Assessment of Liaison XL Murex Chagas diagnostic performance in blood screening for Chagas disease using a reference array of chimeric antigens.

BACKGROUND: Chagas disease (CD) serological screening at blood banks is usually performed by a single highly sensitive serological assay, with chemiluminescent immunoassays (CLIAs) being the method of choice. CLIAs employ recombinant, fusion peptides and/or chimeric antigens that selectively capture anti-Trypanosoma cruzi antibodies. However, despite high sensitivity, the ability of these tests to identify CD-positive cases should be evaluated against T. cruzi strains circulating in specific locales. Herein, we used a latent class analysis (LCA) approach employing an array of four chimeric antigens to assess the diagnostic performance of the Liaison XL Murex Chagas CLIA for the detection of anti-T. cruzi IgG in serum samples. STUDY DESIGN AND METHODS: The study included a panel of 5014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia, submitted to anti-T. cruzi antibody detection using Liaison Chagas CLIA and LCA as a reference test in the absence of a gold standard. RESULTS: LCA classified 4993 samples as negative, while positivity for T. cruzi antibodies was predicted in 21 samples. Compared with LCA, CLIA demonstrated sensitivity and specificity of 76.2% and 99.5%, respectively, providing an overall accuracy of 99.4%. DISCUSSION: In blood banks lacking a de facto highly sensitive screening immunoassay, the low sensitivity offered by Liaison Chagas CLIA renders it unsuitable for standalone use in serological screening procedures for CD. Moreover, blood banks are encouraged to carefully assess the ability of diagnostic methods to identify local T. cruzi strains in circulation.

Mitogen-Activated Protein Kinase Kinase 5 Regulates Proliferation and Biosynthetic Processes in Procyclic Forms of Trypanosoma brucei.

The pathogenic protozoan T. brucei alternates into distinct developmental stages in the mammalian and insect hosts. The mitogen-activated protein kinase (MAPK) signaling pathways transduce extracellular stimuli into a range of cellular responses, which ultimately lead to the adaptation to the external environment. Here, we combined a loss of function approach with stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry (MS) to investigate the role of the mitogen-activated protein kinase kinase 5 (MKK5) in T. brucei. The silencing of MKK5 significantly decreased the proliferation of procyclic forms of T. brucei. To shed light on the molecular alterations associated with this phenotype, we measured the total proteome and phosphoproteome of cells silenced for MKK5. In the total proteome, we observed a general decrease in proteins related to ribosome and translation as well as down-regulation of several components of the fatty acids biosynthesis pathway. In addition, we observed alterations in the protein levels and phosphorylation of key metabolic enzymes, which point toward a suppression of the oxidative metabolism. Taken together, our findings show that the silencing of MKK5 alters cell growth, energy metabolism, protein and fatty acids biosynthesis in procyclic T. brucei.

Surveillance of donated blood during the 2016 arbovirus outbreak in Brazil.

Reports of arboviral transmission via blood transfusion may be a cause of concern among asymptomatic infected donors. This study evaluated the presence of arboviruses in donated blood products during the 2016 outbreak in Vitória da Conquista (Bahia-Brazil). Serum samples (n = 676) were screened for ZIKV, CHIKV, and the four DENV serotypes using a one-step reverse transcriptase-based Real-Time Polymerase Chain Reaction (RT-PCR). No samples tested positive for any of the targets, whereas positive controls performed as expected. The results suggest a low risk of arboviral transmission via transfusion of blood products in the endemic area studied during the 2016 outbreak.

Post-translational Modifications of Trypanosoma cruzi Canonical and Variant Histones.

Chagas disease, caused by Trypanosoma cruzi, still affects millions of people around the world. No vaccines nor treatment for chronic Chagas disease are available, and chemotherapy for the acute phase is hindered by limited efficacy and severe side effects. The processes by which the parasite acquires infectivity and survives in different hosts involve tight regulation of gene expression, mainly post-transcriptionally. Nevertheless, chromatin structure/organization of trypanosomatids is similar to other eukaryotes, including histone variants and post-translational modifications. Emerging evidence suggests that epigenetic mechanisms also play an important role in the biology/pathogenesis of these parasites, making epigenetic targets suitable candidates to drug discovery. Here, we present the first comprehensive map of post-translational modifications of T. cruzi canonical and variant histones and show that its histone code can be as sophisticated as that of other eukaryotes. A total of 13 distinct modification types were identified, including rather novel and unusual ones such as alternative lysine acylations, serine/threonine acetylation, and N-terminal methylation. Some histone marks correlate to those described for other organisms, suggesting that similar regulatory mechanisms may be in place. Others, however, are unique to T. cruzi or to trypanosomatids as a group and might represent good candidates for the development of antiparasitic drugs.

Preliminary multiplex microarray IgG immunoassay for the diagnosis of toxoplasmosis and rubella.

BACKGROUND: During pregnancy, toxoplasmosis and rubella can cause serious damage to the mother and the foetus through vertical transmission. Early diagnosis enables implementation of health measures aimed at preventing vertical transmission and minimising damage caused by these diseases. OBJECTIVE: Here, we report the development of a multiplex assay for simultaneous detection of IgG antibodies produced during toxoplasmosis and rubella infection. METHODS: This assay is based on xMap technology. Initially, by singleplex assays, we evaluated the following antigens: one Toxoplasma gondii lysate; two antigenic extracts of T. gondii (TOX8131 and TOX8122); fragments of T. gondii antigens [SAG-1 (amino acids 45-198), GRA-7 (24-100), GRA-1 (57-149), ROP-4, and MIC-3 (234-306)]; two chimeric antigens composed of fragments of SAG-1, GRA-7, and P35 (CTOX and CTOXH); and fragments of Rubella virus antigens [E-1 (157-176, 213-239, 374-390), E-2 (31-105), and C (1-123)]. FINDINGS: A multiplex assay to simultaneously diagnose toxoplasmosis and rubella was designed with the best-performing antigens in singleplex and multiplex assays, which included CTOXH, T. gondii lysate, TOX8131, E-1, and E-2. The multiplex assay showed 100% sensitivity and specificity for anti-T. gondii IgG detection and 95.6% sensitivity and 100% specificity for anti-R. virus IgG detection. MAIN CONCLUSIONS: We found that, despite the difficulties related to developing multiplex systems, different types of antigens (extracts and recombinant proteins) can be used to develop high-performance diagnostic tests. The assay developed is suitable to screen for prior T. gondii and R. virus infections, because it is a rapid, high-throughput, low-cost alternative to the current standard diagnostic tools, which require multiple individual tests.

In silico analysis of amino acid variation in human respiratory syncytial virus: insights into immunodiagnostics.

BACKGROUND: The highly contagious nature of human respiratory syncytial virus (HRSV) and the gravity of its infection in newborns and vulnerable adults pose a serious public health problem. Thus, a rapid and sensitive diagnostic test for viral detection that can be implemented upon the first appearance of symptoms is needed. The genetic variation of the virus must be considered for immunodiagnostic purposes. OBJECTIVES: To analyse HRSV genetic variation and discuss the possible consequences for capture immunoassay development. METHODS: We performed a wide analysis of N, F and G protein variation based on the HRSV sequences currently available in the GenBank database. We also evaluated their similarity with homologous proteins from other viruses. FINDINGS: The mean amino acid divergences for the N, F, and G proteins between HRSV-A and HRSV-B were determined to be approximately 4%, 10% and 47%, respectively. Due to their high conservation, assays based on the full-length N and F proteins may not distinguish HRSV from human metapneumovirus and other Mononegavirales viruses, and the full-length G protein would most likely produce false negative results due to its high divergence. MAIN CONCLUSIONS: We have identified specific regions in each of these three proteins that have higher potential to produce specific results, and their combined utilisation should be considered for immunoassay development.

Splenic architecture disruption and parasite-induced splenocyte activation and anergy in Plasmodium falciparum-infected Saimiri sciureus monkeys.

BACKGROUND: The understanding of the mechanisms of immunity in malaria is crucial for the rational development of interventions such as vaccines. During blood stage infection, the spleen is considered to play critical roles in both immunity and immunopathology of Plasmodium falciparum infections. METHODS: Saimiri sciureus monkeys were inoculated with blood stages of P. falciparum (FUP strain) and spleens removed during acute disease (days 7 and 13 of infection) and during convalescence (15 days after start of chloroquine treatment). Cytokine (IFNγ, TNFα, IL2, IL6, IL10, and IL12) responses of splenocytes stimulated with P. falciparum-parasitized red blood cells were assessed by real-time PCR using specific Saimiri primers, and histological changes were evaluated using haematoxylin-eosin and Giemsa-stained slides. RESULTS: Early during infection (day 7, 1-2% parasitaemia), spleens showed disruption of germinal centre architecture with heavy B-cell activation (centroblasts), and splenocytes showed increased expression of IFNγ, IL6 and IL12 upon in vitro stimuli by P. falciparum-parasitized red blood cells (pRBC). Conversely, 15 days after treatment of blood stage infection with chloroquine, splenocytes showed spontaneous in vitro expression of TNFα, IL2, IL6, IL10, and IL12, but not IFNγ, and stimulation with P. falciparum pRBC blocked the expression of all these cytokines. During the acute phase of infection, splenic disarray with disorganized germinal centres was observed. During convalescence, spleens of the chloroquine-treated animals showed white pulp hyperplasia with extensive lymphocyte activation and persistency of heavily haemozoin-laden macrophages throughout the red pulp. CONCLUSIONS: Inability to eliminate haemozoin is likely involved in the persistent lymphocyte activation and in the anergic responses of Saimiri splenocytes to P. falciparum pRBC, with important negative impact in immune responses and implications for the design of malaria vaccine.

Towards the phosphoproteome of trypanosomatids.

The identification and localization of protein phosphorylation sites provide clues to what proteins or pathways might be activated in a given condition, helping to improve our understanding about signaling networks. Advances in strategies for enrichment of phosphorylated peptides/proteins, mass spectrometry (MS) instrumentation, and specific MS techniques for identification and quantification of post-translational modifications have allowed for large-scale mapping of phosphorylation sites, promoting the field of phosphoproteomics. The great promise of phosphoproteomics is to unravel the dynamics of signaling networks, a layer of the emerging field of systems biology. Until a few years ago only a small number of phosphorylation sites had been described. Following large-scale trends, recent phosphoproteomic studies have reported the mapping of thousands of phosphorylation sites in trypanosomatids. However, quantitative information about the regulation of such sites in different conditions is still lacking. In this chapter, we provide a historical overview of phosphoproteomic studies for trypanosomatids and discuss some challenges and perspectives in the field.

LM14 defined medium enables continuous growth of Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas disease, alternates between distinct morphological and functional forms during its life cycle. Axenic multiplication and differentiation processes of this protozoan parasite can be reproduced in vitro, enabling the isolation and study of the different evolutionary forms. Although there are several publications attempting the cultivation of T. cruzi under chemically defined conditions, in our experience none of the published media are capable of maintaining T. cruzi in continuous growth. RESULTS: In this work we modified a known chemically defined medium for Trypanosoma brucei growth. The resulting LM14 and LM14B defined media enabled cultivation of five different strains of T. cruzi for more than forty passages until now. The parasite's biological characteristics such as morphology and differentiation to metacyclic trypomastigotes were maintained when defined media is used. CONCLUSIONS: The establishment of a defined medium for T. cruzi cultivation is an important tool for basic biological research allowing several different approaches, providing new perspectives for further studies related to cell biology of this parasite.

Natural Plasmodium infection in monkeys in the state of Rondônia (Brazilian Western Amazon).

BACKGROUND: Simian malaria is still an open question concerning the species of Plasmodium parasites and species of New World monkeys susceptible to the parasites. In addition, the lingering question as to whether these animals are reservoirs for human malaria might become important especially in a scenario of eradication of the disease. To aid in the answers to these questions, monkeys were surveyed for malaria parasite natural infection in the Amazonian state of Rondônia, Brazil, a state with intense environmental alterations due to human activities, which facilitated sampling of the animals. METHODS: Parasites were detected and identified in DNA from blood of monkeys, by PCR with primers for the 18S rRNA, CSP and MSP1 genes and sequencing of the amplified fragments. Multiplex PCR primers for the 18S rRNA genes were designed for the parasite species Plasmodium falciparum and Plasmodium vivax, Plasmodium malariae/Plasmodium brasilianum and Plasmodium simium. RESULTS: An overall infection rate of 10.9% was observed or 20 out 184 monkey specimens surveyed, mostly by P. brasilianum. However, four specimens of monkeys were found infected with P. falciparum, two of them doubly infected with P. brasilianum and P. falciparum. In addition, a species of monkey of the family Aotidae, Aotus nigriceps, is firstly reported here naturally infected with P. brasilianum. None of the monkeys surveyed was found infected with P. simium/P. vivax. CONCLUSION: The rate of natural Plasmodium infection in monkeys in the Brazilian state of Rondônia is in line with previous surveys of simian malaria in the Amazon region. The fact that a monkey species was found that had not previously been described to harbour malaria parasites indicates that the list of monkey species susceptible to Plasmodium infection is yet to be completed. Furthermore, finding monkeys in the region infected with P. falciparum clearly indicates parasite transfer from humans to the animals. Whether this parasite can be transferred back to humans and how persistent the parasite is in monkeys in the wild so to be efficient reservoirs of the disease, is yet to be evaluated. Finding different species of monkeys infected with this parasite species suggests indeed that these animals can act as reservoirs of human malaria.

Molecular characterization of the Trypanosoma cruzi specific RNA binding protein TcRBP40 and its associated mRNAs.

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected disorder that affects millions of people in the Americas. T. cruzi relies mostly upon post-transcriptional regulation to control stage specific gene expression. RNA binding proteins (RBPs) associate with functionally related mRNAs forming ribonucleoprotein complexes that define post-transcriptional operons. The RNA Recognition Motif (RRM) is the most common and ancient family of RBPs. This family of RBPs has been identified in trypanosomatid parasites and only a few of them have been functionally characterized. We describe here the functional characterization of TcRBP40, a T. cruzi specific RBP, and its associated mRNAs. We used a modified version of the recombinant RIP-Chip assay to identify the mRNAs with which it associates and in vivo TAP-tag assays to confirm these results. TcRBP40 binds to an AG-rich sequence in the 3'UTR of the associated mRNAs, which were found to encode mainly putative transmembrane proteins. TcRBP40 is differentially expressed in metacyclogenesis. Surprisingly, in epimastigotes, it is dispersed in the cytoplasm but is concentrated in the reservosomes, a T. cruzi specific organelle, which suggests a putative new function for this parasite organelle.

A comparison of two distinct murine macrophage gene expression profiles in response to Leishmania amazonensis infection.

BACKGROUND: The experimental murine model of leishmaniasis has been widely used to characterize the immune response against Leishmania. CBA mice develop severe lesions, while C57BL/6 present small chronic lesions under L. amazonensis infection. Employing a transcriptomic approach combined with biological network analysis, the gene expression profiles of C57BL/6 and CBA macrophages, before and after L. amazonensis infection in vitro, were compared. These strains were selected due to their different degrees of susceptibility to this parasite. RESULTS: The genes expressed by C57BL/6 and CBA macrophages, before and after infection, differ greatly, both with respect to absolute number as well as cell function. Uninfected C57BL/6 macrophages express genes involved in the deactivation pathway of macrophages at lower levels, while genes related to the activation of the host immune inflammatory response, including apoptosis and phagocytosis, have elevated expression levels. Several genes that participate in the apoptosis process were also observed to be up-regulated in C57BL/6 macrophages infected with L. amazonensis, which is very likely related to the capacity of these cells to control parasite infection. By contrast, genes involved in lipid metabolism were found to be up-regulated in CBA macrophages in response to infection, which supports the notion that L. amazonensis probably modulates parasitophorous vacuoles in order to survive and multiply in host cells. CONCLUSION: The transcriptomic profiles of C57BL/6 macrophages, before and after infection, were shown to be involved in the macrophage pathway of activation, which may aid in the control of L. amazonensis infection, in contrast to the profiles of CBA cells.

Expression profile and subcellular localization of HslV, the proteasome related protease from Trypanosoma cruzi.

Trypanosoma cruzi is a rare example of an eukaryote that has genes for two threonine proteases: HslVU complex and 20S proteasome. HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. In this study, we expressed and obtained specific antibodies to HslU and HslV recombinant proteins and demonstrated the interaction between HslU/HslV by coimmunoprecipitation. To evaluate the intracellular distribution of HslV in T. cruzi we used an immunofluorescence assay and ultrastructural localization by transmission electron microscopy. Both techniques demonstrated that HslV was localized in the kinetoplast of epimastigotes. We also analyzed the HslV/20S proteasome co-expression in Y, Berenice 62 (Be-62) and Berenice 78 (Be-78) T. cruzi strains. Our results showed that HslV and 20S proteasome are differently expressed in these strains. To investigate whether a proteasome inhibitor could modulate HslV and proteasome expressions, epimastigotes from T. cruzi were grown in the presence of PSI, a classical proteasome inhibitor. This result showed that while the level of expression of HslV/20S proteasome is not affected in Be-78 strain, in Y and Be-62 strains the presence of PSI induced a significantly increase in Hslv/20S proteasome expression. Together, these results suggest the coexistence of the protease HslVU and 20S proteasome in T. cruzi, reinforcing the hypothesis that non-lysosomal degradation pathways have an important role in T. cruzi biology.

Lessons from the pandemic: Responding to emerging zoonotic viral diseases-a Keystone Symposia report.

The COVID-19 pandemic caught the world largely unprepared, including scientific and policy communities. On April 10-13, 2022, researchers across academia, industry, government, and nonprofit organizations met at the Keystone symposium "Lessons from the Pandemic: Responding to Emerging Zoonotic Viral Diseases" to discuss the successes and challenges of the COVID-19 pandemic and what lessons can be applied moving forward. Speakers focused on experiences not only from the COVID-19 pandemic but also from outbreaks of other pathogens, including the Ebola virus, Lassa virus, and Nipah virus. A general consensus was that investments made during the COVID-19 pandemic in infrastructure, collaborations, laboratory and manufacturing capacity, diagnostics, clinical trial networks, and regulatory enhancements-notably, in low-to-middle income countries-must be maintained and strengthened to enable quick, concerted responses to future threats, especially to zoonotic pathogens.

Trypanosoma cruzi infection induces a global host cell response in cardiomyocytes.

Chagas' disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, affects millions of people in South and Central America. Chronic chagasic cardiomyopathy, the most devastating manifestation of this disease, occurs in approximately one-third of infected individuals. Events associated with the parasite's tropism for and invasion of cardiomyocytes have been the focus of intense investigation in recent years. In the present study, we use murine microarrays to investigate the cellular response caused by invasion of primary murine cardiomyocytes by T. cruzi trypomastigotes. These studies identified 353 murine genes that were differentially expressed during the early stages of invasion and infection of these cells. Genes associated with the immune response, inflammation, cytoskeleton organization, cell-cell and cell-matrix interactions, apoptosis, cell cycle, and oxidative stress are among those affected during the infection. Our data indicate that T. cruzi induces broad modulations of the host cell machinery in ways that provide insight into how the parasite survives, replicates, and persists in the infected host and ultimately defines the clinical outcome of the infection.

Comparative performance evaluation of hepatitis C virus genotyping based on the 5' untranslated region versus partial sequencing of the NS5B region of brazilian patients with chronic hepatitis C.

BACKGROUND: Genotyping of hepatitis C virus (HCV) has become an essential tool for prognosis and prediction of treatment duration. The aim of this study was to compare two HCV genotyping methods: reverse hybridization line probe assay (LiPA v.1) and partial sequencing of the NS5B region. METHODS: Plasma of 171 patients with chronic hepatitis C were screened using both a commercial method (LiPA HCV Versant, Siemens, Tarrytown, NY, USA) and different primers targeting the NS5B region for PCR amplification and sequencing analysis. RESULTS: Comparison of the HCV genotyping methods showed no difference in the classification at the genotype level. However, a total of 82/171 samples (47.9%) including misclassification, non-subtypable, discrepant and inconclusive results were not classified by LiPA at the subtype level but could be discriminated by NS5B sequencing. Of these samples, 34 samples of genotype 1a and 6 samples of genotype 1b were classified at the subtype level using sequencing of NS5B. CONCLUSIONS: Sequence analysis of NS5B for genotyping HCV provides precise genotype and subtype identification and an accurate epidemiological representation of circulating viral strains.

Trypanosoma cruzi: identification of DNA targets of the nuclear periphery coiled-coil protein TcNUP-1.

The nuclear lamina is a structure that lines the inner nuclear membrane. In metazoans, lamins are the primary structural components of the nuclear lamina and are involved in several processes. Eukaryotes that lack lamins have distinct proteins with homologous functions. Some years ago, a coiled-coil protein in Trypanosoma brucei, NUP-1, was identified as the major filamentous component of its nuclear lamina. However, its precise role has not been determined. We characterized a homologous protein in Trypanosoma cruzi, TcNUP-1, and identified its in vivo DNA binding sites using a chromatin immunoprecipitation assay. We demonstrate for the first time that TcNUP-1 associates with chromosomal regions containing large non-tandem arrays of genes encoding surface proteins. We therefore suggest that TcNUP-1 is a structural protein that plays an essential role in nuclear organization by anchoring T. cruzi chromosomes to the nuclear envelope.

Characterization of TcSTI-1, a homologue of stress-induced protein-1, in Trypanosoma cruzi.

The life cycle of the protozoan Trypanosoma cruzi exposes it to several environmental stresses in its invertebrate and vertebrate hosts. Stress conditions are involved in parasite differentiation, but little is known about the stress response proteins involved. We report here the first characterization of stress-induced protein-1 (STI-1) in T. cruzi (TcSTI-1). This co-chaperone is produced in response to stress and mediates the formation of a complex between the stress proteins HSP70 and HSP90 in other organisms. Despite the similarity of TcSTI-1 to STI-1 proteins in other organisms, its expression profile in response to various stress conditions, such as heat shock, acidic pH or nutrient starvation, is quite different. Neither polysomal mRNA nor protein levels changed in exponentially growing epimastigotes cultured under any of the stress conditions studied. Increased levels of TcSTI-1 were observed in epimastigotes subjected to nutritional stress in the late growth phase. Co-immunoprecipitation assays revealed an association between TcSTI-1 and TcHSP70 in T. cruzi epimastigotes. Immunolocalization demonstrated that TcSTI-1 was distributed throughout the cytoplasm and there was some colocalization of TcSTI-1 and TcHSP70 around the nucleus. Thus, TcSTI-1 associates with TcHSP70 and TcSTI-1 expression is induced when the parasites are subjected to stress conditions during specific growth phase.

Ready-to-use qPCR for detection of Cyclospora cayetanensis or Trypanosoma cruzi in food matrices.

Foodborne outbreaks caused by parasites have long been a public health issue. Among the available contamination detection methods, qPCR is one of the most sensitive and specific. However, it can be cumbersome and error-prone, if used by unexperienced users. Moreover, qPCR reagents usually require freezer temperatures for transportation and storage. We present a gelified reaction format that allows the reagents to be stored at 2-8 °C for up to 90 days without losing performance. The gelification process eliminates most operator mistakes during reaction setup, and renders the qPCR plates ready-to-use. The new reaction makeup was evaluated using artificially contaminated samples of distinct food matrices for sensitivity, specificity, repeatability, reproducibility, and stability. Samples consisted of cilantro leaves and raspberry fruits spiked with Cyclospora cayetanensis oocysts, as well as açai pulp and sugarcane juice tainted with Trypanosoma cruzi trypomastigotes. No significant difference between the gelified and the non-gelified qPCR was found. Our results suggest that gelifying the assay may help to achieve more reproducible qPCR data across laboratories, thus supporting surveillance actions. (170 words).

A high-throughput cloning system for reverse genetics in Trypanosoma cruzi.

BACKGROUND: The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches. RESULTS: We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed. CONCLUSIONS: We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.