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The malaria parasite Plasmodium goes through two life stages in the human host, a non-symptomatic liver stage (LS) followed by a blood stage with all clinical manifestation of the disease. In this study, we investigated a series of 2-alkynoic fatty acids (2-AFAs) with chain lengths between 14 and 18 carbon atoms for dual in vitro activity against both life stages. 2-Octadecynoic acid (2-ODA) was identified as the best inhibitor of Plasmodium berghei parasites with ten times higher potency (IC50 = 0.34 μg/ml) than the control drug. In target determination studies, the same compound inhibited three Plasmodium falciparum FAS-II (PfFAS-II) elongation enzymes PfFabI, PfFabZ, and PfFabG with the lowest IC50 values (0.28-0.80 μg/ml, respectively). Molecular modeling studies provided insights into the molecular aspects underlying the inhibitory activity of this series of 2-AFAs and a likely explanation for the considerably different inhibition potentials. Blood stages of P. falciparum followed a similar trend where 2-ODA emerged as the most active compound, with 20 times less potency. The general toxicity and hepatotoxicity of 2-AFAs were evaluated by in vitro and in vivo methods in mammalian cell lines and zebrafish models, respectively. This study identifies 2-ODA as the most promising antiparasitic 2-AFA, particularly towards P. berghei parasites. © 2014 Elsevier Ltd. All rights reserved.

application/pdf, Malaria is the world's most important parasitic disease, imposing a massive health burden on people living in the tropics, often in the poorest countries. The vast majority of deaths in humans from malaria are caused by one species of the hemoprotozoan, Plasmodium falciparum., against which effective control measures are urgently needed. The global situation has deteriorated in recent times due to increased resistance of the anopheline mosquitoes that transmit P. falciparum to insecticides, and of the parasites themselves to drug therapy. An efficacious and cost-effective vaccine against this parasite is considered a public health priority. A vaccine that targets pre-erythrocytic parasites in the liver could potentially prevent clinical disease by blocking development of the pathogenic erythrocytic stage of the parasite's life cycle. Among around 40 known P. falciparum antigens, liver stage antigen-1 (LSA-1) is the only protein expressed exclusively by liver stage parasites. Independent studies in humans have consistently related immune responses to LSA-1 with resistance to malaria infection or disease, providing a powerful rationale for the development of liver stage vaccines based on LSA-1. By dissecting the mechanism(s) of immunity to this antigen, epitopes associated with protection can be evaluated in different delivery systems as components of a focused and coordinated multi-antigen malaria vaccine strategy.

In Plasmodium vivax and Plasmodium ovale malaria, some of the liver stage parasites remain dormant. The activation of these dormant forms (called hypnozoite) can give rise to relapse weeks, months or years after the initial infection. To prevent relapses, a course of primaquine may be given as terminal prophylaxis to patients. Different strains of Plasmodium vivax vary in their sensitivity to primaquine and, recently, cases of relapse of Plasmodium vivax after this standard primaquine therapy were reported from various countries. We reported a case of primaquine resistant malaria which initially was thought to be relapsed caused by loss of terminal prophylaxis.

Fatty acid biosynthesis has been viewed as an important biological function of and therapeutic target for Plasmodium falciparum asexual blood stage infection. This apicoplast-resident type II pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of the bacterial FabI inhibitor triclosan. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood stage growth. In contrast, mosquito-derived fabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver stage development in vitro. This is characterized by an inability to form intra-hepatic merosomes that normally initiate blood stage infections. These data illuminate key differences between liver and blood stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Summary Malaria starts with the infection of the liver by Plasmodium parasites. Although extensive analysis of Plasmodium has revealed the expression patterns of this parasite in every stage of its life cycle, the liver stage remained unexplored. Recently, Tarun et al. have published the first complete transcriptome and proteome analysis of this intriguing parasite stage, providing a list of potential candidate target genes for antimalarial vaccines and drugs.

The Santa Lucia strain of Plasmodium falciparum was transmitted to Aotus lemurinus griseimembra, A. azarae boliviensis, A. vociferans, and A. nancymaae monkeys by bite and by intravenous inoculation of sporozoites dissected from Anopheles freeborni, An. stephensi, An. gambiae, An. albimanus, and An. maculatus mosquitoes. The data obtained from these infections indicate that A. nancymaae can be considered a suitable host model when combined with the Santa Lucia strain of P. falciparum for the testing of candidate anti-sporozoite and liver stage vaccines.

Plasmodium malariaecauses uncommon benign malaria found in the malaria endemic regions mostly of Sub-Saharan Africa. AsPlasmodium malariaedoes not have a continued liver stage in humans the only way to have reinfection without reexposure is through recrudescence. However, reports of its recrudescence after antimalarials are rare with only a handful of case reports in the literature. Research in this field to date has not been able to establish definitively an emergence of resistance inPlasmodium malariaeto commonly used antimalarials. In the presented case, patient had a recrudescence ofP. malariaeafter full treatment with quinine and clindamycin. This recrudescence was treated with full course of chloroquine with clearance of parasite from blood immediately after treatment and at two months’ follow up. The recrudescence in this case cannot be explained by mechanisms explained in prior articles. We propose that the indolence of some of thePlasmodium malariaetrophozoites in the blood can shield them from the effect of the toxic effects of antimalarials and enable them to produce recrudescence later. However, when recrudescence happens, this should not be considered a case of development of resistance and a course of chloroquine should be considered.

Background: Chimeric mice with humanized livers represent a promising tool for infections with Plasmodium falciparum to evaluate novel methods for prevention and treatment of pre-erythrocytic stages. Adequate assessment of hepatic infections is generally compromised by the limited number of human hepatocytes infected by developing parasites. Methods: A qPCR-based method has been developed that sensitively and reliably detects P. falciparum liver stage infection of humanized mice and quantitatively expresses the results as the number of parasites per human hepatocyte. Results: This assay allows for detection of liver stage parasites after challenging humanized mice with infected mosquito bites or after intravenous injection with sporozoites. The sensitivity of the protocol, which comprises approximately 25% of the total chimeric liver, allows for the detection of a single infected hepatocyte in the analysed tissue. Conclusions: This method allows for the detection and quantification of P. falciparum parasites in chimeric mice repopulated with human hepatocytes. It will be a useful tool when studying the in vivo therapeutic and/or prophylactic qualities of novel compounds, small molecules or antibodies directed against the liver stage of P. falciparum infections.

This digest covers some of the most relevant progress in malaria drug discovery published between 2010 and 2012. There is an urgent need to develop new antimalarial drugs. Such drugs can target the blood stage of the disease to alleviate the symptoms, the liver stage to prevent relapses, and the transmission stage to protect other humans. The pipeline for the blood stage is becoming robust, but this should not be a source of complacency, as the current therapies set a high standard. Drug discovery efforts directed towards the liver and transmission stages are in their infancy but are receiving increasing attention as targeting these stages could be instrumental in eradicating malaria.

We present a novel series of quinolin-4(1H)-imines as dual-stage antiplasmodials, several-fold more active than primaquine in vitro against Plasmodium berghei liver stage. Among those, compounds 5g and 5k presented low nanomolar IC50 values. The compounds are metabolically stable and modulate several drug targets. These results emphasize the value of quinolin-4(1H)-imines as a new chemotype and their suitable properties for further drug development.

Terrestrial plants have proven to be a prolific producer of clinically effective antimalarial drugs, but the antimalarial potential of seaweeds has been little explored. The main aim of this study was to assess the in vitro chemotherapeutical and prophylactic potential of the extracts of twenty-three seaweeds collected from the south coast of England against blood stage (BS) and liver stage (LS) Plasmodium parasites. The majority (14) of the extracts were active against BS of P. falciparum, with brown seaweeds Cystoseira tamariscifolia, C. baccata and the green seaweed Ulva lactuca being the most active (IC(50)s around 3 g/mL). The extracts generally had high selectivity indices (>10). Eight seaweed extracts inhibited the growth of LS parasites of P. berghei without any obvious effect on the viability of the human hepatoma (Huh7) cells, and the highest potential was exerted by U. lactuca and red seaweeds Ceramium virgatum and Halopitys incurvus (IC50 values 14.9 to 28.8 g/mL). The LS-active extracts inhibited one or more key enzymes of the malarial type-II fatty acid biosynthesis (FAS-II) pathway, a drug target specific for LS. Except for the red seaweed Halopitys incurvus, all LS-active extracts showed dual activity versus both malarial intracellular stage parasites. This is the first report of LS antiplasmodial activity and dual stage inhibitory potential of seaweeds.

Although the attenuated sporozoite is the most efficient vaccine to prevent infection with the malaria parasite, the limitation of a source of sterile sporozoites greatly hampers its application. In this study, we found that the whole-killed, blood-stage lysate vaccine could confer protection against the blood stage as well as the liver stage. Although the protective immunity induced by the whole-organism vaccine against the blood stage is dependent on parasite-specific CD4+ T-cell responses and antibodies, in mice immunized with the whole-killed, blood-stage lysate vaccine, CD8+ , but not CD4+ effector T-cell responses greatly contributed to protection against the liver stage. Thus, our data suggested that the whole-killed, blood-stage lysate vaccine could be an alternative promising strategy to prevent malaria infection and to reduce the morbidity and mortality of patients with malaria.

Severe malaria occurs predominantly in young children and immunity to clinical disease is associated with cumulative exposure in holoendemic settings. The relative contribution of immunity against various stages of the parasite life cycle that results in controlling infection and limiting disease is not well understood. Here we analyse the dynamics of Plasmodium falciparum malaria infection after treatment in a cohort of 197 healthy study participants of different ages in order to model naturally acquired immunity. We find that both delayed time-to-infection and reductions in asymptomatic parasitaemias in older age groups can be explained by immunity that reduces the growth of blood stage as opposed to liver stage parasites. We found that this mechanism would require at least two components – a rapidly acting strain-specific component, as well as a slowly acquired cross-reactive or general immunity to all strains. Analysis and modelling of malaria infection dynamics and naturally acquired immunity with age provides important insights into what mechanisms of immune control may be harnessed by malaria vaccine strategists., Author Summary Human malaria infections resulting in serious complications and death occur predominantly in young children, and resistance is gradually acquired with repeated exposure. Malaria parasites have two major stages within the human host during its life cycle: an initial liver stage, and the subsequent blood stage, where parasites replicate in and destroy red blood cells. The mechanisms of acquired resistance to severe malaria may involve immunity to both the liver and blood stage parasites. However the relative contribution of each type of immunity is not yet understood. To gain novel insight, we have analysed data from a malaria exposed cohort from western Kenya. We used mathematical modeling to understand what form of immunity is consistent with the observed rates of reinfection in adults and children seen in the field study data. We found that the reinfection pattern can be completely explained by blood stage immunity. Moreover, the blood stage immunity must consist of rapidly-induced strain-specific immunity that clears individual infections, and general immunity that accumulates slowly and decreases the average parasite growth rate with age. Understanding the dynamics of naturally acquired immunity and infection provides important insights for effective vaccine development.

The Plasmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable, and reproducible in vitro liver-stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum, including the release of infected merozoites and infection of overlaid erythrocytes, as well as the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput antimalarial drug screening and vaccine development., Bill & Melinda Gates Foundation (51066)

Despite strong efforts to control it, malaria remains a serious health problem in Madagascar with episodic unpredictable recrudescence. Local communities, especially those living in remote areas, rely on traditional medicine to treat the disease. Our research on ethnobotany-based malaria chemotherapy began in the 1990s following the sudden resurgence of the disease. Our work was first focused on drugs that could reverse chloroquine resistance in malaria. This led to the discovery of naturally occurring chemosensitizers, namely, the herveline series and the malagashanine series. Moving to the hepatic stage, we isolated a new alkaloid named tazopsine which was selectively active against the liver stage of the malaria parasite. Malagashanine was used as a biochemical tool for the understating of chloroquine resistance and its reversal. The alkaloid strychnobrasiline was used as a starting material for various chemical modifications. These investigations, together with careful examination of structures of naturally occurring and synthetic chemosenitizers, gave clues for the design and synthesis of new chemosensitizers. Gallic acid was found to enhance the antiplasmodial activities of phenolic compounds, and this property led to the identification of chromophores that may interact with the fatty acid II biosynthesis. After a critical analysis of previous investigations on malaria, and based on our preliminary results, we propose a holistic approach which includes killing the parasites, boosting the immune system and managing the inflammatory process.

After the bite of a malaria-infected mosquito, the Plasmodium sporozoite infects liver cells and produces thousands of merozoites, which then infect red blood cells, causing malaria. In malaria-endemic areas, several hundred infected mosquitoes can bite an individual each year, increasing the risk of superinfection. However, in infants that are yet to acquire immunity, superinfections are infrequent. We have recently shown that blood-stage parasitaemia, above a minimum threshold, impairs the growth of a subsequent sporozoite infection of liver cells. Blood-stage parasites stimulate the production of the host iron-regulatory factor hepcidin, which redistributes iron away from hepatocytes, reducing the development of the iron-dependent liver stage. This could explain why Plasmodium superinfection is not often found in young nonimmune children. Here, we discuss the impact that such protection from superinfection might have in epidemiological settings or in programmes for controlling malaria, as well as how the induction of hepcidin and redistribution of iron might influence anaemia and the outcome of non-Plasmodium co-infections. © 2011 European Molecular Biology Organization.

Malaria afflicts 350–500 million people annually, and this debilitating and deadly infectious disease exacts a heavy toll on susceptible populations around the globe. Efforts to find effective, safe, and low-cost drugs for malaria have sharply increased in recent years. Almost all of these efforts have focused on the cyclic blood stage of the disease, partly because the parasites can be easily maintained in culture through addition of human red blood cells to the growth medium, and partly because blood stage infection causes malaria's characteristic symptoms. However, the asymptomatic liver stage, which the parasite goes through only once in its life history, presents the best opportunity for developing drugs that both hit new targets and also could be used in highly desirable eradication campaigns. Recent research, especially on the frequency of differentially expressed genes in blood and liver stage parasites, supports the feasibility of discovering stage-specific drugs. Discovering these drugs will require a high-throughput liver stage phenotypic screen comparable to the existing blood stage screens, and the basic tools for such a screen have recently been created.

Malaria is one of the foremost public health problems in Africa. It is endemic in 90 countries, affecting nearly 40% of the global population. The increasing prevalence of drug-resistant Plasmodium falciparum strains is one of the greatest challenges in malaria control. In order to overcome drug-resistance, new antimalarial drugs are urgently needed. Most of the available antimalarial agents kill blood stage parasites and only a limited number of drugs act on liver stages. In fact, the study of Plasmodium liver stage development (Fig. 1) has been hampered by limitations in the experimental approaches required to quantify hepatocyte infection by the parasite. Therefore, the development of new drugs targeting Plasmodium liver stages represents an important and underexploited site of intervention [1, 2].

Background Human populations that are naturally subjected to Plasmodium infection do not acquire complete protection against the liver stage of this parasite despite prolonged and frequent exposure. However, sterile immunity against Plasmodium liver stage can be achieved after repeated exposure to radiation attenuated sporozoites. The reasons for this different response remain largely unknown, but a suppressive effect of blood stage Plasmodium infection has been proposed as a cause for the lack of liver stage protection. Methods Using Plasmodium yoelii 17XNL, the response generated in mice subjected to daily infective bites from normal or irradiated mosquitoes was compared. The effect of daily-infected mosquito bites on mice that were previously immunized against P. yoelii liver stage was also studied. Results It was observed that while the bites of normal infected mosquitoes do not generate strong antibody responses and protection, the bites of irradiated mosquitoes result in high levels of anti-sporozoite antibodies and protection against liver stage Plasmodium infection. Exposure to daily infected mosquito bites did not eliminate the protection acquired previously with a experimental liver stage vaccine. Conclusions Liver stage immunity generated by irradiated versus normal P. yoelii infected mosquitoes is essentially different, probably because of the blood stage infection that follows normal mosquito bites, but not irradiated. While infective mosquito bites do not induce a protective liver stage response, they also do not interfere with previously acquired liver stage protective responses, even if they induce a complete blood stage infection. Considering that the recently generated anti-malaria vaccines induce only partial protection against infection, it is encouraging that, at least in mouse models, immunity is not negatively affected by subsequent exposure and infection with the parasite.

Protozoan parasites of the genus Plasmodium are the causative agents of malaria. Despite more than 100 years of research, the complex life cycle of the parasite still bears many surprises and it is safe to say that understanding the biology of the pathogen will keep scientists busy for many years to come. Malaria research has mainly concentrated on the pathological blood stage of Plasmodium parasites, leaving us with many questions concerning parasite development within the mosquito and during the exo-erythrocytic stage in the vertebrate host. After the discovery of the Plasmodium liver stage in the middle of the last century, it remained understudied for many years but the realization that it represents a promising target for vaccination approaches has brought it back into focus. The last decade saw many new and exciting discoveries concerning the exo-erythrocytic stage and in this review we will discuss the highlights of the latest developments in the field.

Relapsing human malaria is regarded to be caused by Plasmodium vivax and Plasmodium ovale. P. vivax relapses originate from dormant liver stages: “hypnozoites”. Also, P. ovale, a species considered as closely related to P. vivax, is in analogy assumed to display hypnozoites. A close biologic relationship is, however, not supported by molecular genetic studies. Therefore, original literature published since the description of P. ovale in 1922 was systematically screened for the demonstration of hypnozoites or circumstantial evidence for their existence, i.e. the occurrence of true relapses. In P. ovale infection, hypnozoites have never been demonstrated by biological experiments, and the few reports published on relapses have conflicting results.

Liver enzyme elevations among patients on antiretroviral therapy (ART) were determined by prospectively evaluating aspartate aminotransferase (AST) data in a cohort of patients in Kampala over 36 months. A proportion of patients had hepatitis B virus (HBV) status determined. Hepatotoxicity was graded I to IV according to the AIDS Clinical Trial Group criteria. Of 546 patients, 377 (69%) were women; overall median baseline CD4+ T-cell was 97/μL (interquartile range [IQR] 20–164). Hepatitis B surface antigen (HBsAg) was detected in 42 (9%) of 470 persons. ART included lamivudine, with either nevirapine and d4T (74%) or efavirenz and AZT (26%). Median (IQR) AST level at baseline was 35 (27, 53 IU/L). Over 36 months, only eight patients had grade III AST elevation. Neither HBsAg nor ART regimen influenced AST levels. Male gender and CD4+ change from baseline were correlated with AST elevation. Patients with HIV/HBV co-infection were not at an increased risk of AST elevation, which occurred uncommonly in this setting.

Malaria sporozoites must cross at least two cell barriers to reach their initial site of replication in the mammalian host. After transmission into the skin by an infected mosquito, they migrate towards small dermal capillaries, traverse the vascular endothelial layer,1,2 and rapidly home to the liver. To infect hepatocytes, the parasites must cross the sinusoidal cell layer, composed of specialized highly fenestrated sinusoidal endothelia and Kupffer cells, the resident macrophages of the liver (Fig. 1). The exact route Plasmodium sporozoites take to hepatocytes has been subject of controversial discussions for many years. Recent cell biological, microscopic, and genetic approaches have considerably enhanced our understanding of the initial events leading to the establishment of a malaria infection in the liver (for recent reviews see refs. 3, 4, 5, 6, 7, 8).

Individuals in malaria endemic regions do not develop fully protective immune responses against Plasmodium liver stage infections. In high transmission areas, individuals can be exposed to more than two infective mosquito bites daily. Their exposure to Plasmodium sporozoites, therefore, is in the form of small and frequent doses. This is very different from individuals studied in controlled immunization trials where the delivery of large numbers of radiation-attenuated sporozoites in a limited number of doses can induce sterile protective immunity. Using irradiated mosquitoes infected with Plasmodium yoelii 17XNL, we tested whether daily bites from a few mosquitoes can induce a protective immune response in mice. This immunization strategy successfully induced a protective response, preventing the development of liver stages when mice were challenged with non-irradiated sporozoites. These results provide further support for the development of liver stage vaccines. They are also a call for further study into why fully protective responses against the liver stage are not seen in individuals from endemic regions.

All mammalian malaria parasite species have an initial tissue stage in liver cells. The liver stage produces new parasite forms that can enter and live inside red blood cells. Accordingly, the first place of residence provides parasites with a radically different cellular and molecular environment from their subsequent red blood cell home. Liver stages have remained refractory to reveal their secrets, yet the last few years have seen several advances in elucidating their biology. This review looks at the more recent findings concerning the liver stage-host hepatocyte association, some of which may become powerful weapons in the prevention of malaria infection. We also outline areas of liver stage research and technological development that provide promising foci to accelerate a better understanding of this most elusive of the parasites many life cycle stages.

Plasmodium sporozoites are deposited in the skin of their vertebrate hosts through the bite of an infected female Anopheles mosquito. Most of these parasites find a blood vessel and travel in the peripheral blood circulation until they reach the liver sinusoids. Once there, the sporozoites cross the sinusoidal wall and migrate through several hepatocytes before they infect a final hepatocyte, with the formation of a parasitophorous vacuole, in which the intrahepatic form of the parasite grows and multiplies. During this period, each sporozoite generates thousands of merozoites. As the development of Plasmodium sporozoites inside hepatocytes is an obligatory step before the onset of disease, understanding the parasite's requirements during this period is crucial for the development of any form of early intervention. This Review summarizes our current knowledge on this stage of the Plasmodium life cycle.

The development of a microscale human liver platform that recapitulates the entire liver stage of malaria and could be used to screen antimalarial drugs and vaccines.

A standard method for evaluating inhibitory effects on preerythrocytic parasites in murine malaria models is to measure the time to patency of a blood-stage infection following a sporozoite challenge. The prevention of a blood-stage infection or a delay in patency suggests that preerythrocytic parasites have been eliminated or that liverstage development has been inhibited. However, susceptibility to a challenge with blood-stage parasites must also be determined to conclude that preerythrocytic parasites and not blood-stage parasites were targeted. As a more direct method, liver-parasite burden can be measured. Due to the paucity of liver-stage parasites at physiological challenge doses, 1.0 × 106 sporozoites) (2). Molecular techniques that target Plasmodium-specific small subunit rRNA are much more sensitive, requiring less than 5.0 × 104 sporozoites to quantitate liver stages (3,4). Plasmodium rRNA has been now measured in total liver RNA by spot-blot hybridization with Plasmodium-specific rRNA probes (3) and as described here by quantitativecompetitive reverse transcriptase–polymerase chain reaction (RT-PCR) (4).

Malaria parasite species that infect mammals, including humans, must first take up residence in hepatic host cells as exoerythrocytic forms (EEF) before initiating infection of red blood cells that leads to malaria disease. Despite the importance of hepatic stages for immunity against malaria, little is known about their biology and antigenic composition. Here, we show that sporozoites, the parasites' transmission stage that resides in the mosquito vector salivary glands, can transform into early EEF without intracellular residence in host hepatocytes. The morphological sequence of transformation and the expression of proteins in the EEF appear indistinguishable from parasites that develop within host cells. Transformation depends on temperature elevation to 37°C and serum. Our findings demonstrate that residence in a host hepatocyte or specific host cell–derived factors are not necessary to bring about the profound morphological and biochemical changes of the parasite that occur after its transmission from vector to mammalian host.

We found that infection of a rodent malaria, Plasmodium berghei, occurred when the sporozoites were injected into the skin, the muscle, the peritoneal cavity and the tail end. Mice, which were injected with sporozoites in the tail end and had the site cut 5 min later, did not develop malaria. We also found that mice developed malaria when malaria infective mosquitoes, Anopheles stephensi, were forced not to take blood but only to probe into the skin. Moreover, the mice probed by the infective mosquitoes were protected from malaria infection if the site was treated with Kyu (heat treatment) after the mosquitoes had probed. These findings indicate that malaria infection occurs not only by blood feeding of the infective mosquito but also by probing of the mosquito. Sporozoites injected into the skin remain at the injected site for at least 5 min, then migrate to the blood vessels and invade into the blood stream. At present, the mechanism is not clear, although we propose here the existence of the skin stage of malaria parasites before the liver stage and the blood stage.

The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species

Background: As anti-malarial drug resistance escalates, new safe and effective medications are necessary to prevent and treat malaria infections. The US Army is developing tafenoquine (TQ), an analogue of primaquine (PQ), which is expected to be more effective in preventing malaria in deployed military personnel. Methods: To compare the prophylactic efficacy of TQ and PQ, a transgenic Plasmodium berghei parasite expressing the bioluminescent reporter protein luciferase was utilized to visualize and quantify parasite development in C57BL/6 albino mice treated with PQ and TQ in single or multiple regimens using a real-time in vivo imaging system (IVIS). As an additional endpoint, blood stage parasitaemia was monitored by flow cytometry. Comparative pharmacokinetic (PK) and liver distribution studies of oral and intravenous PQ and TQ were also performed. Results: Mice treated orally with three doses of TQ at 5 mg/kg three doses of PQ at 25 mg/kg demonstrated no bioluminescence liver signal and no blood stage parasitaemia was observed suggesting both drugs showed 100% causal activity at the doses tested. Single dose oral treatment with 5 mg TQ or 25 mg of PQ, however, yielded different results as only TQ treatment resulted in causal prophylaxis in P. berghei sporozoite-infected mice. TQ is highly effective for causal prophylaxis in mice at a minimal curative single oral dose of 5 mg/kg, which is a five-fold improvement in potency versus PQ. PK studies of the two drugs administered orally to mice showed that the absolute bioavailability of oral TQ was 3.5-fold higher than PQ, and the AUC of oral TQ was 94-fold higher than oral PQ. The elimination half-life of oral TQ in mice was 28 times longer than PQ, and the liver tissue distribution of TQ revealed an AUC that was 188-fold higher than PQ. Conclusions: The increased drug exposure levels and longer exposure time of oral TQ in the plasma and livers of mice highlight the lead quality attributes that explain the much improved efficacy of TQ when compared to PQ.

US Department of Health and Human Services. Public Health Service. Centers for Disease Control and Prevention. National Center for Infectious Diseases. Division of Parasitic Diseases. Immunology Branch. Clifion Road, Atlanta, USA. US Department of Health and Human Services. Public Health Service. Centers for Disease Control and Prevention. National Center for Infectious Diseases . Division of Parasitic Diseases. Immunology Branch. Clifion Road, Atlanta, USA. US Department of Health and Human Services. Public Health Service. Centers for Disease Control and Prevention. National Center for Infectious Diseases . Division of Parasitic Diseases. Immunology Branch. Clifion Road, Atlanta, USA. Papua New Guinea Institute of Medical Research. Goroka, Papua, New Guinea. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. US Department of Health and Human Services. Public Health Service. Centers for Disease Control and Prevention. National Center for Infectious Diseases - Division of Parasitic Diseases. Immunology Branch. Clifion Road, Atlanta, USA. US Department of Health and Human Services. Public Health Service. Centers for Disease Control and Prevention. National Center for Infectious Diseases - Division of Parasitic Diseases. Immunology Branch. Clifion Road, Atlanta, USA. US Department of Health and Human Services. Public Health Service. Centers for Disease Control and Prevention. National Center for Infectious Diseases - Division of Parasitic Diseases. Immunology Branch. Clifion Road, Atlanta, USA.