Frequency Variation and Dose Modification of Benznidazole Administration for the Treatment of Trypanosoma cruzi Infection in Mice, Dogs, and Nonhuman Primates.

Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and nonhuman primates (NHP). Collectively, these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ~2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

Frequency variation and dose modification of benznidazole administration for the treatment of Trypanosoma cruzi infection in mice, dogs and non-human primates.

Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and non-human primates (NHP). Collectively these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ∻2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

Prophylactic low-dose, bi-weekly benznidazole treatment fails to prevent Trypanosoma cruzi infection in dogs under intense transmission pressure.

Trypanosoma cruzi naturally infects a wide variety of wild and domesticated mammals, in addition to humans. Depending on the infection dose and other factors, the acute infection can be life-threatening, and in all cases, the risk of chagasic heart disease is high in persistently infected hosts. Domestic, working, and semi-feral dogs in the Americas are at significant risk of T. cruzi infection and in certain settings in the southern United States, the risk of new infections can exceed 30% per year, even with the use of vector control protocols. In this study, we explored whether intermittent low-dose treatment with the trypanocidal compound benznidazole (BNZ) during the transmission season, could alter the number of new infections in dogs in an area of known, intense transmission pressure. Preliminary studies in mice suggested that twice-weekly administration of BNZ could prevent or truncate infections when parasites were delivered at the mid-point between BNZ doses. Pre-transmission season screening of 126 dogs identified 53 dogs (42.1%) as T. cruzi infection positive, based upon blood PCR and Luminex-based serology. Serial monitoring of the 67 uninfected dogs during the high transmission season (May to October) revealed 15 (22.4%) new infections, 6 in the untreated control group and 9 in the group receiving BNZ prophylaxis, indicating no impact of this prophylaxis regimen on the incidence of new infections. Although these studies suggest that rigorously timed and more potent dosing regimen may be needed to achieve an immediate benefit of prophylaxis, additional studies would be needed to determine if drug prophylaxis reduced disease severity despite this failure to prevent new infections.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs.

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease's complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

Identification of Trypanosoma cruzi Growth Inhibitors with Activity In Vivo within a Collection of Licensed Drugs.

Chagas disease, caused by the parasite Trypanosoma cruzi (T. cruzi), affects more than six million people worldwide, with its greatest burden in Latin America. Available treatments present frequent toxicity and variable efficacy at the chronic phase of the infection, when the disease is usually diagnosed. Hence, development of new therapeutic strategies is urgent. Repositioning of licensed drugs stands as an attractive fast-track low-cost approach for the identification of safer and more effective chemotherapies. With this purpose we screened 32 licensed drugs for different indications against T. cruzi. We used a primary in vitro assay of Vero cells infection by T. cruzi. Five drugs showed potent activity rates against it (IC50 < 4 µmol L-1), which were also specific (selectivity index >15) with respect to host cells. T. cruzi inhibitory activity of four of them was confirmed by a secondary anti-parasitic assay based on NIH-3T3 cells. Then, we assessed toxicity to human HepG2 cells and anti-amastigote specific activity of those drugs progressed. Ultimately, atovaquone-proguanil, miltefosine, and verapamil were tested in a mouse model of acute T. cruzi infection. Miltefosine performance in vitro and in vivo encourages further investigating its use against T. cruzi.

A modified drug regimen clears active and dormant trypanosomes in mouse models of Chagas disease.

A major contributor to treatment failure in Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is that current treatment regimens do not address the drug insensitivity of transiently dormant T. cruzi amastigotes. Here, we demonstrated that use of a currently available drug in a modified treatment regimen of higher individual doses, given less frequently over an extended treatment period, could consistently extinguish T. cruzi infection in three mouse models of Chagas disease. Once per week administration of benznidazole at a dose 2.5 to 5 times the standard daily dose rapidly eliminated actively replicating parasites and ultimately eradicated the residual, transiently dormant parasite population in mice. This outcome was initially confirmed in "difficult to cure" mouse infection models using immunological, parasitological, and molecular biological approaches and ultimately corroborated by whole organ analysis of optically clarified tissues using light sheet fluorescence microscopy (LSFM). This tool was effective for monitoring pathogen load in intact organs, including detection of individual dormant parasites, and for assessing treatment outcomes. LSFM-based analysis also suggested that dormant amastigotes of T. cruzi may not be fully resistant to trypanocidal compounds such as benznidazole. Collectively, these studies provide important information on the phenomenon of dormancy in T. cruzi infection in mice, demonstrate methods to therapeutically override dormancy using a currently available drug, and provide methods to monitor alternative therapeutic approaches for this, and possibly other, low-density infectious agents.

Repurposing bioenergetic modulators against protozoan parasites responsible for tropical diseases.

Malaria, leishmaniasis and trypanosomiasis are arthropod-borne, parasitic diseases that constitute a major global health problem. They are generally found in developing countries, where lack of access to preventive tools and treatment hinders their management. Because these parasites share an increased demand on glucose consumption with most cancer cells, six compounds used in anti-tumoral research were selected to be tested as antiparasitic agents in in vitro models of Leishmania infantum, Trypanosoma brucei, T. cruzi, and Plasmodium falciparum: dichloroacetic acid (DCA), 3-bromopyruvic acid (3BP), 2-deoxy-D-glucose (2DG), lonidamine (LND), metformin (MET), and sirolimus (SIR). No parasite-killing activity was found in L. infantum promastigotes, whereas DCA and 3BP reduced the burden of intra-macrophagic amastigotes. For T. brucei all selected compounds, but 2DG, decreased parasite survival. DCA, 2DG, LND and MET showed parasite-killing activity in T. cruzi. Finally, anti-plasmodial activity was found for DCA, 2DG, LND, MET and SIR. These results reinforce the hypothesis that drugs with proven efficacy in the treatment of cancer by interfering with ATP production, proliferation, and survival cell strategies might be useful in treating threatening parasitic diseases and provide new opportunities for their repurposing.

Trypanosoma cruzi 13C-labeled O-Glycan standards for mass spectrometry.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a debilitating condition that affects over 10 million humans in the American continents. In addition to its traditional mode of human entry via the "kissing bug" in endemic areas, the infection can also be spread in non-endemic countries through blood transfusion, organ transplantation, eating food contaminated with the parasites, and from mother to fetus. Previous NMR-based studies established that the parasite expresses a variety of strain-specific and developmentally-regulated O-glycans that may contribute to virulence. In this report, we describe five synthetic O-glycan analytical standards and show their potential to enable a more facile analysis of native O-glycan isomers based on mass spectrometry.

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins.

Trypanosomatids (order Kinetoplastida), including the human pathogens Trypanosoma cruzi (agent of Chagas disease), Trypanosoma brucei, (African sleeping sickness), and Leishmania (leishmaniasis), affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP) complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9), but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9), and in vitro-transcribed single guide RNAs (sgRNAs) results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi, as well as in T. brucei and Leishmania major RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens.IMPORTANCE Protozoan parasites remain some of the highest-impact human and animal pathogens, with very limited treatment and prevention options. The development of improved therapeutics and vaccines depends on a better understanding of the unique biology of these organisms, and understanding their biology, in turn, requires the ability to track and manipulate the products of genes. In this work, we describe new methods that are available to essentially any laboratory and applicable to any parasite isolate for easily and rapidly editing the genomes of kinetoplastid parasites. We demonstrate that these methods provide the means to quickly assess function, including that of the products of essential genes and potential targets of drugs, and to tag gene products at their endogenous loci. This is all achieved without gene cloning or drug selection. We expect this advance to enable investigations, especially in Trypanosoma cruzi and Leishmania spp., that have eluded investigators for decades.

Long-Term Immunity to Trypanosoma cruzi in the Absence of Immunodominant trans-Sialidase-Specific CD8+ T Cells.

Trypanosoma cruzi infection drives the expansion of remarkably focused CD8(+) T cell responses targeting epitopes encoded by variant trans-sialidase (TS) genes. Infection of C57BL/6 mice with T. cruzi results in up to 40% of all CD8(+) T cells committed to recognition of the dominant TSKB20 and subdominant TSKB18 TS epitopes. However, despite this enormous response, these mice fail to clear T. cruzi infection and subsequently develop chronic disease. One possible reason for the failure to cure T. cruzi infection is that immunodomination by these TS-specific T cells may interfere with alternative CD8(+) T cell responses more capable of complete parasite elimination. To address this possibility, we created transgenic mice that are centrally tolerant to these immunodominant epitopes. Mice expressing TSKB20, TSKB18, or both epitopes controlled T. cruzi infection and developed effector CD8(+) T cells that maintained an activated phenotype. Memory CD8(+) T cells from drug-cured TSKB-transgenic mice rapidly responded to secondary T. cruzi infection. In the absence of the response to TSKB20 and TSKB18, immunodominance did not shift to other known subdominant epitopes despite the capacity of these mice to expand epitope-specific T cells specific for the model antigen ovalbumin expressed by engineered parasites. Thus, CD8(+) T cell responses tightly and robustly focused on a few epitopes within variant TS antigens appear to neither contribute to, nor detract from, the ability to control T. cruzi infection. These data also indicate that the relative position of an epitope within a CD8(+) immunodominance hierarchy does not predict its importance in pathogen control.

Potential new clinical therapies for Chagas disease.

Chagas disease is the highest impact parasitic disease in the Americas but often goes untreated due to the shortcomings of currently available therapeutics. Thus there is an urgent need for new treatment options and growing interest in drug development for the infection. This review summarizes some of the recent advances and failures in this realm, with particular emphasis on recently published studies and unpublished results presented at a recent Chagas Drug Discovery Consortium meeting.

New, combined, and reduced dosing treatment protocols cure Trypanosoma cruzi infection in mice.

The development of treatment protocols with reduced toxicity and equivalent or improved efficacy for Trypanosoma cruzi infection is a priority. We tested the effectiveness of benznidazole (BZ), nifurtimox (NFX), other prospective drugs in intermittent and combined treatment protocols to cure T. cruzi infection initiated with susceptible and drug-resistant parasite strains. A 40-day course of BZ, NFX, or the oxaborale AN4169 cured 100% of mice, whereas posaconazole (POS), and NTLA-1 (a nitro-triazole) cured approximately 90% and 20% of mice, respectively. Reducing the overall dosage of BZ or NFX by using an intermittent (once every 5 days) schedule or combining 5 daily doses of POS with 7 intermittent doses of BZ also provided approximately 100% cure. T. cruzi strains resistant to BZ were also found to be resistant to other drugs (POS), and extending the time of treatment or combining drugs did not increase cure rates with these isolates. Thus, dosing schedules for anti-T. cruzi compounds should be determined empirically, and compounds targeting different pathways may be combined to yield effective therapies with reduced toxicity. This work also suggests that standard treatment protocols using BZ and NFX may be significantly overdosing patients, perhaps contributing to the adverse events.

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence.

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PPi ) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF-hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PPi ) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP-OE) resulted in a significant decrease in cytosolic PPi , and short and long-chain polyP levels. Additionally, the TcVSP-OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PPi and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.

Methodological advances in drug discovery for Chagas disease.

INTRODUCTION: Chagas disease is the highest impact human infectious disease in Latin America, and the leading worldwide cause of myocarditis. Despite the availability of several compounds that have demonstrated efficacy in limiting the effects of T. cruzi, these compounds are rarely used due to their variable efficacy, substantial side effects and the lack of methodologies for confirming their effectiveness. Furthermore, the development of more efficacious compounds is challenged by limitations of systems for assessing drug efficacy in vitro and in vivo. AREAS COVERED: Herein, the authors review the development of Chagas disease drug discovery methodology, focusing on recent developments in high throughput screening, in vivo testing methods and assessments of efficacy in humans. Particularly, this review documents the significant progress that has taken place over the last 5 years that have paved the way for both target-focused and high-throughput screens of compound libraries. EXPERT OPINION: The tools for in vitro and in vivo screening of anti-T. cruzi compounds have improved dramatically in the last few years and there are now a number of excellent in vivo testing models available; this somewhat alleviates the bottleneck issue of quickly and definitively demonstrating in vivo efficacy in a relevant host animal system. These advances emphasize the potential for additional progress resulting in new treatments for Chagas disease in the coming years. That being said, national and international agencies must improve the coordination of research and development efforts in addition to cultivating the funding sources for the development of these new treatments.

Oral exposure to Trypanosoma cruzi elicits a systemic CD8⁺ T cell response and protection against heterotopic challenge.

Trypanosoma cruzi infects millions of people in Latin America and often leads to the development of Chagas disease. T. cruzi infection can be acquired at or near the bite site of the triatomine vector, but per os infection is also a well-documented mode of transmission, as evidenced by recent microepidemics of acute Chagas disease attributed to the consumption of parasite-contaminated foods and liquids. It would also be convenient to deliver vaccines for T. cruzi by the oral route, particularly live parasite vaccines intended for the immunization of reservoir hosts. For these reasons, we were interested in better understanding immunity to T. cruzi following oral infection or oral vaccination, knowing that the route of infection and site of antigen encounter can have substantial effects on the ensuing immune response. Here, we show that the route of infection does not alter the ability of T. cruzi to establish infection in muscle tissue nor does it impair the generation of a robust CD8(+) T cell response. Importantly, oral vaccination with attenuated parasites provides protection against wild-type (WT) T. cruzi challenge. These results strongly support the development of whole-organism-based vaccines targeting reservoir species as a means to alleviate the burden of Chagas disease in affected regions.

Report of the 2nd Chagas Drug Discovery Consortium meeting, held on 3 November 2010; Atlanta GA, USA.

Chagas disease is an infectious disease with the highest impact in Latin America and a growing worldwide problem. Chagas disease is the result of long-term, persistent infection with the protozoan parasite Trypanosoma cruzi. The current therapies for treating T. cruzi infection and thus preventing Chagas disease often have adverse effects, unpredictable efficacy and require long courses of treatment. Development of new therapies has been very limited, in part due to lack of interest but also as a result of poor support and inappropriate models for discovering and evaluating candidate drugs. The Chagas Drug Discovery Consortium (CDDC) was created with funding from the US National Institutes of Health to help address some of these issues. The goals of the CDDC are to discover and evaluate new candidate drugs and develop rigorous assays of drug efficacy. This report summarizes the second meeting of the CDDC in November 2010.

In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds.

BACKGROUND: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential. METHODS: In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC(50) lower than that of BZ. FINDINGS: This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course. CONCLUSION: These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

Trypanoside, anti-tuberculosis, leishmanicidal, and cytotoxic activities of tetrahydrobenzothienopyrimidines.

The synthesis of 2-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)hydrazone-derivatives (BTPs) and their in vitro evaluation against Trypanosoma cruzi trypomastigotes, Mycobacterium tuberculosis, Leishmania amazonensis axenic amastigotes, and six human cancer cell lines is described. The in vivo activity of the most active and least toxic compounds against T. cruzi and L. amazonensis was also studied. BTPs constitute a new family of drug leads with potential activity against infectious diseases. Due to their drug-like properties, this series of compounds can potentially serve as templates for future drug-optimization and drug-development efforts for use as therapeutic agents in developing countries.

CD8+ T cells in Trypanosoma cruzi infection.

CD8(+) T cells have emerged as crucial players in the control of a number of protozoan pathogens, including Trypanosoma cruzi, the agent of human Chagas disease. The recent identification of the dominant targets of T. cruzi-specific T cells has allowed investigators to follow the generation of and document the functionality of T cell responses in both mice and humans. Although slow to develop in the early stages of the infection, T. cruzi-specific CD8(+) T cells reach prodigious levels and remain highly functional throughout chronic infections in mice. Following drug-induced cure during either the acute or chronic stage, these immunodominant T cells persist as stable, antigen-independent memory populations. T. cruzi-specific CD8(+) T cells in humans are less-well-studied but appear to lose functionality and decline in numbers in these decades-long infections. Changes in the frequency of parasite-specific T cell upon therapeutic treatment in humans may provide a new metric for determining treatment efficacy.

Drug-induced cure drives conversion to a stable and protective CD8+ T central memory response in chronic Chagas disease.

In this study, we document the development of stable, antigen-independent CD8+ T cell memory after drug-induced cure of a chronic infection. By establishing a system for drug cure of chronic Trypanosoma cruzi infection, we present the first extensively documented case of total parasite clearance after drug treatment of this infection. Cure resulted in the emergence of a stable, parasite-specific CD8+ T cell population with the characteristics of central memory cells, based upon expression of CD62L, CCR7, CD127, CD122, Bcl-2 and a reduced immediate in vivo CTL function. CD8+ T cells from treated and cured mice also expanded more rapidly and provided greater protection following challenge than those from chronically infected mice. These results show that complete pathogen clearance results in stable, antigen-independent and protective T cell memory, despite the potentially exhausting effects of prior long-term exposure to antigen in this chronic infection.