Protective efficacy of the oral vaccine Tc24:Co1 produced in Schizochytrium sp. against Trypanosoma cruzi infection in a mouse model.

Trypanosoma cruzi parasite - causal Chagas disease agent - affects about 7 million people; no vaccine is available, and current medications have not been entirely effective. Multidisciplinary efforts are necessary for developing clinical vaccine prototypes. Thus, this research study aims to assess the expressed and whole-cell administration protection of the oral vaccine prototype Tc24:Co1 using Schizochytrium sp. microalga. High recombinant protein expression yields (675 µg/L) of algal culture were obtained. Additionally, Schizochytrium sp.-Tc24:Co1 resulted stable at 4 °C for up to six months and at 25 °C for three months. After receiving four oral doses of the vaccine, the mice showed a significant humoral immune response and a parasitemia reduction associated with a lack of heart inflammatory damage compared with the unvaccinated controls. The Schizochytrium sp.-Tc24:Co1 vaccine demonstrates to be promising as a prototype for further development showing protective effects against a T. cruzi challenge in a mouse model.

Immunoglobulin and T cell receptor repertoire changes induced by a prototype vaccine against Chagas disease in naïve rhesus macaques.

BACKGROUND: A vaccine against Trypanosoma cruzi, the agent of Chagas disease, would be an excellent additional tool for disease control. A recombinant vaccine based on Tc24 and TSA1 parasite antigens was found to be safe and immunogenic in naïve macaques. METHODS: We used RNA-sequencing and performed a transcriptomic analysis of PBMC responses to vaccination of naïve macaques after each vaccine dose, to shed light on the immunogenicity of this vaccine and guide the optimization of doses and formulation. We identified differentially expressed genes and pathways and characterized immunoglobulin and T cell receptor repertoires. RESULTS: RNA-sequencing analysis indicated a clear transcriptomic response of PBMCs after three vaccine doses, with the up-regulation of several immune cell activation pathways and a broad non-polarized immune profile. Analysis of the IgG repertoire showed that it had a rapid turnover with novel IgGs produced following each vaccine dose, while the TCR repertoire presented several persisting clones that were expanded after each vaccine dose. CONCLUSIONS: These data suggest that three vaccine doses may be needed for optimum immunogenicity and support the further evaluation of the protective efficacy of this vaccine.

Monkeypox Virus Evolution before 2022 Outbreak.

Phylogenetic analysis of monkeypox virus genomes showed statistically significant divergence and nascent subclades during the 2022 mpox outbreak. Frequency of G>A/C>T transitions has increased in recent years, probably resulting from apolipoprotein B mRNA editing enzyme catalytic polypeptide 3G (APOBEC3) deaminase editing. This microevolutionary pattern most likely reflects community spread of the virus and adaptation to humans.

Genetic diversity of Trypanosoma cruzi infecting raccoons (Procyon lotor) in 2 metropolitan areas of southern Louisiana: implications for parasite transmission networks.

Trypanosoma cruzi, the aetiological agent of Chagas disease, exists as an anthropozoonosis in Louisiana. Raccoons are an important reservoir, as they demonstrate high prevalence and maintain high parasitaemia longer than other mammals. Given the complex nature of parasite transmission networks and importance of raccoons as reservoirs that move between sylvatic and domestic environments, detailing the genetic diversity of T. cruzi in raccoons is crucial to assess risk to human health. Using a next-generation sequencing approach targeting the mini-exon, parasite diversity was assessed in 2 metropolitan areas of Louisiana. Sequences were analysed along with those previously identified in other mammals and vectors to determine if any association exists between ecoregion and parasite diversity. Parasites were identified from discrete typing units (DTUs) TcI, TcII, TcIV, TcV and TcVI. DTUs TcII, TcV and TcVI are previously unreported in raccoons in the United States (US). TcI was the most abundant DTU, comprising nearly 80% of all sequences. All but 1 raccoon harboured multiple haplotypes, some demonstrating mixed infections of different DTUs. Furthermore, there is significant association between DTU distribution and level III ecoregion in Louisiana. Finally, while certain sequences were distributed across multiple tissues, others appeared to have tissue-specific tropism. Taken together, these findings indicate that ongoing surveillance of T. cruzi in the US should be undertaken across ecoregions to fully assess risk to human health. Given potential connections between parasite diversity and clinical outcomes, deep sequencing technologies are crucial and interventions targeting raccoons may prove useful in mitigating human health risk.

Geographic Variations in Test Reactivity for the Serological Diagnosis of Trypanosoma cruzi Infection.

Chagas disease is a neglected disease caused by Trypanosoma cruzi parasites. Most diagnosis is based on serological tests, but the lack of a gold standard test complicates the measurement of test performance. To overcome this limitation, we used samples from a cohort of well-characterized T. cruzi-infected women to evaluate the reactivity of two rapid diagnostic tests and one enzyme-linked immunosorbent assay (ELISA). Our cohort was derived from a previous study on congenital transmission of T. cruzi and consisted of 481 blood/plasma samples from Argentina (n = 149), Honduras (n = 228), and Mexico (n = 104), with at least one positive T. cruzi PCR. Reactivity of the three tests ranged from 70.5% for the Wiener ELISA to 81.0% for the T-Detect and 90.4% for the Stat-Pak rapid tests. Test reactivity varied significantly among countries and was highest in Argentina and lowest in Mexico. When considering at least two reactive serological tests to confirm seropositivity, over 12% of T. cruzi infection cases from Argentina were missed by serological tests, over 21% in Honduras, and an alarming 72% in Mexico. Differences in test performance among countries were not due to differences in parasitemia, but differences in antibody levels against ELISA antigens were observed. Geographic differences in T. cruzi parasite strains as well as genetic differences among human populations both may contribute to the discrepancies in serological testing. Improvements in serological diagnostics for T. cruzi infections are critically needed to ensure an optimum identification of cases.

Risk factors for infestation by Triatoma dimidiata in a rural locality of Veracruz, Mexico, with active transmission of Trypanosoma cruzi: weather and rain as factors.

OBJECTIVE: To analyse the ecological and social factors involved in infestation of houses by Triatoma dimidiata in a rural locality of Veracruz, Mexico, where active transmission of the parasite is occurring. METHODS: A survey was applied to the households of the locality to obtain sociodemographic data. In parallel, T. dimidiata insects were collected during one year through community participation. Using PCR, the insects were genotyped, their infection status was assessed, and parasite genotypes infecting the insects were identified. The vector's blood meal sources were identified using a polymerase-heteroduplex chain reaction assay. RESULTS: Seasonal variations in the patterns of infestation by T. dimidiata were observed. An overall infestation rate of 19.46%, a colonisation index of 9.09%, a dispersion rate of 22.15% and a synanthropy index of 80.6% were found. The collected insects were identified as ITS-2 group 2 insects, and a natural infection with T. cruzi of 54.35% was found. TcI and no-TcI genotypes of T. cruzi were found in infected insects. Factors such as rain (P = 0.0006) and temperature (P < 0.0001) were associated with infestation. Analysis of the blood meal sources indicated frequent feeding upon humans and mice. Furthermore, house materials and peridomiciles were found to play an important role in the dynamics of infestation. CONCLUSIONS: The contribution of this study is important for understanding the epidemiology of Chagas disease in rural areas of the state of Veracruz and will help to the establishment of an entomological surveillance system and implementation of prevention and control measures in accordance with the reality of the area.

Sequence of Trypanosoma cruzi reference strain SC43 nuclear genome and kinetoplast maxicircle confirms a strong genetic structure among closely related parasite discrete typing units.

Chagas disease is a zoonotic, parasitic, vector-borne neglected tropical disease that affects the lives of over 6 million people throughout the Americas. Trypanosoma cruzi, the causative agent, presents extensive genetic diversity. Here we report the genome sequence of reference strain SC43cl1, a hybrid strain belonging to the TcV discrete typing unit (DTU). The assembled diploid genome was 79 Mbp in size, divided into 1236 contigs with an average coverage reaching 180×. There was extensive synteny of SC43cl1 genome with closely related TcV and TcVI genomes, with limited sequence rearrangements. TcVI genomes included several expansions not present in TcV strains. Comparative analysis of both nuclear and kinetoplast sequences clearly separated TcV from TcVI strains, which strongly supports the current DTU classification.

Shelter cats host infections with multiple Trypanosoma cruzi discrete typing units in southern Louisiana.

Trypanosoma cruzi is a zoonotic parasite endemic in the southern US and the Americas, which may frequently infect dogs, but limited information is available about infections in cats. We surveyed a convenience sample of 284 shelter cats from Southern Louisiana to evaluate T. cruzi infection using serological and PCR tests. Parasites from PCR positive cats were also genotyped by PCR and deep sequencing to assess their genetic diversity. We detected a seropositivity rate for T. cruzi of at least 7.3% (17/234), and 24.6% of cats (70/284) were PCR positive for the parasite. Seropositivity increased with cat age (R2 = 0.91, P = 0.011), corresponding to an incidence of 7.2% ± 1.3 per year, while PCR positivity decreased with age (R2 = 0.93, P = 0.007). Cats were predominantly infected with parasites from TcI and TcVI DTUs, and to a lesser extent from TcIV and TcV DTUs, in agreement with the circulation of these parasite DTUs in local transmission cycles. These results indicate that veterinarians should have a greater awareness of T. cruzi infection in pets and that it would be important to better evaluate the risk for spillover infections in humans.

Fibronectin degradation as biomarker for Trypanosoma cruzi infection and treatment monitoring in mice.

Biomarkers (coming from host or parasite) to monitor Chagas disease (CD) progression as well as the therapeutic response in chronic CD are critically needed, since seronegativization, which may be considered the best indicator of therapeutic cure, takes several years to be observed in adults. Several molecules have been suggested as biomarkers for CD, however, they have to be validated. Taking advantage of mouse models of Trypanosoma cruzi infection, we investigated changes in the degradation profile of fibronectin in plasma. The degradation profile of fibronectin was different in the acute phase compared to the chronic phase of the infection. Fibronectin fragments of approximately 150, 100, 40 and 30 kDa were identified. Furthermore, those degradation profiles correlated with acute parasitaemia as well as with cardiac parasite burden and tissue damage during the infection. The usefulness of fibronectin degradation as a biomarker for therapeutic response following drug treatment and immunotherapeutic vaccination also was evaluated and a decreased fibronectin degradation profile was observed upon benznidazole or a vaccine candidate treatment.

Interactions among Triatoma sanguisuga blood feeding sources, gut microbiota and Trypanosoma cruzi diversity in southern Louisiana.

Integrating how biodiversity and infectious disease dynamics are linked at multiple levels and scales is highly challenging. Chagas disease is a vector-borne disease, with specificities of the triatomine vectors and Trypanosoma cruzi parasite life histories resulting in a complex multihost and multistrain life cycle. Here, we tested the hypothesis that T. cruzi transmission cycles are shaped by triatomine host communities and gut microbiota composition by comparing the integrated interactions of Triatoma sanguisuga in southern Louisiana with feeding hosts, T. cruzi parasite and bacterial microbiota in two habitats. Bugs were collected from resident's houses and animal shelters and analysed for genetic structure, blood feeding sources, T. cruzi parasites, and bacterial diversity by PCR amplification of specific DNA markers followed by next-generation sequencing, in an integrative metabarcoding approach. T. sanguisuga feeding host communities appeared opportunistic and defined by host abundance in each habitat, yielding distinct parasite transmission networks among hosts. The circulation of a large diversity of T. cruzi DTUs was also detected, with TcII and TcV detected for the first time in triatomines in the US. The bacterial microbiota was highly diverse and varied significantly according to the DTU infecting the bugs, indicating specific interactions among them in the gut. Expanding such studies to multiple habitats and additional triatomine species would be key to further refine our understanding of the complex life cycles of multihost, multistrain parasites such as T. cruzi, and may lead to improved disease control strategies.

Short-course Benznidazole treatment to reduce Trypanosoma cruzi parasitic load in women of reproductive age (BETTY): a non-inferiority randomized controlled trial study protocol.

BACKGROUND: Retrospective observational studies suggest that transmission of Trypanosoma cruzi does not occur in treated women when pregnant later in life. The level of parasitemia is a known risk factor for congenital transmission. Benznidazole (BZN) is the drug of choice for preconceptional treatment to reduce parasitic load. The fear of treatment-related side effects limits the implementation of the Argentine guideline recommending BZN 60d/300 mg (or equivalent) treatment of T. cruzi seropositive women during the postpartum period to prevent transmission in a future pregnancy. A short and low dose BZN treatment might reduce major side effects and increase compliance, but its efficacy to reduce T. cruzi parasitic load compared to the standard 60d/300 mg course is not yet established. Clinical trials testing alternative BZN courses among women of reproductive age are urgently needed. METHODS AND DESIGN: We are proposing to perform a double-blinded, non-inferiority randomized controlled trial comparing a short low dose 30-day treatment with BZN 150 mg/day (30d/150 mg) vs. BZN 60d/300 mg. We will recruit not previously treated T. cruzi seropositive women with a live birth during the postpartum period in Argentina, randomize them at 6 months postpartum, and follow them up with the following specific aims: Specific aim 1: to measure the effect of BZN 30d/150 mg compared to 60d/300 mg preconceptional treatment on parasitic load measured by the frequency of positive Polymerase Chain Reaction (PCR) (primary outcome) and by real-time quantitative PCR (qPCR), immediately and 10 months after treatment. Specific aim 2: to measure the frequency of serious adverse events and/or any adverse event leading to treatment interruption. TRIAL REGISTRATION: ClinicalTrials.gov . Identifier: NCT03672487 . Registered 14 September 2018.

Molecular Genotyping of Trypanosoma cruzi by Next-Generation Sequencing of the Mini-Exon Gene Reveals Infections With Multiple Parasite Discrete Typing Units in Chagasic Patients From Yucatan, Mexico.

The diversity of Trypanosoma cruzi parasites infecting humans is still poorly understood. We used deep sequencing to analyze this diversity in chagasic patients from Mexico. Such information is crucial to understand transmission cycles and to identify determinants of epidemiological and clinical characteristics of the infection. We analyzed parasite mini-exon spliced-leader sequences following amplification of blood DNA by polymerase chain reaction and deep sequencing. Chagasic patients presented a diverse assemblage of parasite haplotypes covering TcI, TcII, TcV, and TcVI discrete typing units, with a mean (±SEM) of 3.9 ± 0.7 haplotypes/patient, and 47% harbored infections with multiple discrete typing units. Most parasite haplotypes from patients were identical or similar to those for Triatoma dimidiata from the same region, confirming their local circulation. Infection with multiple T. cruzi strains may influence serological diagnostic test results and disease progression in patients and should be taken into account to evaluate associations between parasite diversity and clinical aspects of T. cruzi infections.

Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 µM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate.

Seroprevalence of Trypanosoma cruzi (TC) and risk factors in Colima, Mexico.

INTRODUCTION: The present study was conducted to estimate the incidence of seropositivity to anti-Trypanosoma cruzi antibodies and analyze potential risk factors in Colima, on the western coast of Mexico. METHODOLOGY: Longitudinal studies of 209 subjects with negative serology in 1999 for anti-Trypanosoma cruzi antibodies by hemagglutination inhibition test were tested again in 2005. At the same time, 716 children under six years of age were surveyed serologically (total n = 925); the history of Trypanosoma cruzi infection was determined by the same hemagglutination inhibition test. The variables analyzed were age, sex, living in triatomine-infested places, type of community, quality of housing, presence of pets, and number of inhabitants per house. RESULTS: Trypanosoma cruzi seropositivity in the period of six years was 22/925 cases, with a point prevalence of 2.73% and an adjusted rate of 7.3/1,000 person-years. The variable living in triatomine-infested areas showed association with seropositivity anti-Trypanosoma cruzi antibodies (RR: 5.5; 95% CI: 1.28-23.5). The remaining variables showed no significant association. CONCLUSIONS: This study confirms the active transmission of Chagas disease in Mexico´s western-central region, which merits greater epidemiological surveillance and vector control, particularly in localities infested with triatomines.

Ten years (2004-2014) of Chagas disease surveillance and vector control in Ecuador: successes and challenges.

OBJECTIVE: To describe the current situation of Chagas disease in Ecuador and to evaluate the impact of vector control for the period 2004-2014. METHODS: Since 2004, the Ministry of Public Health has formalized activities for the surveillance and control of Chagas disease and we analyzed here available records. RESULTS: More than 200 000 houses were surveyed, and 2.6% were found to be infested (95% CI: 2.6-2.7), and more than 51 000 houses were sprayed with residual insecticide, with important yearly variations. A total of 915 cases of T. cruzi infection were registered. The Amazon region is emerging as a high priority area, where nearly half of T. cruzi infection cases originate. The costal region and the southern highland valleys remain important high-risk area. Vector control efforts over the past 10 years have been effective in the coastal region, where T. dimidiata predominates, and resulted in important reductions in house infestation indices in many areas, even reaching negligible levels in some parishes. CONCLUSION: Vector efforts need to be sustained and expanded for the elimination of T. dimidiata to be feasible. Novel vector control interventions need to be designed to reduce intrusion by several triatomine species present in the Amazon region and southern Ecuador. Strong political commitment is needed to sustain current achievements and improve the national coverage of these programmes.

Spatial extent of an outbreak in animal epidemics.

Characterizing the spatial extent of epidemics at the outbreak stage is key to controlling the evolution of the disease. At the outbreak, the number of infected individuals is typically small, and therefore, fluctuations around their average are important: then, it is commonly assumed that the susceptible-infected-recovered mechanism can be described by a stochastic birth-death process of Galton-Watson type. The displacements of the infected individuals can be modeled by resorting to brownian motion, which is applicable when long-range movements and complex network interactions can be safely neglected, like in the case of animal epidemics. In this context, the spatial extent of an epidemic can be assessed by computing the convex hull enclosing the infected individuals at a given time. We derive the exact evolution equations for the mean perimeter and the mean area of the convex hull, and we compare them with Monte Carlo simulations.

From genome screening to creation of vaccine against Trypanosoma cruzi by use of immunoinformatics.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and activation of CD8(+) T cells is crucial for a protective immune response. Therefore, the identification of antigens with major histocompatibility complex class I epitopes is a crucial step for vaccine development against T. cruzi. Our aim was to identify novel antigens and epitopes by immunoinformatics analysis of the parasite proteome (12 969 proteins) and to validate their immunotherapeutic potential in infected mice. We identified 172 predicted epitopes, using NetMHC and RANKPEP. The corresponding protein sequences were reanalyzed to generate a consensus prediction, and 26 epitopes were selected for in vivo validation. The interferon γ (IFN-γ) recall response of splenocytes from T. cruzi-infected mice confirmed that 10 of 26 epitopes (38%) induced IFN-γ production. The immunotherapeutic potential of a mixture of all 10 peptides was evaluated in infected mice. The therapeutic vaccine was able to control T. cruzi infection, as evidenced by reduced parasitemia, cardiac tissue inflammation, and parasite burden and increased survival. These findings illustrate the benefits of this approach for the rapid development of a vaccine against pathogens with large genomes. The identified peptides and the proteins from which they are derived are excellent candidates for the development of a vaccine against T. cruzi.

Intrusive versus domiciliated triatomines and the challenge of adapting vector control practices against Chagas disease.

Chagas disease prevention remains mostly based on triatomine vector control to reduce or eliminate house infestation with these bugs. The level of adaptation of triatomines to human housing is a key part of vector competence and needs to be precisely evaluated to allow for the design of effective vector control strategies. In this review, we examine how the domiciliation/intrusion level of different triatomine species/populations has been defined and measured and discuss how these concepts may be improved for a better understanding of their ecology and evolution, as well as for the design of more effective control strategies against a large variety of triatomine species. We suggest that a major limitation of current criteria for classifying triatomines into sylvatic, intrusive, domiciliary and domestic species is that these are essentially qualitative and do not rely on quantitative variables measuring population sustainability and fitness in their different habitats. However, such assessments may be derived from further analysis and modelling of field data. Such approaches can shed new light on the domiciliation process of triatomines and may represent a key tool for decision-making and the design of vector control interventions.

Trypanosoma cruzi amastigote transcriptome analysis reveals heterogenous populations with replicating and dormant parasites.

Trypanosoma cruzi is a protozoan parasite causing Chagas disease, with a complex life cycle involving different stages in insect vectors and mammalian hosts. Amastigotes are an intracellular form that replicates in the cytoplasm of host cells, and recent studies suggested that dormant forms may be contributing to parasite persistence, suggesting cellular heterogeneity among amastigotes. We investigated here if a transcriptomic approach could identify some heterogeneity in intracellular amastigotes and identify a dormant population. We used gene expression data derived from bulk RNA-sequencing of T. cruzi infection of human fibroblasts for deconvolution using CDSeq, which allows to simultaneously estimate amastigote cell-type proportions and cell-type-specific expression profiles. Six amastigote subpopulations were identified, confirming intracellular amastigotes heterogeneity, and one population presented characteristics of non-replicative dormant parasites, based on replication markers and TcRAD51 expression. Transcriptomic approaches appear to be powerful to understand T. cruzi cell differentiation and expansion of these studies could provide further insight on the role different cell types in parasite persistence and Chagas disease pathogenesis.

Identification of highly conserved Trypanosoma cruzi antigens for the development of a universal serological diagnostic assay.

Chagas Disease is an important neglected tropical disease caused by Trypanosoma cruzi. There is no gold standard for diagnosis and commercial serological tests perform poorly in certain locations. By aligning T. cruzi genomes covering parasite genetic and geographic diversity, we identified highly conserved proteins that could serve as universal antigens for improved diagnosis. Their antigenicity was tested in high-density peptide microarrays using well-characterized plasma samples, including samples presenting true infections but discordant serology. Individual and combination of epitopes were also evaluated in peptide-ELISAs. We identified >1400 highly conserved T. cruzi proteins evaluated in microarrays. Remarkably, T. cruzi positive controls had a different epitope recognition profile compared to serologically discordant samples. In particular, multiple T. cruzi antigens used in current tests and their strain-variants, and novel epitopes thought to be broadly antigenic failed to be recognized by discordant samples. Nonetheless, >2000 epitopes specifically recognized by IgGs from both positive controls and discordant samples were identified. Evaluation of selected peptides in ELISA further illustrated the extensive variation in antibody profiles among subjects and a peptide combination could outperform a commercial ELISA, increasing assay sensitivity from 52.3% to 72.7%. Individual variation in antibody profiles rather than T. cruzi diversity appears to be the main factor driving differences in serological diagnostic performance according to geography, which will be important to further elucidate. ELISA with a combination of peptides recognized by a greater number of individuals could better capture infections, and further development may lead to an optimal antigen mixture for a universal diagnostic assay.