Optimization of DNA Extraction from Field-Collected Mammalian Whole Blood on Filter Paper for Trypanosoma cruzi (Chagas Disease) Detection.
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ABSTRACT:
SUBMITTER: Gulas-Wroblewski BE
PROVIDER: S-EPMC8398220 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundTo deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients.MethodsImmunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications.FindingsA2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system.InterpretationOur findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.
| S-EPMC7809174 | biostudies-literature
Project description: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.
| S-EPMC6722143 | biostudies-literature
Project description:Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and causes potentially life-threatening disease of the heart and gastrointestinal tract. The southern half of the United States contains enzootic cycles of T. cruzi, involving 11 recognized triatomine vector species. The greatest vector diversity and density occur in the western United States, where woodrats are the most common reservoir; other rodents, raccoons, skunks, and coyotes are also infected with T. cruzi. In the eastern United States, the prevalence of T. cruzi is highest in raccoons, opossums, armadillos, and skunks. A total of 7 autochthonous vector-borne human infections have been reported in Texas, California, Tennessee, and Louisiana; many others are thought to go unrecognized. Nevertheless, most T. cruzi-infected individuals in the United States are immigrants from areas of endemicity in Latin America. Seven transfusion-associated and 6 organ donor-derived T. cruzi infections have been documented in the United States and Canada. As improved control of vector- and blood-borne T. cruzi transmission decreases the burden in countries where the disease is historically endemic and imported Chagas' disease is increasingly recognized outside Latin America, the United States can play an important role in addressing the altered epidemiology of Chagas' disease in the 21st century.
| S-EPMC3194829 | biostudies-literature
Project description: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.
| S-EPMC7023793 | biostudies-literature
Project description:A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally-infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen's kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77-99) and 100% (95% CI: 80-100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62-1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations. The T. cruzi Loopamp kit appears potentially useful for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection.
| S-EPMC7458301 | biostudies-literature
Project description:Currently available drugs against Trypanosoma cruzi infection, which causes 12000 deaths annually, have limitations in their efficacy, safety and tolerability. The evaluation of therapeutic responses to available and new compounds is based on parasite detection in the bloodstream but remains challenging because a substantial proportion of infected individuals have undetectable parasitemia even when using diagnostic tools with the highest accuracy. We characterize parasite dynamics which might impact drug efficacy assessments in chronic Chagas by analyzing pre- and post-treatment quantitative-PCR data obtained from blood samples collected regularly over a year. We show that parasitemia remains at a steady-state independently of the diagnostic sensitivity. This steady-state can be probabilistically quantified and robustly predicted at an individual level. Furthermore, individuals can be assigned to categories with distinct parasitological status, allowing a more detailed evaluation of the efficacy outcomes and adjustment for potential biases. Our analysis improves understanding of parasite dynamics and provides a novel background for optimizing future drug efficacy trials in Chagas disease. Trial Registration: original trial registered with ClinicalTrials.gov, number NCT01489228.
| S-EPMC9721471 | biostudies-literature
Project description:Trypanosoma cruzi, the aetiological agent of Chagas disease, invades nucleated mammalian cells including macrophages. In this study, we investigated the crosstalk between T. cruzi-induced immune activation of reactive oxygen species (ROS) and pro-inflammatory responses, and their role in myocardial pathology. Splenocytes of infected mice (C3H/HeN) responded to Tc-antigenic stimulus by more than a two-fold increase in NADPH oxidase (NOX) activity, ROS generation, cytokine production (IFN-? > IL-4 > TNF? > IL1-?? IL6), and predominant expansion of CD4(+) and CD8(+) T cells. Inhibition of NOX, but not of myeloperoxidase and xanthine oxidase, controlled the ROS (>98%) and cytokine (70-89%) release by Tc-stimulated splenocytes of infected mice. Treatment of infected mice with apocynin (NOX inhibitor) in drinking water resulted in a 50-90% decline in endogenous NOX/ROS and cytokine levels, and splenic phagocytes' proliferation. The splenic percentage of T cells was maintained, though more than a 40% decline in splenic index (spleen weight/body weight) indicated decreased T-cell proliferation in apocynin-treated/infected mice. The blood and tissue parasite burden were significantly increased in apocynin-treated/infected mice, yet acute myocarditis, ie inflammatory infiltrate consisting of macrophages, neutrophils, and CD8(+) T cells, and tissue oxidative adducts (eg 8-isoprostanes, 3-nitrotyrosine, and 4-hydroxynonenal) were diminished in apocynin-treated/infected mice. Consequently, hypertrophy (increased cardiomyocytes' size and ?-MHC, BNP, and ANP mRNA levels) and fibrosis (increased collagen, glycosaminoglycans, and lipid contents) of the heart during the chronic phase were controlled in apocynin-treated mice. We conclude that NOX/ROS is a critical regulator of the splenic response (phagocytes, T cells, and cytokines) to T. cruzi infection, and bystander effects of heart-infiltrating phagocytes and CD8(+) T cells resulting in cardiac remodelling in chagasic mice.
| S-EPMC4378678 | biostudies-literature
Project description:This opinion piece presents an approach to standardisation of an important aspect of Chagas disease drug discovery and development: selecting Trypanosoma cruzi strains for in vitro screening. We discuss the rationale for strain selection representing T. cruzi diversity and provide recommendations on the preferred parasite stage for drug discovery, T. cruzi discrete typing units to include in the panel of strains and the number of strains/clones for primary screens and lead compounds. We also consider experimental approaches for in vitro drug assays. The Figure illustrates the current Chagas disease drug-discovery and development landscape.
| S-EPMC4238778 | biostudies-other
Project description:Acetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD(+)-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation of Trypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion by T. cruzi at concentrations that did not affect host cell viability. In addition, in vivo infection was partially controlled upon administration of salermide. There are two sirtuins in T. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed in Escherichia coli with a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 ?M. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.
| S-EPMC4505258 | biostudies-literature
Project description:The protozoan Trypanosoma cruzi almost invariably establishes life-long infections in humans and other mammals, despite the development of potent host immune responses that constrain parasite numbers. The consistent, decades-long persistence of T. cruzi in human hosts arises at least in part from the remarkable level of genetic diversity in multiple families of genes encoding the primary target antigens of anti-parasite immune responses. However, the highly repetitive nature of the genome-largely a result of these same extensive families of genes-have prevented a full understanding of the extent of gene diversity and its maintenance in T. cruzi. In this study, we have combined long-read sequencing and proximity ligation mapping to generate very high-quality assemblies of two T. cruzi strains representing the apparent ancestral lineages of the species. These assemblies reveal not only the full repertoire of the members of large gene families in the two strains, demonstrating extreme diversity within and between isolates, but also provide evidence of the processes that generate and maintain that diversity, including extensive gene amplification, dispersion of copies throughout the genome and diversification via recombination and in situ mutations. Gene amplification events also yield significant copy number variations in a substantial number of genes presumably not required for or involved in immune evasion, thus forming a second level of strain-dependent variation in this species. The extreme genome flexibility evident in T. cruzi also appears to create unique challenges with respect to preserving core genome functions and gene expression that sets this species apart from related kinetoplastids.
| S-EPMC7872254 | biostudies-literature