: Under-detection of Trypanosoma cruzi (Kinetoplastea: Trypanosomatida) infections in Triatoma dimidiata s.l. (Hemiptera: Reduviidae) from Central America.

Assays for parasite detection in insect vectors provide important information for disease control. American Trypanosomiasis (Chagas disease) is the most devastating vector-borne illness and the fourth most common in Central America behind HIV/AIDS and acute respiratory and diarrheal infections (Peterson et al., 2019). Under-detection of parasites is a general problem which may be influenced by parasite genetic variation; however, little is known about the genetic variation of the Chagas parasite, especially in this region. In this study we compared six assays for detecting the Chagas parasite, Trypanosoma cruzi: genomic reduced representation sequencing (here referred to as genotype-by-sequencing or GBS), two with conventional PCR (i.e., agarose gel detection), two with qPCR, and microscopy. Our results show that, compared to GBS genomic analysis, microscopy and PCR under-detected T. cruzi in vectors from Central America. Of 94 samples, 44% (50/94) were positive based on genomic analysis. The lowest detection, 9% (3/32) was in a subset assayed with microscopy. Four PCR assays, two with conventional PCR and two with qPCR showed intermediate levels of detection. Both qPCR tests and one conventional PCR test targeted the 195 bp repeat of satellite DNA while the fourth test targeted the 18S gene. Statistical analyses of the genomic and PCR results indicate that the PCR assays significantly under detect infections of Central American T. cruzi genotypes.

"Kissing bugs": potential disease vectors and cause of anaphylaxis.

Physicians in the United States should familiarize themselves with "kissing bugs" endemic to their area of practice and appreciate the medical implications of their bites. Bite victims often seek advice from physicians about allergic reactions as well as the risk of contracting Chagas disease. Physicians are generally knowledgeable about the role of kissing bugs in the transmission of Trypanosoma cruzi in Latin America. However, they may be unaware of (1) severe allergic reactions to kissing bug salivary antigens, (2) the widespread occurrence of T. cruzi amongst vertebrate hosts of kissing bugs, and (3) the incidence of T. cruzi among kissing bugs (T. cruzi may infect >50% of sampled bugs). Despite the potential for Chagas disease transmission, the major concern regarding kissing bugs in the United States is anaphylactic reactions to their bites resulting in frequent emergency department visits, especially in areas of endemicity in the Southwest.

Kissing Bugs Harboring Trypanosoma cruzi, Frequently Bite Residents of the US Southwest But Do Not Cause Chagas Disease.

BACKGROUND: Kissing bugs are common household pests in the Desert Southwest of the United States. These hematophagous bugs enter homes and suck blood from resident humans and pets. They are vectors of Trypanosoma cruzi, an enzootic parasite in small mammals and the cause of Chagas disease in humans. Autochthonous cases of Chagas disease are rare in the United States despite the presence of the vector and parasite. Environmental and biological factors accounting for this phenomenon need studying. METHODS: Homeowners in Bisbee and Tucson, Arizona captured kissing bugs inside homes during 2017-2018. Bugs were tested for presence of T. cruzi by polymerase chain reaction. Residents bitten by kissing bugs were tested for Chagas disease by serology. We evaluated invaded homes in the 2 cities. RESULTS: Three species of kissing bugs (n = 521) were collected in or near homes. Triatoma rubida was the most common triatomine in Tucson; T. recurva in Bisbee. T. protracta was uncommon. Seventeen percent of bugs captured in Bisbee and 51.1% in Tucson harbored T. cruzi. Bite victims (n = 105) recalled more than 2200 bites. Reactions to bites were common, including 32 episodes of anaphylaxis in 11 people (10.5%). Tests for Chagas disease (n = 116) were negative. Median age of houses was 91 years in Bisbee and 7 years in Tucson. Bisbee houses had pier and beam foundations. Tucson houses were built on concrete slabs. CONCLUSIONS: Kissing bugs harboring T. cruzi readily entered new and old homes. Bites of humans caused severe, life-threatening reactions. There was no serological evidence of Chagas disease among those bitten.

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.

Second-Best Is Better Than Nothing: Cockroaches as a Viable Food Source for the Kissing Bug Triatoma recurva (Hemiptera: Reduviidae).

Kissing bugs in the genus Triatoma are obligate blood feeders that feed mainly on vertebrate blood and have lost the predatory lifestyle found in other reduviid bugs. They occasionally also feed on the hemolymph of arthropods, especially during the first and second instar stages. The largest kissing bug species in the United States, Triatoma recurva (Stål) (Hemiptera: Reduviidae), is poorly known and was chosen to investigate its ability to feed and develop on a diet of cockroach hemolymph. Molting from first instar individuals to second instars readily occurred at approximately the same rate reported for the species feeding on mammalian blood. Subsequent instars also fed on and survived on cockroach hemolymph with some individuals maturing to adults. In the larger instars, development time and survival rates were reduced relative to the results reported in the literature for mammalian-blood-fed individuals. Two other species of kissing bugs, Triatoma protracta (Uhler) and T. rubida (Uhler) failed to survive on cockroach hemolymph with most individuals failing to molt from the first instar stage. Although T. recurva does not thrive on a diet limited to hemolymph of cockroaches, it appears to be an unusual species in which cockroaches might be a primary source of nutrition for smaller individuals and are a viable exclusive source of nutrition for all immatures. At a minimum during times of limited availability of vertebrate blood sources, the presence of cockroaches enhances survival opportunities. Efforts to control populations of this kissing bug species likely will be improved with additional control of cockroach populations in the environment.

Description of Triatomahuehuetenanguensis sp. n., a potential Chagas disease vector (Hemiptera, Reduviidae, Triatominae).

A new species of the genus Triatoma Laporte, 1832 (Hemiptera, Reduviidae) is described based on specimens collected in the department of Huehuetenango, Guatemala. Triatomahuehuetenanguensis sp. n. is closely related to T.dimidiata (Latreille, 1811), with the following main morphological differences: lighter color; smaller overall size, including head length; and width and length of the pronotum. Natural Trypanosomacruzi (Chagas, 1909) infection, coupled with its presence in domestic habitats, makes this species a potentially important vector of Trypanosomacruzi in Guatemala.

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.

Salivary protein profiles distinguish triatomine species and populations of Triatoma dimidiata (Hemiptera: Reduviidae).

Seven Triatoma dimidiata (Latreille, 1811) populations from different provinces of Guatemala were compared along with three related triatomine species using the electrophoretic profiles of salivary proteins. The analysis of salivary proteins allowed the separation of two of the species into their respective complexes, phyllosoma (T. pallidipennis) and protracta (T. nitida) (Lent and Wygodzinsky, 1979), whereas T. dimidiata seems slightly separated from either of these. Based on salivary protein profiles, T. dimidiata is most closely related to the cluster including T ryckmani and T. nitida (protracta) and more diverged from T. pallidipennis (phyllosoma). Among Guatemalan T. dimidiata populations, the cave population from Lanquin is separated from the rest of populations analyzed, suggesting that it is in the process of speciation. No difference in protein banding pattern was observed among populations from domestic and peridomestic ecotopes from the same region.