The course of malaria in mice: major histocompatibility complex (MHC) effects, but no general MHC heterozygote advantage in single-strain infections.

A general MHC-heterozygote advantage in parasite-infected organisms is often assumed, although there is little experimental evidence for this. We tested the response of MHC-congenic mice (F2 segregants) to malaria and found the course of infection to be significantly influenced by MHC haplotype, parasite strain, and host gender. However, the MHC heterozygotes did worse than expected from the average response of the homozygotes.

No impact of malaria infection or immunisation on the expression of the immune GTPase GIMAP1 at the protein or mRNA levels (Article no. 53)

GIMAP (GTPase of the immunity-associated protein family) proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi. Methods A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. Results GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK), in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. Conclusion The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

The severity of malarial anaemia in Plasmodium chabaudi infections of BALB/c mice is determined independently of the number of circulating parasites

Background: Severe malarial anaemia is a major complication of malaria infection and is multifactorial resulting from loss of circulating red blood cells (RBCs) from parasite replication, as well as immune-mediated mechanisms. An understanding of the causes of severe malarial anaemia is necessary to develop and implement new therapeutic strategies to tackle this syndrome of malaria infection. Methods: Using analysis of variance, this work investigated whether parasite-destruction of RBCs always accounts for the severity of malarial anaemia during infections of the rodent malaria model Plasmodium chabaudi in mice of a BALB/c background. Differences in anaemia between two different clones of P. chabaudi were also examined. Results: Circulating parasite numbers were not correlated with the severity of anaemia in either BALB/c mice or under more severe conditions of anaemia in BALB/c RAG2 deficient mice (lacking T and B cells). Mice infected with P. chabaudi clone CB suffered more severe anaemia than mice infected with clone AS, but this was not correlated with the number of parasites in the circulation. Instead, the peak percentage of parasitized RBCs was higher in CB-infected animals than in AS-infected animals, and was correlated with the severity of anaemia, suggesting that the availability of uninfected RBCs was impaired in CB-infected animals. Conclusion: This work shows that parasite numbers are a more relevant measure of parasite levels in P. chabaudi infection than % parasitaemia, a measure that does not take anaemia into account. The lack of correlation between parasite numbers and the drop in circulating RBCs in this experimental model of malaria support a role for the host response in the impairment or destruction of uninfected RBC in P. chabaudi infections, and thus development of acute anaemia in this malaria model.

The pathology of Plasmodium chabaudi infection is not ameliorated by the secreted filarial nematode immunomodulatory molecule, ES-62

ES-62 is a phosphorylcholine-containing glycoprotein secreted by filarial nematodes. This molecule has been shown to reduce the severity of inflammation in collagen-induced arthritis (CIA) in mice, a model of rheumatoid arthritis, via down-regulation of anti-collagen type 1 immune responses. Malaria parasites induce a pro-inflammatory host immune response and many of the symptoms of malaria are immune system-mediated. Therefore we have asked whether the immunomodulatory properties of ES-62 can down-regulate the severity of malaria infection in BALB/c mice infected with Plasmodium chabaudi. We have found that ES-62 has no significant effect on the course of P. chabaudi parasitaemia, and does not significantly affect any of the measures of malaria-induced pathology taken throughout infection.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Melatonin and IP(3)-induced Ca(2+) Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum-infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle-like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake by P. falciparum-infected erythrocytes shows that at R and S stages, a time-increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time-increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

Liver accumulation of Plasmodium chabaudi-infected red blood cells and modulation of regulatory T Cell and dendritic cell responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (PciRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4+ T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-a, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

Potencies of human immunodeficiency virus protease inhibitors in vitro against Plasmodium falciparum and in vivo against murine malaria

Parasite resistance to antimalarial drugs is a serious threat to human health, and novel agents that act on enzymes essential for parasite metabolism, such as proteases, are attractive targets for drug development. Recent studies have shown that clinically utilized human immunodeficiency virus (HIV) protease inhibitors can inhibit the in vitro growth of Plasmodium falciparum at or below concentrations found in human plasma after oral drug administration. The most potent in vitro antimalarial effects have been obtained for parasites treated with saquinavir, ritonavir, or lopinavir, findings confirmed in this study for a genetically distinct P. falciparum line (3D7). To investigate the potential in vivo activity of antiretroviral protease inhibitors (ARPIs) against malaria, we examined the effect of ARPI combinations in a murine model of malaria. In mice infected with Plasmodium chabaudi AS and treated orally with ritonavir-saquinavir or ritonavir-lopinavir, a delay in patency and a significant attenuation of parasitemia were observed. Using modeling and ligand docking studies we examined putative ligand binding sites of ARPIs in aspartyl proteases of P. falciparum (plasmepsins II and IV) and P. chabaudi (plasmepsin) and found that these in silico analyses support the antimalarial activity hypothesized to be mediated through inhibition of these enzymes. In addition, in vitro enzyme assays demonstrated that P. falciparum plasmepsins II and IV are both inhibited by the ARPIs saquinavir, ritonavir, and lopinavir. The combined results suggest that ARPIs have useful antimalarial activity that may be especially relevant in geographical regions where HIV and P. falciparum infections are both endemic.

Studies on the protective immune response to Plasmodium chabaudi in mice

Inbred strains of C5731 and NIH nice infected with the A/S strain of Plasmodium chaubaudi usually developed high parasitaemias but infections were rarely fatal in immunocompetent mice and in most mice the parasites could be eradicated within 53 days or less. The immune response of C57B1 and NTH mice to infection with the A/S strain of P. chabaudi was studied. The principle method used in this study for investigating the immune response of the mice was to examine the immunity conferred on syngeneic mice, either X-irradiated or non-irradiated, by transferring to them lymphoid cells or serum from immune or semi-immune donors. The lymphoid cell populations examined were unfractionated spleen cells, nylon wool column enriched subpopulations of thymus-derived lymphocytes (T cells) and the so-called bursa-derived lymphocytes (B cells), bone marrow cells and phagocytic cells. In the course of these experiments observations were made on the effect of X-irradiation on the subsequent growth and multiplication of the parasite. In addition, an in vitro assay for antibody-dependent cell mediated cytotoxicity was used to investigate the activity of splenic K cells during malaria infection. K cells are lymphoid cells which may include lymphocytes of an undefined category, but possess receptors for the Fc portion of antibody on their surface and have the ability to non-specifically lyse target cells coated in antibodies. a) The adoptive transfer of immunity to P.chabaudi with immune spleen cells. Spleen cells from mice which had previously been infected with P.chabaudi were able to confer some immunity on syngeneic mice which had been irradiated with 600 or 800 rads. The protection was detected as a shortened patent parasitaemia in immune cell recipients compared to controls. The early experiments indicated the value of using irradiated recipients rather than non-irradiated recipients. In irradiated mice, a) smaller numbers of immune cells were required to promote detectable immunity than in non-irradiated mice, b) there was an amplification of the difference in the duration of primary parasitaemias in recipients of immune cells and normal cells compared to non-irradiated mice and c) as the irradiated host is immunodepressed, the protective effect of donor cells can be examined with a reduced contribution by the hosts own immune system. An initial non-specific resistance to P.chabaudi infection was observed in irradiated mice, although the infection in most of these mice was subsequently more severe than in non-irradiated mice. The non-specific resistance could be reduced or abolished by injecting lymphoid cells into mice shortly after irradiation or by infecting irradiated mice more than 15 days after irradiation. Other workers suggest that following irradiation, the reticulo-endothelial system is stimulated at the time that the non-specific resistance to P.chabaudi was observed. b) the adoptive transfer of immunity in syngeneic mice with enriched subpopulations of splenic immune T cells, B. cells, bone marrow cells and phagocytes. Immunity to P.chabaudi could be adoptively transferred with enriched spleen subpopulations of immune T cells or immune B cells in mice which had been irradiated 600 or 300 rads. The protective effects of unfractionated immune cells was, however, usually better than that of either immune T or F cell subpopulations. In most experiments enriched immune T cell recipients were more likely to suffer relapsing patent parasitaemias than either enriched immune B cell recipients or unfractionated immune cell recipients. In one experiment a comparison was made of the course of P.chabaudi infection in mice which had been irradiated with either 600 rads or 300 rads and which received injections of different immune cells. A dose of 600 rads permits the immune system of mice to recover from the effects of irradiation, but a dose of 800 rads is lethal to mice unless lymphoid cells are injected after irradiation. It was found that in recipients of enriched immune T or B cells, which had been irradiated with 600 rads, the parasitaemia became subpatent before their equivalents irradiated with 800 rads, but that there was little difference in parasitaemias between recipients of unfractionated immune cells given 600 or 800 rads. Experiments in which enriched immune T cells and B cells were recombined and injected into syngeneic mice gave inconclusive results as to whether the immune subpopulations acted synergistically. Similar experiments in which immune subpopulations of lymphoid cells were recombined with normal subpopulations of lymphoid cells demonstrated that the latter cells did not enhance the protective effect of the former cells. Bone marrow cells from immune mice were able to confer some protection on syngeneic recipients, but were not as protective as enriched immune T cells or B cells. The results obtained in adoptive transfer experiments using phagocytic cells from the spleen of immune mice depended on the length of time spleen cells were incubated in petri-dishes at 37° C before harvesting the phagocytes. Using C57B1 mice, phagocytes harvested after 15 hours incubation were as protective as unfractionated immune cells in a cell transfer experiment, but phagocytes harvested after 16 hours incubation were not protective. Examination of NIH phagocytic cells after 2.5 hours incubation at 37°C, which were as protective as unfractionated immune spleen cells in a cell transfer experiment, demonstrated that the petri-dish adherent cells may have contained B lymphocytes. c) The passive transfer of immunity with serum from P.chabaudi infected mice. The passive transfer of serum from C57B1 mice which had been previously infected with P.chabaudi to normal or irradiated syngeneic mice demonstrated that the serum recipients were initially protected from infection. Irradiated mice, however, were delayed longer in the onset of parasitaemia compared to non-irradiated mice. Using NIH mice, sera were collected from unfractionated immune spleen cell recipients, enriched immune T cell recipients and normal spleen recipients on the 11th day of a P.chabaudi infection, just after peak parasitaemia, and also on the 14th day of infection. On day 14, all immune cells recipients and most of the enriched immune T cell recipients had become subpatent but all normal cell recipients still had patent infections. Sera collected from the different spleen cell recipients on the 11th day of infection and passively transferred to irradiated mice demonstrated little protection. Sera collected on the 14th day of infect ion, however, reflected the immune status of the donors in their protective properties in mice infected with P.chabaudi. The serum from unfractionated immune cell recipients was the most protective of the 3 sera when compared to normal NIH serum and the serum from enriched immune T cell recipients was slightly protective, but the serum from normal cell recipients produced an enhanced infection in mice infected with P.chabaudi. d) Antibody-dependent cell-mediated cytotoxicity of spleen cells in P.chabaudi infected mice. In a preliminary investigation of K cell activity in the spleens of P.chabaudi infected mice, it was found that there was an increased activity of K cells collected at around peak parasitaemia compared to the activity of K cells in non-infected mice, and that this increased activity could also be found in mice which had recently become subpatent. As the target cell for antibody-dependent cell-mediated cytotoxicity employed was the thick red blood cell, it is not known whether the K cell is involved in the killing of P.chabaudi parasites. These results suggest that both T cells and B cells and antibody may be important in the immune response to P.chabaudi in mice. Primed T cells may act as helper cells in the production of malarial antibodies, but, as enriched primed T cells could confer protection on immunodepressed mice, it is possible that a cell-mediated mechanism of immunity may also exist.

Imported Malaria In A Non-endemic Area: The Experience Of The University Of Campinas Hospital In The Brazilian Southeast.

Although malaria in Brazil almost exclusively occurs within the boundaries of the Amazon Region, some concerns are raised regarding imported malaria to non-endemic areas of the country, notably increased incidence of complications due to delayed diagnoses. However, although imported malaria in Brazil represents a major health problem, only a few studies have addressed this subject. A retrospective case series is presented in which 263 medical charts were analysed to investigate the clinical and epidemiological characterization of malaria cases that were diagnosed and treated at Hospital & Clinics, State University of Campinas between 1998 and 2011. Amongst all medical charts analysed, 224 patients had a parasitological confirmed diagnosis of malaria. Plasmodium vivax and Plasmodium falciparum were responsible for 67% and 30% of the infections, respectively. The majority of patients were male (83%) of a productive age (median, 37 years old). Importantly, severe complications did not differ significantly between P. vivax (14 cases, 9%) and P. falciparum (7 cases, 10%) infections. Severe malaria cases were frequent among imported cases in Brazil outside of the Amazon area. The findings reinforce the idea that P. vivax infections in Brazil are not benign, regardless the endemicity of the area studied. Moreover, as the hospital is located in a privileged site, it could be used for future studies of malaria relapses and primaquine resistance mechanisms. Finally, based on the volume of cases treated and the secondary complications, referral malaria services are needed in the non-endemic areas of Brazil for a rapid and efficient and treatment.

A Decade Of Malaria During Pregnancy In Brazil: What Has Been Done Concerning Prevention And Management.

In Brazil, malaria remains a disease of major epidemiological importance because of the high number of cases in the Amazonian Region. Plasmodium spp infections during pregnancy are a significant public health problem with substantial risks for the pregnant woman, the foetus and the newborn child. In Brazil, the control of malaria during pregnancy is primarily achieved by prompt and effective treatment of the acute episodes. Thus, to assure rapid diagnosis and treatment for pregnant women with malaria, one of the recommended strategy for low transmission areas by World Health Organization and as part of a strategy by the Ministry of Health, the National Malaria Control Program has focused on integrative measures with woman and reproductive health. Here, we discuss the approach for the prevention and management of malaria during pregnancy in Brazil over the last 10 years (2003-2012) using morbidity data from Malaria Health Information System. Improving the efficiency and quality of healthcare and education and the consolidation of prevention programmes will be challenges in the control of malaria during pregnancy in the next decade.

Photodynamic antimicrobial chemotherapy (PACT) for the treatment of malaria, leishmaniasis and trypanosomiasis

A photodynamic effect occurs when photosensitiser molecules absorb light and dissipate the absorbed energy by transferring it to biological acceptors (usually oxygen), generating an excess of reactive species that are able to force cells into death pathways. Several tropical diseases present physiopathological aspects that are accessible to the application of a photosensitiser and local illumination. In addition, disease may be transmitted through infected blood donations, and many of the aetiological agents associated with tropical diseases have been shown to be susceptible to the photodynamic approach. However, there has been no systematic investigation of the application of photoantimicrobial agents in the various presentations, whether to human disease or to the disinfection of blood products or even as photo-insecticides. We aim in this review to report the advances in the photoantimicrobial approach that are beneficial to the field of anti-parasite therapy and also have the potential to facilitate the development of low-cost/high-efficiency protocols for underserved populations.