Correction: Vector mapping and bloodmeal metabarcoding demonstrate risk of urban Chagas disease transmission in Caracas, Venezuela.

[This corrects the article DOI: 10.1371/journal.pntd.0010613.].

Vector mapping and bloodmeal metabarcoding demonstrate risk of urban Chagas disease transmission in Caracas, Venezuela.

Chagas disease is a significant public health risk in rural and semi-rural areas of Venezuela. Triatomine infection by the aetiological agent Trypanosoma cruzi is also observed in the Metropolitan District of Caracas (MDC), where foodborne T. cruzi outbreaks occasionally occur but active vector-to-human transmission (infection during triatomine bloodmeal) is considered absent. Citizen science-based domiciliary triatomine collection carried out between 2007 and 2013 in the MDC has advanced understanding of urban T. cruzi prevalence patterns and represents an important public awareness-building tool. The present study reports on the extension of this triatomine collection program from 2014 to 2019 and uses mitochondrial metabarcoding to assess feeding behavior in a subset of specimens. The combined, thirteen-year dataset (n = 4872) shows a high rate of T. cruzi infection (75.2%) and a predominance of Panstrongylus geniculatus (99.01%) among triatomines collected in domiciliary areas by MDC inhabitants. Collection also involved nymphal stages of P. geniculatus in 18 of 32 MDC parishes. Other collected species included Triatoma nigromaculata, Triatoma maculata, Rhodnius prolixus, and Panstrongylus rufotuberculatus. Liquid intestinal content indicative of bloodmeal was observed in 53.4% of analyzed specimens. Dissection pools representing 108 such visually blooded P. geniculatus specimens predominantly tested positive for human cytochrome b DNA (22 of 24 pools). Additional bloodmeal sources detected via metabarcoding analysis included key sylvatic T. cruzi reservoirs (opossum and armadillo), rodents, and various other synanthropic and domesticated animals. Results suggest a porous sylvatic-domiciliary transmission interface and ongoing adaptation of P. geniculatus to the urban ecotope. Although P. geniculatus defecation traits greatly limit the possibility of active T. cruzi transmission for any individual biting event, the cumulation of this low risk across a vast metropolitan population warrants further investigation. Efforts to prevent triatomine contact with human food sources also clearly require greater attention to protect Venezuela's capital from Chagas disease.

Intraspecific and Interspecific Phenotypic Differences Confirm the Absence of Cryptic Speciation in Triatoma sordida (Hemiptera, Triatominae).

Triatoma sordida is an endemic Chagas disease vector in South America, distributed in Brazil, Bolivia, Paraguay, and Uruguay. Chromosomal, molecular, isoenzymatic, and cuticular hydrocarbon pattern studies indicate cryptic speciation in T. sordida. Recently, T. rosai was described from specimens from Argentina initially characterized as T. sordida. Although several authors assume that the speciation process that supports this differentiation in T. sordida is the result of cryptic speciation, further morphological and/or morphometric studies are necessary to prove the application of this evolutionary event, because the only morphological intraspecific comparison performed in T. sordida is based on geometric morphometry and the only interspecific comparison made is between T. rosai and T. sordida from Brazil that evaluated morphological and morphometric differences. Based on this, morphological analyses of thorax and abdomen using Scanning Electron Microscopy and morphometric analyses of the head, thorax, and abdomen among T. sordida from Brazil, Bolivia, and Paraguay, as well as T. rosai, were performed to assess whether the evolutionary process responsible for variations is the cryptic speciation phenomenon. Morphological differences in the thorax and female external genitalia, as well as morphometric differences in the head, thorax, abdomen, pronotum, and scutellum structures, were observed. Based on this, the evolutionary process that supports, so far, these divergences observed for T. sordida populations/T. sordida subcomplex is not cryptic speciation. Moreover, we draw attention to the necessity for morphological/morphometric studies to correctly apply the cryptic species/speciation terms in triatomines.

Chagas Disease Vectors of Paraguay: Entomoepidemiological Aspects of Triatoma sordida (Stål, 1859) and Development of an Identification Key for Paraguayan Triatomines Based on Cytogenetics Data.

Approximately 150,000 people are living with Chagas disease in Paraguay. Although the country has been since 2008 considered as one of the countries that succeeded in interrupted the vector transmission of Chagas by Triatoma infestans in houses of the eastern region, there are nine other species notified in the country that are potential vectors and also deserve attention from vector control programs. Thus, we carried out an entomoepidemiological study of T. sordida in the eastern and western regions of the country and we developed an identification key for Paraguay's triatomines based on cytogenetic data. Between the years 2003 to 2004, 271 specimens of T. sordida were captured in domestic, peridomestic, and wild ecotopes, with 131 insects caught in the eastern (Alto Paraguay, Boquerón and Pte. Hayes) and 140 in the western region of Paraguay (Guairá and Paraguarí). High rates of peridomicillary infestation were observed for both regions. Besides that, the natural infection of the captured insects was detected by optical microscopy in 12% and 10%, and by PCR in 21% and 20% in the eastern and western regions, respectively. Based on cytogenetic data from nine of ten species notified in Paraguay, an identification key was developed to differentiate all taxa. Thus, given the vectorial importance of T. sordida, we highlight the need for continued attention from Paraguay's vector control programs for this species. Further, we provide a taxonomic key that assists in the correct classification of Paraguayan triatomines.

Repeat-Driven Generation of Antigenic Diversity in a Major Human Pathogen, Trypanosoma cruzi.

Trypanosoma cruzi, a zoonotic kinetoplastid protozoan parasite, is the causative agent of American trypanosomiasis (Chagas disease). Having a very plastic, repetitive and complex genome, the parasite displays a highly diverse repertoire of surface molecules, with pivotal roles in cell invasion, immune evasion and pathogenesis. Before 2016, the complexity of the genomic regions containing these genes impaired the assembly of a genome at chromosomal level, making it impossible to study the structure and function of the several thousand repetitive genes encoding the surface molecules of the parasite. We here describe the genome assembly of the Sylvio X10/1 genome sequence, which since 2016 has been used as a reference genome sequence for T. cruzi clade I (TcI), produced using high coverage PacBio single-molecule sequencing. It was used to analyze deep Illumina sequence data from 34 T. cruzi TcI isolates and clones from different geographic locations, sample sources and clinical outcomes. Resolution of the surface molecule gene distribution showed the unusual duality in the organization of the parasite genome, a synteny of the core genomic region with related protozoa flanked by unique and highly plastic multigene family clusters encoding surface antigens. The presence of abundant interspersed retrotransposons in these multigene family clusters suggests that these elements are involved in a recombination mechanism for the generation of antigenic variation and evasion of the host immune response on these TcI strains. The comparative genomic analysis of the cohort of TcI strains revealed multiple cases of such recombination events involving surface molecule genes and has provided new insights into T. cruzi population structure.

Genetic variability of Panstrongylus geniculatus (Reduviidae: Triatominae) in the Metropolitan District of Caracas, Venezuela.

Panstrongylus geniculatus has become the most frequently registered vector of Chagas disease in the metropolitan area of Caracas, Venezuela. This triatomine species has invaded urban areas in recent years and has been implicated in multiple oral outbreaks of Chagas disease in the region. The study of genetic variability and spatial structure in P. geniculatus populations can provide information about possible events of domiciliation and aid intervention programs against triatomine species rapidly adapting to urban ecotopes. We sequenced a region of the cytochrome-b gene in 114 specimens of P. geniculatus from the Metropolitan District of Caracas and assessed patterns of gene flow and phylogenetic relationships among these individuals. A total of 29 haplotypes were detected in the two sampled municipalities, Sucre and Libertador. Though high genetic connectivity was observed between the municipalities (FST = 0.10796; Nm = 11.20), subtle genetic structuring was also observed in particular geographic sub regions. Based on neutrality tests and the observed allele-frequency distribution, the Panstrongylus geniculatus population appears to be expanding and adapting to different microhabitats present in the study area. Our findings affirm the capacity of this insect to adapt to different environments and emphasize its principal role in the epidemiology of Chagas disease in northern Venezuela.

Proliferación de trypanosoma cruzi en la membrana peritoneal y líquido ascítico de ratones con infección aguda / Proliferation of trypanosoma cruzi in the peritoneal membrane and ascitic liquid of mice with acute infection

En este estudio fue analizada la parasitemia y el parasitismo en el líquido ascítico (LA), membrana peritoneal (MP) y en otros tejidos de 40 ratones machos NMRI durante la infección aguda. Los ratones (20 ratones por grupo) fueron inoculados por vía intradérmica con tripomastigotos metacíclicos linaje T.cr I de las cepas P6 y P11 obtenidos de Rhodnius prolixus. Formas delgadas y gruesas de tripomastigotos fueron encontradas en la sangre de todos los ratones. En los ratones infectados con la cepa P6 los tripomastigotos aparecieron primero en él LA a los 13 días pi y en la sangre a los 18 días pi, en los ratones infectados con la cepa P11 los parásitos se observaron primero en la sangre a los 15 días pi y en él LA a los 22 días pi. Los ratones desarrollaron emaciación, disnea, hirsutismo, pérdida de la actividad motora de las patas posteriores y hepatoesplenomegalia. Los ratones fueron sacrificados a los 39 días pi. El estudio histológico mostró que T. cruzi prolifera formando nidos de amastigotos y tripomastigotos en la MP. Los parásitos también fueron encontrados en el músculo esquelético y en el corazón de los ratones infectados con la cepa P6. La inmunotinción con PAP reveló antígeno de T. cruzi en las secciones de esófago, estómago, intestino delgado y grueso, bazo, riñón, hígado, próstata y pene de los ratones. Estos resultados confirmaron que las cepas P6 y P11 desarrollaron anormalidades histopatológicas en el tracto gastrointestinal, renal y órgano reproductivo. La localización intra-peritoneal de los parásitos y la acumulación de fluido peritoneal, reveló ascitis y peritonitis causada por el incremento de líquido en la cavidad peritoneal y destrucción del tejido peritoneal de los ratones. El presente estudio reporta por primera vez la proliferación de tripomastigotos en la cavidad peritoneal cinco días antes de encontrarse en la sangre periférica para la cepa P6 causando daño intraperitoneal y muerte del modelo murino utilizado.

In this study we analyzed the parasitemia and parasitism in the ascitic fluid (AF), peritoneal membrane (PM) and in other tissues of 40 NMRI male mice during acute infection. Mice (20 mice per group) were inoculated by intradermal route with metacyclic trypomastigotes T.cI lineage of P6 or P11 strains obtained from Rhodnius prolixus. Slender and stout forms were observed in the blood of all mice. In infected mice with P6 strain the trypomastigotes were observed first in the AF at day 13 pi and in blood at day 18 pi. Meanwhile in infected mice with P11 strain trypomastigotes were observed first in the blood at day 15 pi and in the AF at day 22 pi. Infected mice showed emaciation, dyspnea, bristled hair, loss of motor activities in the rear limbs and hepatosplenomegaly. Mice were sacrificed at day 39 pi. Histological finding indicated that T. cruzi proliferates forming amastigotes and trypomastigotes nests in the PM. Parasites were also observed in skeletal muscle of the mice and in the heart of infected mice with P6 strain. Imunostaining with PAP revealed T. cruzi antigen in esophagus, stomach, thin and thick intestine, spleen, and kidney, liver, prostate and penis. The results show that P6 and P11 colonized and produced abnormalities in the gastrointestinal tract, renal and reproductive organs. Intra-peritoneal localization of parasites and accumulation of peritoneal fluid, revealed ascites and peritonitis caused by increase of fluid in the peritoneal cavity and destruction of the membrane peritoneal of the mice. This study reports for the first time the proliferation of trypomastigotes in the peritoneal cavity five days earlier than in peripheral blood for the P6 strain causing intra-peritoneal damage and death in the murine model used.

Electrocardiography repolarization abnormalities are characteristic signs of acute chagasic cardiomyopathy / Los trastornos de la repolarización ventricular son signos característicos de la cardiomiopatía chagásica aguda

Chagas disease is a tropical parasitic disease caused by the protozoan Trypanosoma cruzi (T. cruzi), whose reemergence as oral outbreaks is currently a public health problem in Venezuela. T. cruzi infection induces myocardial damage; which according to the microvascular theory, is derived from parasite-mediated disruption of the endothelium, inducing platelet aggregation and ischemia. In order to determine whether ventricular repolarization disorders observed in human patients are characteristic signs of the disease that can be reproduced in NMRI mice; we studied 12 patients with a well documented diagnosis of acute Chagas disease, based on epidemiological, clinical, parasitological and molecular data. Also, T. cruzi isolates from the blood of human patients from other Venezuelan geographical regions were characterized and inoculated in albino NMRI mice. A standard 12-lead and bipolar electrocardiogram configuration were done in human patients during the acute phase of the disease and in mice, after three weeks of infection. Results in human showed repolarization disorders, characterized by: negative, bimodal or biphasic T waves, ST segment depression or elevation and early repolarization. In mice a significant increase in T wave amplitude, increased QT interval duration and elevation or depression of ST segment were observed. These findings were evidenced in all infected mice, suggesting that electrocardiographic repolarization abnormalities in a well documented clinical and epidemiological context are signs that increase the sensitivity for the diagnosis of acute Chagas´ disease.

La enfermedad de Chagas es una hemoparasitosis causada por Trypanosoma cruzi (T. cruzi), cuya re-emergencia como epidemias por contaminación oral es actualmente un problema de salud pública en Venezuela. La infección por T. cruzi causa miocarditis; que de acuerdo con la teoría microvascular deriva del daño del endotelio vascular, al inducir agregación plaquetaria e isquemia. Con el objetivo de demostrar que los trastornos de repolarización son signos propios de la miocarditis chagásica aguda (MChA) reproducibles en modelos animales, estudiamos 12 pacientes humanos con diagnostico bien documentado de MChA, basado en datos epidemiológicos, clínicos, parasitológicos y moleculares. A partir de la sangre de los pacientes obtuvimos los aislados de T cruzi, los caracterizamos molecularmente y los inoculamos en ratones albinos NMRI; paralelamente, aislados de T cruzi provenientes de otras regiones de Venezuela fueron también ensayados. Tanto en los pacientes humanos como en los ratones con Chagas agudo, se realizaron estudios electrocardiográficos en 12 derivaciones estándares y en configuración bipolar, respectivamente. En humanos observamos trastornos de la repolarización ventricular caracterizados por: onda T negativa, bimodal o bifásica; elevación o depresión del segmento ST y despolarizaciones tempranas. En ratones observamos incrementos en la amplitud de la onda T, aumento en la duración del intervalo QT y elevación o depresión del segmento ST. Estos hallazgos fueron evidenciados en todos los ratones infectados con los diferentes aislados, sugiriendo que los trastornos de repolarización, en un adecuado y bien documentado contexto epidemiológico y clínico, son signos que aumentan la sensibilidad para el diagnóstico de MChA.

Electrocardiography repolarization abnormalities are characteristic signs of acute chagasic cardiomyopathy.

Chagas disease is a tropical parasitic disease caused by the protozoan Trypanosoma cruzi (T. cruzi), whose reemergence as oral outbreaks is currently a public health problem in Venezuela. T. cruzi infection induces myocardial damage; which according to the microvascular theory, is derived from parasite-mediated disruption of the endothelium, inducing platelet aggregation and ischemia. In order to determine whether ventricular repolarization disorders observed in human patients are characteristic signs of the disease that can be reproduced in NMRI mice; we studied 12 patients with a well documented diagnosis of acute Chagas disease, based on epidemiological, clinical, parasitological and molecular data. Also, T. cruzi isolates from the blood of human patients from other Venezuelan geographical regions were characterized and inoculated in albino NMRI mice. A standard 12-lead and bipolar electrocardiogram configuration were done in human patients during the acute phase of the disease and in mice, after three weeks of infection. Results in human showed repolarization disorders, characterized by: negative, bimodal or biphasic T waves, ST segment depression or elevation and early repolarization. In mice a significant increase in T wave amplitude, increased QT interval duration and elevation or depression of ST segment were observed. These findings were evidenced in all infected mice, suggesting that electrocardiographic repolarization abnormalities in a well documented clinical and epidemiological context are signs that increase the sensitivity for the diagnosis of acute Chagas' disease.

Dietary fiber analysis of cassava using gravimetric methods

We report the application of a method which combines digestion with pancreatin and neutral detergent treatment in the analytical study of dietary fiber from cassava. The use of pancreatin previous to the detergent extraction enabled rapid filtration, thus giving more reproducible results for neutral detergent fiber (NDF). Acid detergent fiber (ADF), hemicellulose, lignin and pectin were also determined. The values obtained for NDF (4.65 per cent ) and pectin (1.17 per cent ) are very important, considering their role in the digestive process