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.

Comparative proteomic analysis of Trypanosoma cruzi TcI lineage epimastigotes unveils metabolic and phenotypic differences between fast- and slow-dividing strains.

Trypanosoma cruzi, the causative agent of Chagas disease, is a genetically and phenotypically diverse species, divided into 5 main phylogenetic lineages (TcI to TcVI). TcI is the most widespread lineage in the Americas. Proteomics is a suitable tool to study the global protein expression dynamics in pathogens. Previous proteomic studies have revealed a link between (i) the genetic variability; (ii) the protein expression; and (iii) the biological characteristics of T. cruzi. Here, two-dimensional electrophoresis (2DE) and mass spectrometry were used to characterize the overall protein expression profiles of epimastigotes from four distinct TcI strains displaying different growth kinetics. Ascending hierarchical clustering analysis based on the global 2DE protein expression profiles grouped the strains under study into two clusters that were congruent with their fast or slow growth kinetics. A subset of proteins differentially expressed by the strains in each group were identified by mass spectrometry. Biological differences between the two groups, including use of glucose as an energy source, flagellum length, and metabolic activity, were predicted by proteomic analysis and confirmed by metabolic tests and microscopic measurements performed on the epimastigotes of each strain. Our results show that protein expression profiles are correlated with parasite phenotypes, which may in turn influence the parasite's virulence and transmission capacity.

Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity.

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/µl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

COVID-19 Vaccine Hesitancy in Three Latin American Countries: Reasons Given for Not Becoming Vaccinated in Colombia, Ecuador, and Venezuela.

Although vaccines have been developed to prevent COVID-19, vaccine hesitancy is a significant barrier for vaccination programs. Most research on COVID-19 vaccine hesitancy has blamed misinformation and misstated concerns about effectiveness, safety, and side effects of these vaccines. The preponderance of these studies has been performed in the Global North. Although Latin American has been substantially and negatively impacted by COVID-19, few studies have examined COVID-19 vaccine hesitancy there. We explored reasons volunteered for COVID-19 vaccine hesitancy from a sample of 1,173 Colombians, Ecuadorians, and Venezuelans. Overall, COVID-19 vaccine hesitancy in these three countries is higher than desirable, but most people who are COVID-19 vaccine hesitant offered one reason or fewer. The reasons offered are diverse, including myths and exaggerations, but also individual-level contraindications for vaccination and structural barriers. Because of the diversity of reasons, single-issue mass campaigns are unlikely to bring about large shifts in COVID-19 vaccine hesitancy in Colombia, Ecuador, and Venezuela. Our data suggest that interpersonal communication, particularly in Ecuador, and addressing structural concerns, particularly in Venezuela, are likely to have the greatest impact on vaccine uptake.

Fatal acute Chagas disease by Trypanosoma cruzi DTU TcI, Ecuador.

BACKGROUND: Chagas disease is caused by the haemoflagellate protozoan Trypanosoma cruzi. Currently, T. cruzi recognizes seven discrete typing units (DTUs): TcI to TcVI and Tcbat. The genetic diversity of T. cruzi is suspected to influence the clinical outcome. Acute clinical manifestations, which include myocarditis and meningoencephalitis, are sometimes fatal; occur most frequently in children and in immunocompromised individuals. Acute disease is often overlooked, leading to a poor prognosis. CASE PRESENTATION: A 38-year-old man from a subtropical area of the Andes mountains of Ecuador was hospitalized after 3 weeks of evolution with high fever, chills, an enlarged liver, spleen, and lymph nodes, as well as facial edema. ECG changes were also observed. T. cruzi was identified in blood smears, culture and amplification of DNA by PCR. Tests for anti-T. cruzi IgG and IgM and HIV were negative. Molecular typing by restriction fragment length polymorphism (PCR-RFLP) determined the parasite to DTU TcI. In the absence of a timely anti-T. cruzi medication, the patient died. CONCLUSIONS: This is a case of severe pathogenicity and the virulence of a DTU TcI strain in an adult patient. The severe acute Chagas disease was probably overlooked due to limited awareness and its low incidence. Our findings suggest that T. cruzi DTU TcI strains circulating in Ecuador are capable of causing fatal acute disease. Early diagnosis and prompt treatment is of paramount importance to avoid fatalities in acute infections.

Knowledge, Attitudes, and Practices Towards COVID-19 Among Ecuadorians During the Outbreak: An Online Cross-Sectional Survey.

Preventing the transmission of SARS-CoV-2 (causative agent for COVID-19) requires implementing contact and respiratory precautions. Modifying human behavior is challenging and requires understanding knowledge, attitudes, and practices (KAPs) regarding health threats. This study explored KAPs among people in Ecuador. A cross-sectional, internet-based questionnaire was used to assess knowledge about COVID-19, attitudes toward ability to control COVID-19, self-reported practices related to COVID-19, and demographics. A total of 2399 individuals participated. Participants had moderate to high levels of knowledge. Participants expressed mixed attitudes about the eventual control of COVID-19 in Ecuador. Participants reported high levels of adoption of preventive practices. Binomial regression analysis suggests unemployed individuals, househusbands/housewives, or manual laborers, as well as those with an elementary school education, have lower levels of knowledge. Women, people over 50 years of age, and those with higher levels of schooling were the most optimistic. Men, individuals 18-29, single, and unemployed people took the riskiest behaviors. Generally, knowledge was not associated with optimism or with practices. Our findings indicate knowledge about COVID-19 is insufficient to prompt behavioral change among Ecuadorians. Since current COVID-19 control campaigns seek to educate the public, these efforts' impacts are likely to be limited. Given attitudes determine people's actions, further investigation into the factors underlying the lack of confidence in the ability of the world, and of Ecuador, to overcome COVID-19, is warranted. Edu-communicational campaigns should be accompanied by efforts to provide economically disadvantaged populations resources to facilitate adherence to recommendations to prevent the spread of the virus.

[Social factors associated with use of prenatal care in Ecuador]. / Factores sociales asociados con la utilización de los servicios de atención prenatal en Ecuador.

OBJECTIVES: Prenatal care is a pillar of public health, enabling access to interventions including prevention of mother-to-child transmission of HIV and congenital syphilis. This paper describes social factors related to use of prenatal care in Ecuador. METHODS: In 2011 and 2012, participant clinical history and interview information was analyzed from a national probability sample of 5 998 women presenting for delivery or miscarriage services in 15 healthcare facilities in Ecuador, to estimate prevalence of HIV, syphilis, and Chagas disease, and prenatal care coverage. RESULTS: The study found that 94.1% of women had attended at least one prenatal visit, but that attendance at no less than four visits was 73.1%. Furthermore, lower educational level, greater number of pregnancies, occupation in the agriculture or livestock sector, and membership in ethnic indigenous, Afro-Ecuadorian, or other minority groups were factors associated with lack of use (no prenatal visits) or insufficient use of prenatal care (fewer than four visits or first visit at >20 weeks gestation) in Ecuador. CONCLUSIONS: These results point to persistence of marked inequalities in access to and use of prenatal health services attributable to socioeconomic factors and to the need to strengthen strategies to address them, to reach the goal of universal prenatal care coverage.

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.

High prevalence of Zika virus infection in populations of Aedes aegypti from South-western Ecuador.

We performed an arboviral survey in mosquitoes from four endemic Ecuadorian cities (Huaquillas, Machala, Portovelo and Zaruma) during the epidemic period 2016-2018. Collections were performed during the pre-rainy season (2016), peak transmission season (2017) and post-rainy season (2018). Ae. aegypti mosquitoes were pooled by date, location and sex. Pools were screened by RT-PCR for the presence of ZIKV RNA, and infection rates (IRs) per 1,000 specimens were calculated. A total of 2,592 pools (comprising 6,197 mosquitoes) were screened. Our results reveal high IRs in all cities and periods sampled. Overall IRs among female mosquitoes were highest in Machala (89.2), followed by Portovelo (66.4), Zaruma (47.4) and Huaquillas (41.9). Among male mosquitoes, overall IRs were highest in Machala (35.6), followed by Portovelo (33.1), Huaquillas (31.9) and Zaruma (27.9), suggesting that alternative transmission routes (vertical/venereal) can play important roles for ZIKV maintenance in the vector population of these areas. Additionally, we propose that the stabilization of ZIKV vertical transmission in the vector population could help explain the presence of high IRs in field-caught mosquitoes during inter-epidemic periods.

Bioactivity of synthetic peptides from Ecuadorian frog skin secretions against Leishmania mexicana, Plasmodium falciparum, and Trypanosoma cruzi.

Chagas disease, leishmaniasis, and malaria are major parasitic diseases disproportionately affecting the underprivileged population in developing nations. Finding new, alternative anti-parasitic compounds to treat these diseases is crucial because of the limited number of options currently available, the side effects they cause, the need for long treatment courses, and the emergence of drug-resistant parasites. Anti-microbial peptides (AMPs) derived from amphibian skin secretions are small bioactive molecules capable of lysing the cell membrane of pathogens while having low toxicity against human cells. Here, we report the anti-parasitic activity of five AMPs derived from skin secretions of three Ecuadorian frogs: cruzioseptin-1, cruzioseptin-4 (CZS-4), and cruzioseptin-16 from Cruziohyla calcarifer; dermaseptin-SP2 from Agalychnis spurrelli; and pictuseptin-1 from Boana picturata. These five AMPs were chemically synthesized. Initially, the hemolytic activity of CZS-4 and its minimal inhibitory concentration against Escherichia coli, Staphylococcus aureus, and Candida albicans were determined. Subsequently, the cytotoxicity of the synthetic AMPs against mammalian cells and their anti-parasitic activity against Leishmania mexicana promastigotes, erythrocytic stages of Plasmodium falciparum and mammalian stages of Trypanosoma cruzi were evaluated in vitro. The five AMPs displayed activity against the pathogens studied, with different levels of cytotoxicity against mammalian cells. In silico molecular docking analysis suggests this bioactivity may occur via pore formation in the plasma membrane, resulting in microbial lysis. CZS-4 displayed anti-bacterial, anti-fungal, and anti-parasitic activities with low cytotoxicity against mammalian cells. Further studies about this promising AMP are required to gain a better understanding of its activity.IMPORTANCEChagas disease, malaria, and leishmaniasis are major tropical diseases that cause extensive morbidity and mortality, for which available treatment options are unsatisfactory because of limited efficacy and side effects. Frog skin secretions contain molecules with anti-microbial properties known as anti-microbial peptides. We synthesized five peptides derived from the skin secretions of different species of tropical frogs and tested them against cultures of the causative agents of these three diseases, parasites known as Trypanosoma cruzi, Plasmodium falciparum, and Leishmania mexicana. All the different synthetic peptides studied showed activity against one of more of the parasites. Peptide cruzioseptin-4 is of special interest since it displayed intense activity against parasites while being innocuous against cultured mammalian cells, which indicates it does not simply hold general toxic properties; rather, its activity is specific against the parasites.

High growth hormone serum partially protects mice against Trypanosoma cruzi infection.

Chagas disease (CD) is one of the most devasting parasitic diseases in the Americas, affecting 7-8 million people worldwide. In vitro and in vivo experiments have demonstrated that growth hormone (GH) serum levels decrease as CD progresses. Interestingly, inactivating mutations in the GH receptor in humans result in Laron syndrome (LS), a clinical entity characterized by increased serum levels of GH and decreased insulin growth factor-1 (IGF-1). The largest cohort of LS subjects lives in the southern provinces of Ecuador. Remarkably, no clinical CD cases have been reported in these individuals despite living in highly endemic areas. In the current ex vivo study, we employed serum from GHR-/- mice, also known as LS mice (a model of GH resistance with high GH and low IGF-1 levels), and serum from bovine GH (bGH) transgenic mice (high GH and IGF-1), to test the effect on Trypanosoma cruzi infection. We infected mouse fibroblast L-cells with T. cruzi (etiological CD infectious agent) and treated them with serum from each mouse type. Treatment with GHR-/- serum (LS mice) significantly decreased L-cell infection by 28% compared with 48% from control wild-type mouse serum (WT). Treatment with bGH mouse serum significantly decreased infection of cells by 41% compared with 54% from WT controls. Our results suggest that high GH and low IGF-1 in blood circulation, as typically seen in LS individuals, confer partial protection against T. cruzi infection. This study is the first to report decreased T. cruzi infection using serum collected from two modified mouse lines with altered GH action (GHR-/- and bGH).

Growth hormone modulates Trypanosoma cruzi infection in vitro.

OBJECTIVE: Chagas disease (CD) is caused by the protozoan parasite, Trypanosoma cruzi. It affects 7 to 8 million people worldwide and leads to approximately 50,000 deaths per year. In vitro and in vivo studies had demonstrated that Trypanosoma cruziinfection causes an imbalance in the hypothalamic-pituitary-adrenal (HPA) axis that is accompanied by a progressive decrease in growth hormone (GH) and prolactin (PRL) production. In humans, inactivating mutations in the GH receptor gene cause Laron Syndrome (LS), an autosomal recessive disorder. Affected subjects are short, have increased adiposity, decreased insulin-like growth factor-I (IGFI), increased serum GH levels, are highly resistant to diabetes and cancer, and display slow cognitive decline. In addition, CD incidence in these individuals is diminished despite living in highly endemic areas. Consequently, we decided to investigate the in vitro effect of GH/IGF-I on T. cruzi infection. DESIGN: We first treated the parasite and/or host cells with different peptide hormones including GH, IGFI, and PRL. Then, we treated cells using different combinations of GH/IGF-I attempting to mimic the GH/IGF-I serum levels observed in LS subjects. RESULTS: We found that exogenous GH confers protection against T. cruzi infection. Moreover, this effect is mediated by GH and not IGFI. The combination of relatively high GH (50 ng/ml) and low IGF-I (20 ng/ml), mimicking the hormonal pattern seen in LS individuals, consistently decreased T. cruzi infection in vitro. CONCLUSIONS: The combination of relatively high GH and low IGF-I serum levels in LS individuals may be an underlying condition providing partial protection against T. cruzi infection.

In vitro susceptibility of Trypanosoma cruzi discrete typing units (DTUs) to benznidazole: A systematic review and meta-analysis.

BACKGROUND: Chagas disease, a neglected tropical disease endemic to Latin America caused by the parasite Trypanosoma cruzi, currently affects 6-7 million people and is responsible for 12,500 deaths each year. No vaccine exists at present and the only two drugs currently approved for the treatment (benznidazole and nifurtimox), possess serious limitations, including long treatment regimes, undesirable side effects, and frequent clinical failures. A link between parasite genetic variability and drug sensibility/efficacy has been suggested, but remains unclear. Therefore, we investigated associations between T. cruzi genetic variability and in vitro benznidazole susceptibility via a systematic article review and meta-analysis. METHODOLOGY/PRINCIPAL FINDINGS: In vitro normalized benznidazole susceptibility indices (LC50 and IC50) for epimastigote, trypomastigote and amastigote stages of different T. cruzi strains were recorded from articles in the scientific literature. A total of 60 articles, which include 189 assays, met the selection criteria for the meta-analysis. Mean values for each discrete typing unit (DTU) were estimated using the meta and metaphor packages through R software, and presented in a rainforest plot. Subsequently, a meta-regression analysis was performed to determine differences between estimated mean values by DTU/parasite stage/drug incubation times. For each parasite stage, some DTU mean values were significantly different, e.g. at 24h of drug incubation, a lower sensitivity to benznidazole of TcI vs. TcII trypomastigotes was noteworthy. Nevertheless, funnel plots detected high heterogeneity of the data within each DTU and even for a single strain. CONCLUSIONS/SIGNIFICANCE: Several limitations of the study prevent assigning DTUs to different in vitro benznidazole sensitivity groups; however, ignoring the parasite's genetic variability during drug development and evaluation would not be advisable. Our findings highlight the need for establishment of uniform experimental conditions as well as a screening of different DTUs during the optimization of new drug candidates for Chagas disease treatment.

Parasite-Mediated Remodeling of the Host Microfilament Cytoskeleton Enables Rapid Egress of Trypanosoma cruzi following Membrane Rupture.

Chagas' disease arises as a direct consequence of the lytic cycle of Trypanosoma cruzi in the mammalian host. While invasion is well studied for this pathogen, study of egress has been largely neglected. Here, we provide the first description of T. cruzi egress documenting a coordinated mechanism by which T. cruzi engineers its escape from host cells in which it has proliferated and which is essential for maintenance of infection and pathogenesis. Our results indicate that this parasite egress is a sudden event involving coordinated remodeling of host cell cytoskeleton and subsequent rupture of host cell plasma membrane. We document that host cells maintain plasma membrane integrity until immediately prior to parasite release and report the sequential transformation of the host cell's actin cytoskeleton from normal meshwork in noninfected cells to spheroidal cages-a process initiated shortly after amastigogenesis. Quantification revealed gradual reduction in F-actin over the course of infection, and using cytoskeletal preparations and electron microscopy, we were able to observe disruption of the F-actin proximal to intracellular trypomastigotes. Finally, Western blotting experiments suggest actin degradation driven by parasite proteases, suggesting that degradation of cytoskeleton is a principal component controlling the initiation of egress. Our results provide the first description of the cellular mechanism that regulates the lytic component of the T. cruzi lytic cycle. We show graphically how it is possible to preserve the envelope of host cell plasma membrane during intracellular proliferation of the parasite and how, in cells packed with amastigotes, differentiation into trypomastigotes may trigger sudden egress. IMPORTANCE Understanding how Trypanosoma cruzi interacts with host cells has been transformed by high-quality studies that have examined in detail the mechanisms of T. cruzi host cell invasion. In contrast, little is known about the latter stages of the parasite's lytic cycle: how parasites egress and thereby sustain round after round of infection. Our results show that once in the host cell cytosol and having undergone amastigogenesis, T. cruzi begins to alter the host cell cytoskeleton, remodeling normal F-actin meshworks into encapsulating spheroidal cages. Filamentous actin diminishes over the course of the lytic cycle, and just prior to egress, the filaments comprising the cages are severely degraded where adjacent to the parasites. We conclude that sudden egress follows breach of the containment afforded by the actin cytoskeleton and subsequent plasma membrane rupture-a process that when understood in molecular detail may serve as a target for future novel therapeutic interventions.

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.

Remarkable genetic diversity of Trypanosoma cruzi and Trypanosoma rangeli in two localities of southern Ecuador identified via deep sequencing of mini-exon gene amplicons.

BACKGROUND: Trypanosoma cruzi, the causative agent of Chagas disease, and T. rangeli are kinetoplastid parasites endemic to Latin America. Although closely related to T. cruzi and capable of infecting humans, T. rangeli is non-pathogenic. Both parasite species are transmitted by triatomine bugs, and the presence of T. rangeli constitutes a confounding factor in the study of Chagas disease prevalence and transmission dynamics. Trypanosoma cruzi possesses high molecular heterogeneity: seven discrete typing units (DTUs) are currently recognized. In Ecuador, T. cruzi TcI and T. rangeli KP1(-) predominate, while other genetic lineages are seldom reported. METHODS: Infection by T. cruzi and/or T. rangeli in different developmental stages of triatomine bugs from two communities of southern Ecuador was evaluated via polymerase chain reaction product size polymorphism of kinetoplast minicircle sequences and the non-transcribed spacer region of the mini-exon gene (n = 48). Forty-three mini-exon amplicons were also deep sequenced to analyze single-nucleotide polymorphisms within single and mixed infections. Mini-exon products from ten monoclonal reference strains were included as controls. RESULTS: Trypanosoma cruzi genetic richness and diversity was not significantly greater in adult vectors than in nymphal stages III and V. In contrast, instar V individuals showed significantly higher T. rangeli richness when compared with other developmental stages. Among infected triatomines, deep sequencing revealed one T. rangeli infection (3%), 8 T. cruzi infections (23.5%) and 25 T. cruzi + T. rangeli co-infections (73.5%), suggesting that T. rangeli prevalence has been largely underestimated in the region. Furthermore, deep sequencing detected TcIV sequences in nine samples; this DTU had not previously been reported in Loja Province. CONCLUSIONS: Our data indicate that deep sequencing allows for better parasite identification/typing than amplicon size analysis alone for mixed infections containing both T. cruzi and T. rangeli, or when multiple T. cruzi DTUs are present. Additionally, our analysis showed extensive overlap among the parasite populations present in the two studied localities (c.28 km apart), suggesting active parasite dispersal over the study area. Our results highlight the value of amplicon sequencing methodologies to clarify the population dynamics of kinetoplastid parasites in endemic regions and inform control campaigns in southern Ecuador.

Cytokine-dependent and-independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling.

BACKGROUND: The requirements for growth and survival of the intracellular pathogen Trypanosoma cruzi within mammalian host cells are poorly understood. Transcriptional profiling of the host cell response to infection serves as a rapid read-out for perturbation of host physiology that, in part, reflects adaptation to the infective process. Using Affymetrix oligonucleotide array analysis we identified common and disparate host cell responses triggered by T. cruzi infection of phenotypically diverse human cell types. RESULTS: We report significant changes in transcript abundance in T. cruzi-infected fibroblasts, endothelial cells and smooth muscle cells (2852, 2155 and 531 genes respectively; fold-change > or = 2, p-value < 0.01) 24 hours post-invasion. A prominent type I interferon response was observed in each cell type, reflecting a secondary response to secreted cytokine in infected cultures. To identify a core cytokine-independent response in T. cruzi-infected fibroblasts and endothelial cells transwell plates were used to distinguish cytokine-dependent and -independent gene expression profiles. This approach revealed the induction of metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding as common themes in T. cruzi-infected cells. In addition, the downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection may impede host cell cycle progression. The observation of impaired cytokinesis in T. cruzi-infected cells, following nuclear replication, confirmed this prediction. CONCLUSION: Metabolic pathways and cellular processes were identified as significantly altered at the transcriptional level in response to T. cruzi infection in a cytokine-independent manner. Several of these alterations are supported by previous studies of T. cruzi metabolic requirements or effects on the host. However, our methods also revealed a T. cruzi-dependent block in the host cell cycle, at the level of cytokinesis, previously unrecognized for this pathogen-host cell interaction.

Congenital Chagas Disease in the Ecuadorian Amazon: Maternal Screening at Delivery and Evaluation of Risk Factors Associated with Vector Exposure.

Congenital infection with Trypanosoma cruzi remains a major route for Chagas disease transmission in endemic and non-endemic regions. We evaluated an intervention strategy aimed to detect congenital Chagas disease cases at a major hospital in the Ecuadorian Amazon via cord blood analysis at the time of delivery. All women giving birth at the hospital during the study period (191) were invited to participate. Among them, two (1.0%) did not adjust to the inclusion criteria and four (2.1%) declined to participate in the study, showing the intervention had good acceptability among the mothers. It was possible to obtain cord blood samples during 146 of the deliveries, and only one woman was found to be seropositive, without evidence of transmission to the newborn at delivery or 8 months later. In addition, sociodemographic and economic characterization of the study population revealed that few women had previous knowledge about Chagas disease (16.1%) whereas more than half (62.5%) recognized the vector. Recognizing the vector and having seen it indoors were associated with women from rural families, involved in agriculture, and hunting in the forest. Interestingly, most women (87.3%) reported having easy access to Ecuador's national health system, suggesting serological screening during prenatal visits would be of value in this province. With a proper prenatal screening system in place, cord blood screening would allow for timely detection of T. cruzi infection in newborns from both seropositive women and the minority (2.1%) of women who do not comply with prenatal care visits.

Meiotic sex in Chagas disease parasite Trypanosoma cruzi.

Genetic exchange enables parasites to rapidly transform disease phenotypes and exploit new host populations. Trypanosoma cruzi, the parasitic agent of Chagas disease and a public health concern throughout Latin America, has for decades been presumed to exchange genetic material rarely and without classic meiotic sex. We present compelling evidence from 45 genomes sequenced from southern Ecuador that T. cruzi in fact maintains truly sexual, panmictic groups that can occur alongside others that remain highly clonal after past hybridization events. These groups with divergent reproductive strategies appear genetically isolated despite possible co-occurrence in vectors and hosts. We propose biological explanations for the fine-scale disconnectivity we observe and discuss the epidemiological consequences of flexible reproductive modes. Our study reinvigorates the hunt for the site of genetic exchange in the T. cruzi life cycle, provides tools to define the genetic determinants of parasite virulence, and reforms longstanding theory on clonality in trypanosomatid parasites.

Venezuela's humanitarian crisis, resurgence of vector-borne diseases, and implications for spillover in the region.

In the past 5-10 years, Venezuela has faced a severe economic crisis, precipitated by political instability and declining oil revenue. Public health provision has been affected particularly. In this Review, we assess the impact of Venezuela's health-care crisis on vector-borne diseases, and the spillover into neighbouring countries. Between 2000 and 2015, Venezuela witnessed a 359% increase in malaria cases, followed by a 71% increase in 2017 (411 586 cases) compared with 2016 (240 613). Neighbouring countries, such as Brazil, have reported an escalating trend of imported malaria cases from Venezuela, from 1538 in 2014 to 3129 in 2017. In Venezuela, active Chagas disease transmission has been reported, with seroprevalence in children (<10 years), estimated to be as high as 12·5% in one community tested (n=64). Dengue incidence increased by more than four times between 1990 and 2016. The estimated incidence of chikungunya during its epidemic peak is 6975 cases per 100 000 people and that of Zika virus is 2057 cases per 100 000 people. The re-emergence of many vector-borne diseases represents a public health crisis in Venezuela and has the possibility of severely undermining regional disease elimination efforts. National, regional, and global authorities must take action to address these worsening epidemics and prevent their expansion beyond Venezuelan borders.