Improving in vitro screening compounds anti-Trypanosoma cruzi by GFP-expressing parasites.

BACKGROUND: Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. OBJECTIVES: In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. METHODS: Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. FINDINGS: The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. MAIN CONCLUSIONS: Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.

Comparative analysis of metacyclogenesis and infection curves in different discrete typing units of Trypanosoma cruzi.

Chagas disease (CD), caused by the complex life cycle parasite Trypanosoma cruzi, is a global health concern and impacts millions globally. T. cruzi's genetic variability is categorized into discrete typing units (DTUs). Despite their widespread presence in the Americas, a comprehensive understanding of their impact on CD is lacking. This study aims to analyze life cycle traits across life cycle stages, unraveling DTU dynamics. Metacyclogenesis curves were generated, inducing nutritional stress in epimastigotes of five DTUs (TcI (MG), TcI (DA), TcII(Y), TcIII, TcIV, and TcVI), resulting in metacyclic trypomastigotes. Infection dynamics in Vero cells from various DTUs were evaluated, exploring factors like amastigotes per cell, cell-derived trypomastigotes, and infection percentage. Statistical analyses, including ANOVA tests, identified significant differences. Varying onset times for metacyclogenesis converged on the 7th day. TcI (MG) exhibited the highest metacyclogenesis potential. TcI (DA) stood out, infecting 80% of cells within 24 h. TcI demonstrated the highest potential in both metacyclogenesis and infection among the strains assessed. Intra-DTU diversity was evident among TcI strains, contributing to a comprehensive understanding of Trypanosoma cruzi dynamics and genetic diversity.

Dynamics of the Trypanosoma cruzi infection in adipose tissue: Assessing gene expression of PNPLA2, FASN, and ACAT1 under Benzonidazole treatment and indirect mononuclear immune cells interaction.

Trypanosoma cruzi is a parasite with a high capacity to adapt to the host. Animal models have already demonstrated that the tropism of this parasite occurs not only in cardiac/digestive tissues but also in adipose tissue (AT). That said, the consequences ofT. cruziinfection for AT and the implications of treatment with Benzonidazole in this tissue are under discussion. Here, we tested the hypothesis that T. cruzi infection in adipose tissue upon treatment with Benzonidazole (Bz) and the interaction of mononuclear immune cells (PBMC) influences the relative expression of ACAT1, FASN, and PNPLA2 genes. Thus, stem cells derived from adipose tissue (ADSC) after adipogenic differentiation were indirectly cultivated with PBMC after infection with the T. cruzi Y strain and treatment with Bz. We use the TcSAT-IAM system and RT-qPCR to evaluate the parasite load and the relative quantification (ΔCt) of the ACAT1, FASN, and PNPLA2 genes. Our results demonstrate that treatment with Bz did not reduce adipocyte infection in the presence (p-value: 0.5796) or absence (p-value: 0.1854) of cultivation with PBMC. In addition, even though there is no statistical difference when compared to the control group (AT), T. cruzi induces the FASN expression (Rq: 14.00). However, treatment with Bz in AT suggests the increases of PNPLA2 expression levels (Rq: 12.58), even in the absence of T. cruzi infection. During indirect cultivation with PBMC, T. cruzi smooths the expression of PNPLA2 (Rq: 0.824) and instigates the expression of ACAT1 (Rq: 1.632) and FASN (Rq: 1.394). Furthermore, the treatment with Bz during infection induces PNPLA2 expression (Rq: 1.871), maintaining FASN expression levels (Rq: 1.334). Given this, our results indicate that treatment with Benzonidazole did not decrease T. cruzi infection in adipose tissue. However, treating the adipocyte cells with Bz during the interaction with PBMC cells influences the lipid pathways scenario, inducing lipolytic metabolism through the expression of PNPLA2.

New insights into Trypanosoma cruzi genetic diversity, and its influence on parasite biology and clinical outcomes.

Chagas disease, caused by Trypanosoma cruzi, remains a serious public health problem worldwide. The parasite was subdivided into six distinct genetic groups, called "discrete typing units" (DTUs), from TcI to TcVI. Several studies have indicated that the heterogeneity of T. cruzi species directly affects the diversity of clinical manifestations of Chagas disease, control, diagnosis performance, and susceptibility to treatment. Thus, this review aims to describe how T. cruzi genetic diversity influences the biology of the parasite and/or clinical parameters in humans. Regarding the geographic dispersion of T. cruzi, evident differences were observed in the distribution of DTUs in distinct areas. For example, TcII is the main DTU detected in Brazilian patients from the central and southeastern regions, where there are also registers of TcVI as a secondary T. cruzi DTU. An important aspect observed in previous studies is that the genetic variability of T. cruzi can impact parasite infectivity, reproduction, and differentiation in the vectors. It has been proposed that T. cruzi DTU influences the host immune response and affects disease progression. Genetic aspects of the parasite play an important role in determining which host tissues will be infected, thus heavily influencing Chagas disease's pathogenesis. Several teams have investigated the correlation between T. cruzi DTU and the reactivation of Chagas disease. In agreement with these data, it is reasonable to suppose that the immunological condition of the patient, whether or not associated with the reactivation of the T. cruzi infection and the parasite strain, may have an important role in the pathogenesis of Chagas disease. In this context, understanding the genetics of T. cruzi and its biological and clinical implications will provide new knowledge that may contribute to additional strategies in the diagnosis and clinical outcome follow-up of patients with Chagas disease, in addition to the reactivation of immunocompromised patients infected with T. cruzi.

Comparative proteomic analysis of the hemolymph and salivary glands of Rhodnius prolixus and R. colombiensis reveals candidates associated with differential lytic activity against Trypanosoma cruzi Dm28c and T. cruzi Y.

BACKGROUND: Immune response of triatomines plays an important role in the success or failure of transmission of T. cruzi. Studies on parasite-vector interaction have shown the presence of trypanolytic factors and have been observed to be differentially expressed among triatomines, which affects the transmission of some T. cruzi strains or DTUs (Discrete Typing Units). METHODOLOGY/PRINCIPAL FINDINGS: Trypanolytic factors were detected in the hemolymph and saliva of R. prolixus against epimastigotes and trypomastigotes of the Y strain (T. cruzi II). To identify the components of the immune response that could be involved in this lytic activity, a comparative proteomic analysis was carried out, detecting 120 proteins in the hemolymph of R. prolixus and 107 in R. colombiensis. In salivary glands, 1103 proteins were detected in R. prolixus and 853 in R. colombiensis. A higher relative abundance of lysozyme, prolixin, nitrophorins, and serpin as immune response proteins was detected in the hemolymph of R. prolixus. Among the R. prolixus salivary proteins, a higher relative abundance of nitrophorins, lipocalins, and triabins was detected. The higher relative abundance of these immune factors in R. prolixus supports their participation in the lytic activity on Y strain (T. cruzi II), but not on Dm28c (T. cruzi I), which is resistant to lysis by hemolymph and salivary proteins of R. prolixus due to mechanisms of evading oxidative stress caused by immune factors. CONCLUSIONS/SIGNIFICANCE: The lysis resistance observed in the Dm28c strain would be occurring at the DTU I level. T. cruzi I is the DTU with the greatest geographic distribution, from the south of the United States to central Chile and Argentina, a distribution that could be related to resistance to oxidative stress from vectors. Likewise, we can say that lysis against strain Y could occur at the level of DTU II and could be a determinant of the vector inability of these species to transmit T. cruzi II. Future proteomic and transcriptomic studies on vectors and the interactions of the intestinal microbiota with parasites will help to confirm the determinants of successful or failed vector transmission of T. cruzi DTUs in different parts of the Western Hemisphere.

First report of mixed Trypanosoma cruzi discrete typing units infection in Triatoma phyllosoma in the peri-urban environment of Oaxaca, Mexico.

BACKGROUND: Chagas disease, a zoonosis transmitted mainly by hematophagous insects of the subfamily Triatominae, is caused by Trypanosoma cruzi, classified into six discrete typing units (DTUs: TcI-TcVI and Tcbat). METHODS: Insect vectors were collected from 84 human dwellings in the municipality of Santo Domingo Tehuantepec, Oaxaca, Mexico; 4.76% were infested. DTUs were determined using conventional and nested PCR. RESULTS: The infection rate was 43.6%. All insects were infected with TcI while one specimen showed mixed infection with TcII. CONCLUSIONS: This is the first report of T. cruzi mixed infection in Triatoma phyllosoma, its main vector in the study region.

Integrating high-throughput analysis to create an atlas of replication origins in Trypanosoma cruzi in the context of genome structure and variability.

Trypanosoma cruzi is the etiologic agent of the most prevalent human parasitic disease in Latin America, Chagas disease. Its genome is rich in multigenic families that code for virulent antigens and are present in the rapidly evolving genomic compartment named Disruptive. DNA replication is a meticulous biological process in which flaws can generate mutations and changes in chromosomal and gene copy numbers. Here, integrating high-throughput and single-molecule analyses, we were able to identify Predominant, Flexible, and Dormant Orc1Cdc6-dependent origins as well as Orc1Cdc6-independent origins. Orc1Cdc6-dependent origins were found in multigenic family loci, while independent origins were found in the Core compartment that contains conserved and hypothetical protein-coding genes, in addition to multigenic families. In addition, we found that Orc1Cdc6 density is related to the firing of origins and that Orc1Cdc6-binding sites within fired origins are depleted of a specific class of nucleosomes that we previously categorized as dynamic. Together, these data suggest that Orc1Cdc6-dependent origins may contribute to the rapid evolution of the Disruptive compartment and, therefore, to the success of T. cruzi infection and that the local epigenome landscape is also involved in this process.IMPORTANCETrypanosoma cruzi, responsible for Chagas disease, affects millions globally, particularly in Latin America. Lack of vaccine or treatment underscores the need for research. Parasite's genome, with virulent antigen-coding multigenic families, resides in the rapidly evolving Disruptive compartment. Study sheds light on the parasite's dynamic DNA replication, discussing the evolution of the Disruptive compartment. Therefore, the findings represent a significant stride in comprehending T. cruzi's biology and the molecular bases that contribute to the success of infection caused by this parasite.

High-throughput analysis of the Trypanosoma cruzi minicirculome (mcDNA) unveils structural variation and functional diversity.

Trypanosoma cruzi causes Chagas disease and has a unique extranuclear genome enclosed in a structure called the kinetoplast, which contains circular genomes known as maxi- and minicircles. While the structure and function of maxicircles are well-understood, many aspects of minicircles remain to be discovered. Here, we performed a high-throughput analysis of the minicirculome (mcDNA) in 50 clones isolated from Colombia's diverse T. cruzi I populations. Results indicate that mcDNA comprises four diverse subpopulations with different structures, lengths, and numbers of interspersed semi-conserved (previously termed ultra-conserved regions mHCV) and hypervariable (mHVPs) regions. Analysis of mcDNA ancestry and inter-clone differentiation indicates the interbreeding of minicircle sequence classes is placed along diverse strains and hosts. These results support evidence of the multiclonal dynamics and random bi-parental segregation. Finally, we disclosed the guide RNA repertoire encoded by mcDNA at a clonal scale, and several attributes of its abundance and function are discussed.

Vaccination with parasite-specific TcTASV proteins combined with recombinant baculovirus as a delivery platform protects against acute and chronic Trypanosoma cruzi infection.

Chagas' is a neglected disease caused by the eukaryotic kinetoplastid parasite, Trypanosoma cruzi. Currently, approximately 8 million people are infected worldwide, most of whom are in the chronic phase of the disease, which involves cardiac, digestive, or neurologic manifestations. There is an urgent need for a vaccine because treatments are only effective in the initial phase of infection, which is generally underdiagnosed. The selection and combination of antigens, adjuvants, and delivery platforms for vaccine formulations should be designed to trigger mixed humoral and cellular immune responses, considering that T. cruzi has a complex life cycle with both intracellular and bloodstream circulating parasite stages in vertebrate hosts. Here, we report the effectiveness of vaccination with a T. cruzi-specific protein family (TcTASV), employing both recombinant proteins with aluminum hydroxide and a recombinant baculovirus displaying a TcTASV antigen at the capsid. Vaccination stimulated immunological responses by producing lytic antibodies and antigen-specific CD4+ and CD8+ IFNÉ£ secreting lymphocytes. More than 90% of vaccinated animals survived after lethal challenges with T. cruzi, whereas all control mice died before 30 days post-infection. Vaccination also induced a strong decrease in chronic tissue parasitism and generated immunological memory that allowed vaccinated and infected animals to control both the reactivation of the infection after immunosuppression and a second challenge with T. cruzi. Interestingly, inoculation with wild-type baculovirus partially protected the mice against T. cruzi. In brief, we demonstrated for the first time that the combination of the baculovirus platform and the TcTASV family provides effective protection against Trypanosoma cruzi, which is a promising vaccine for Chagas disease.

First Report of Chagas Disease Vector Species Triatoma sanguisuga (Hemiptera: Reduviidae) Infected with Trypanosoma cruzi in Delaware.

In July and October 2023, two live triatomine bugs were found inside a home in New Castle County, Delaware. The bugs were identified as Triatoma sanguisuga, the most widespread triatomine bug species in the United States. Triatoma sanguisuga is a competent vector of Trypanosoma cruzi, the causative agent of Chagas disease. The two specimens were tested via real-time PCR (qPCR) for infection with T. cruzi, and one of the specimens was positive. Despite T. sanguisuga being endemic to the area, attainment of accurate species identification and T. cruzi testing of the bugs required multiple calls to federal, state, private, and academic institutions over several months. This constitutes the first report of T. sanguisuga infected with T. cruzi in Delaware. In addition, this is the first published report of T. sanguisuga in New Castle County, the northernmost and most densely populated county in Delaware. New Castle County still conforms to the described geographic range of T. sanguisuga, which spans from Texas to the East Coast of the United States. The T. cruzi infection prevalence of the species has not been studied in the northeastern United States, but collections in southern states have found prevalences as high as 60%. The Delaware homeowner's lengthy pursuit of accurate information about the vector highlights the need for more research on this important disease vector in Delaware.

Enhancing Trypanosomatid Identification and Genotyping with Oxford Nanopore Sequencing: Development and Validation of an 18S rRNA Amplicon-Based Method.

Trypanosomatids, including Trypanosoma and Leishmania species, present significant medical and veterinary challenges, causing substantial economic losses, health complications, and even fatalities. Diagnosing and genotyping these species and their genotypes is often complex, involving multiple steps. This study aimed to develop an amplicon-based sequencing (ABS) method using Oxford Nanopore long-read sequencing to enhance Trypanosomatid detection and genotyping. The 18S rDNA gene was targeted for its inter-species conservation. The Trypanosomatid-ABS method effectively distinguished between 11 Trypanosoma species (including Trypanosoma evansi, Trypanosoma theileri, Trypanosoma vivax, and Trypanosoma rangeli) and 6 Trypanosoma cruzi discrete typing units (TcI to TcVI and TcBat), showing strong concordance with conventional methods (κ index of 0.729, P < 0.001). It detected co-infections between Trypanosomatid genera and T. cruzi, with a limit of detection of one parasite per mL. The method was successfully applied to human, animal, and triatomine samples. Notably, TcI predominated in chronic Chagas samples, whereas TcII and TcIV were found in the acute stage. Triatomine vectors exhibited diverse Trypanosomatid infections, with Triatoma dimidiata mainly infected with TcI and occasional TcBat co-infections, and Rhodnius prolixus showing TcI and TcII infections, along with T. rangeli co-infections and mixed TcII infections. Animals were infected with T. vivax, T. theileri, and T. evansi. The ABS method's high resolution, sensitivity, and accuracy make it a valuable tool for understanding Trypanosomatid dynamics, enhancing disease control strategies, and enabling targeted interventions.

Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras.

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.

Transmission ecology of Trypanosoma cruzi by Rhodnius prolixus (Reduviidae: Triatominae) infesting palm-tree species in the Colombian Orinoco, indicates risks to human populations.

BACKGROUND: Chagas disease, affecting approximately eight million individuals in tropical regions, is primarily transmitted by vectors. Rhodnius prolixus, a triatomine vector, commonly inhabits in ecotopes with diverse palm tree species, creating optimal conditions for vector proliferation. This study aims to explore the transmission ecology of Trypanosoma cruzi, the causative parasite of Chagas disease, by investigating the feeding patterns and natural infection rates of R. prolixus specimens collected from various wild palm species in the Colombian Orinoco region. MATERIALS AND METHODS: To achieve this objective, we sampled 35 individuals from three palm species (Attalea butyracea, Acrocomia aculeata, and Mauritia flexuosa) in a riparian forest in the Casanare department of eastern Colombia, totaling 105 sampled palm trees. DNA was extracted and analyzed from 115 R. prolixus specimens at different developmental stages using quantitative PCR (qPCR) for T. cruzi detection and identification of discrete typing units. Feeding preferences were determined by sequencing the 12S rRNA gene amplicon through next-generation sequencing. RESULTS: A total of 676 R. prolixus specimens were collected from the sampled palms. The study revealed variation in population densities and developmental stages of R. prolixus among palm tree species, with higher densities observed in A. butyracea and lower densities in M. flexuosa. TcI was the exclusive T. cruzi discrete typing unit (DTU) found, with infection frequency positively correlated with R. prolixus abundance. Insects captured in A. butyracea exhibited higher abundance and infection rates than those from other palm species. The feeding sources comprised 13 mammal species, showing no significant differences between palm species in terms of blood sources. However, Didelphis marsupialis and Homo sapiens were present in all examined R. prolixus, and Dasypus novemcinctus was found in 89.47% of the insects. CONCLUSION: This study highlights the significance of wild palms, particularly A. butyracea, as a substantial risk factor for T. cruzi transmission to humans in these environments. High population densities and infection rates of R. prolixus were observed in each examined palm tree species.

Trypanosomatid diversity in a bat community of an urban area in Campo Grande, Mato Grosso do Sul, Brazil.

Bats have a long evolutionary history with trypanosomatids, but the role of these flying mammals on parasite transmission cycles in urban areas, especially for Trypanosoma and Leishmania species, remains poorly known. The objective of this study was to evaluate the species richness of trypanosomatids parasitizing a bat community in Campo Grande (CG), a state capital within the Cerrado of the Brazilian Midwest. We evaluated 237 bats of 13 species by means of hemoculture and molecular detection in spleen samples. The bat community of CG appears to participate in the transmission cycles of various species of trypanosomatids. We report an overall trypanosomatid detection rate of 34.2% (n = 81), involving 11 out of 13 sampled bat species. We identified six species of trypanosomatids from 61 bats by analyzing SSU rRNA and/or kDNA: Trypanosoma cruzi DTU TcI, T. c. marinkellei, T. dionisii, Leishmania infantum, L. amazonensis, and T. janseni, with this latter being detected by hemoculture for the first time in a bat species. We also detected a Molecular Operational Taxonomic Unit, Trypanosoma sp. DID, in the phyllostomids Glossophaga soricina and Platyrrhinus lineatus. The highest trypanosomatid richness was observed for Sturnira lilium, which hosted three species: L. infantum, T. dionisii and T. janseni. Given that visceral leishmaniasis is endemic in CG, special focus should be placed on L. infantum. Moreover, L. amazonensis and T. cruzi warrant attention, since these are zoonotic parasites responsible for human cases of tegumentary leishmaniasis and Chagas disease, respectively. In this respect, we discuss how bat communities may influence the Leishmania spp. transmission in endemic areas.

Population genetics and genomics of Triatoma brasiliensis (Hemiptera, Reduviidae) in an area of high pressure of domiciliary infestation in Northeastern Brazil.

Understanding the population dynamics of vectors is crucial for effective control of vector-borne diseases. In the Northeastern Brazilian semi-arid region, Triatoma brasiliensis persists as the most significant Chagas disease vector, frequently displaying recurrent domiciliary infestations. This situation raises relevant public health concerns in the municipality of Currais Novos in the state of Rio Grande do Norte. This area has experienced a high prevalence of peridomiciliary re-infestations by T. brasiliensis, coupled with elevated rates of Trypanosoma cruzi infection. Therefore, we assessed the distribution of genetic variation via mitochondrial Cytochrome b gene (MT-CYB) sequencing (n = 109) and single nucleotide polymorphisms (SNPs, n = 86) to assess the gene flow among distinct populations distributed in varied geographic spots and environments, mainly sylvatic and peridomiciliary. Insects were collected from rural communities at Currais Novos, enclosed within a 16 km radius. Sampling included 13 populations: one intradomiciliary, eight peridomiciliary, and four sylvatic. Furthermore, an external population located 220 km from Currais Novos was also included in the study. The method employed to obtain SNP information relied on ddRAD-seq genotyping-by-sequencing (GBS), enabling a genome-wide analysis to infer genetic variation. Through AMOVA analysis of MT-CYB gene variation, we identified four distinct population groups with statistical significance (FCT= 0.42; p<0.05). We identified a total of 3,013 SNPs through GBS, with 11 loci showing putative signs of being under selection. The variation based on 3,002 neutral loci evidenced low genetic structuration based on low FST values (p>0.05), indicating local panmixia. However, resampling algorithms pointed out that three samples from the external population were assigned (>98 %) in a cluster contrasting from the ones putatively under local panmixia - validating the newly applied genome-wide marker for studies on the population genetics at finer-scale resolution for T. brasiliensis. The presence of population structuring in some of the sampled points, as suggested by the mitochondrial marker, leads us to assume that infestations were probably initiated by small populations of females - demographic event poses a risk for rapid re-infestations. The local panmictic pattern revealed by the GBS marker poses a challenge for vector control measures, as re-infestation foci may be distributed over a wide geographical and ecological range. In such instances, vectors exhibit reduced susceptibility to conventional insecticide spraying operations since sylvatic populations are beyond the reach of these interventions. The pattern of infestation exhibited by T. brasiliensis necessitates integrating innovative strategies into the existing control framework, holding the potential to create a more resilient and adaptive vector control program. In our dataset, the results demonstrated that the genetic signals from both markers were complementary. Therefore, it is essential to consider the nature and inheritance pattern of each marker when inferring the pattern of re-infestations.

IgG Isotypes Targeting a Recombinant Chimeric Protein of Trypanosoma cruzi in Different Clinical Presentations of Chronic Chagas Disease.

Chagas disease (CD) is caused by the protozoan Trypanosoma cruzi, which leads to a spectrum of clinical presentations that range from asymptomatic to severe cardiac involvement. The host immune response plays a pivotal role in disease progression. Ig isotypes may contribute to disease pathogenesis. Investigating these components can provide insights into the immunopathogenic mechanisms underlying CD. This cross-sectional study aims to establish a correlation between the Ig profile of individuals infected with T. cruzi with the clinical forms of chronic CD. Serum samples were collected from partner institutions in different states of Brazil. Individuals diagnosed with chronic CD were categorized based on the clinical form of the disease. The indirect ELISA method using the recombinant chimeric Molecular Biology Institute of Paraná membrane protein 8.4 as the antigen was used to determine the Ig profile, including total IgG, IgG1, IgG2, IgG3, and IgG4. Ninety-seven serum samples from patients classified as negative (NEG, n = 38), indeterminate (IND, n = 24), mild cardiac (MC, n = 20), and severe cardiac (SC, n = 15) forms were analyzed. IgG1 exhibited greater levels compared with the other isotypes, showing a significant difference between the MC and IND groups. IgG3 levels were greater in individuals from the MC group compared with the SC group. IgG1 and IgG3 isotypes can serve as biomarkers to evaluate the progression of CD because they exhibit variations across clinical groups. Additional longitudinal studies are necessary to explore the relationship between antibody kinetics and the development of tissue damage.

Quantitative PCR as a marker for preemptive therapy and its role in therapeutic control in Trypanosoma cruzi/HIV coinfection.

BACKGROUND: Trypanosoma cruzi and HIV coinfection can evolve with depression of cellular immunity and increased parasitemia. We applied quantitative PCR (qPCR) as a marker for preemptive antiparasitic treatment to avoid fatal Chagas disease reactivation and analyzed the outcome of treated cases. METHODOLOGY: This mixed cross-sectional and longitudinal study included 171 Chagas disease patients, 60 coinfected with HIV. Of these 60 patients, ten showed Chagas disease reactivation, confirmed by parasites identified in the blood, cerebrospinal fluid, or tissues, 12 exhibited high parasitemia without reactivation, and 38 had low parasitemia and no reactivation. RESULTS: We showed, for the first time, the success of the timely introduction of benznidazole in the non-reactivated group with high levels of parasitemia detected by qPCR and the absence of parasites in reactivated cases with at least 58 days of benznidazole. All HIV+ patients with or without reactivation had a 4.0-5.1 higher chance of having parasitemia than HIV seronegative cases. A positive correlation was found between parasites and viral loads. Remarkably, treated T. cruzi/HIV-coinfected patients had 77.3% conversion from positive to negative parasitemia compared to 19.1% of untreated patients. Additionally, untreated patients showed ~13.6 times higher Odds Ratio of having positive parasitemia in the follow-up period compared with treated patients. Treated and untreated patients showed no differences regarding the evolution of Chagas disease. The main factors associated with all-cause mortality were higher parasitemia, lower CD4 counts/µL, higher viral load, and absence of antiretroviral therapy. CONCLUSION: We recommend qPCR prospective monitoring of T. cruzi parasitemia in HIV+ coinfected patients and point out the value of pre-emptive therapy for those with high parasitemia. In parallel, early antiretroviral therapy introduction is advisable, aiming at viral load control, immune response restoration, and increasing survival. We also suggest an early antiparasitic treatment for all coinfected patients, followed by effectiveness analysis alongside antiretroviral therapy.

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.

Mapping the transporter-substrate interactions of the Trypanosoma cruzi NB1 nucleobase transporter reveals the basis for its high affinity and selectivity for hypoxanthine and guanine and lack of nucleoside uptake.

Trypanosoma cruzi is a protozoan parasite and the etiological agent of Chagas disease, a debilitating and sometimes fatal disease that continues to spread to new areas. Yet, Chagas disease is still only treated with two related nitro compounds that are insufficiently effective and cause severe side effects. Nucleotide metabolism is one of the known vulnerabilities of T. cruzi, as they are auxotrophic for purines, and nucleoside analogues have been shown to have genuine promise against this parasite in vitro and in vivo. Since purine antimetabolites require efficient uptake through transporters, we here report a detailed characterisation of the T. cruzi NB1 nucleobase transporter with the aim of elucidating the interactions between TcrNB1 and its substrates and finding the positions that can be altered in the design of novel antimetabolites without losing transportability. Systematically determining the inhibition constants (Ki) of purine analogues for TcrNB1 yielded their Gibbs free energy of interaction, ΔG0. Pairwise comparisons of substrate (hypoxanthine, guanine, adenine) and analogues allowed us to determine that optimal binding affinity by TcrNB1 requires interactions with all four nitrogen residues of the purine ring, with N1 and N9, in protonation state, functioning as presumed hydrogen bond donors and unprotonated N3 and N7 as hydrogen bond acceptors. This is the same interaction pattern as we previously described for the main nucleobase transporters of Trypanosoma brucei spp. and Leishmania major and makes it the first of the ENT-family genes that is functionally as well as genetically conserved between the three main kinetoplast pathogens.

Detection and Genotyping of Trypanosoma cruzi Samples in Species of Genus Rhodnius from Different Environments in the Brazilian Amazon.

Background: In the Amazon region, several species of triatomines occur in the natural environments. Among them, species of the genus Rhodnius are a risk to human populations due to their high rates of infection with Trypanosoma cruzi. The aim of this study was to identify the T. cruzi genotypes in Rhodnius specimens and their relationship with sylvatic hosts from different environments in the Brazilian Amazon. Methods: A total of 492 triatomines were collected from the municipalities of Monte Negro, Rondônia state, and Humaitá, Amazonas state, 382 of them being nymphs and 110 adults. Genotyping of T. cruzi in six discrete typing units (DTUs) was performed using conventional multilocus PCR. The triatomines that were positive for T. cruzi and engorged with blood were also targeted for amplification of the cytochrome B (cytB) gene to identify bloodmeal sources. Results: Of the 162 positive samples, the identified DTUs were TcI (87.65%) and TcIV (12.35%). It was observed that 102 specimens were engorged with a variety of bloodmeals. Triatomines infected with TcI were associated with DNA of all identified vertebrates, except Plecturocebus brunneus. TcIV was detected in triatomines that fed on Coendou prehensilis, Didelphis marsupialis, Mabuya nigropunctata, P. brunneus, Pithecia irrorata, Sapajus apella, and Tamandua tetradactyla. Conclusion: Results highlight the need to understand the patterns of T. cruzi genotypes in Rhodnius spp. and their association with sylvatic hosts to better elucidate their role in the transmission of Chagas disease in the Amazon region.