Promoter regions of Plasmodium vivax are poorly or not recognized by Plasmodium falciparum

Abstract Background Heterologous promoter analysis in Plasmodium has revealed the existence of conserved cis regulatory elements as promoters from different species can drive expression of reporter genes in heterologous transfection assays. Here, the functional characterization of different Plasmodium vivax promoters in Plasmodium falciparum using luciferase as the reporter gene is presented. Methods Luciferase reporter plasmids harboring the upstream regions of the msp1, dhfr, and vir3 genes as well as the full-length intergenic regions of the vir23/24 and ef-1α genes of P. vivax were constructed and transiently transfected in P. falciparum. Results Only the constructs with the full-length intergenic regions of the vir23/24 and ef-1α genes were recognized by the P. falciparum transcription machinery albeit to values approximately two orders of magnitude lower than those reported by luc plasmids harbouring promoter regions from P. falciparum and Plasmodium berghei. A bioinformatics approach allowed the identification of a motif (GCATAT) in the ef-1α intergenic region that is conserved in five Plasmodium species but is degenerate (GCANAN) in P. vivax. Mutations of this motif in the P. berghei ef-1α promoter region decreased reporter expression indicating it is active in gene expression in Plasmodium. Conclusion Together, this data indicates that promoter regions of P. vivax are poorly or not recognized by the P. falciparum transcription machinery suggesting the existence of P. vivax-specific transcription regulatory elements.

Pattern of humoral immune response to Plasmodium falciparum blood stages in individuals presenting different clinical expressions of malaria

Abstract Background The development of protective immunity against malaria is slow and to be maintained, it requires exposure to multiple antigenic variants of malaria parasites and age-associated maturation of the immune system. Evidence that the protective immunity is associated with different classes and subclasses of antibodies reveals the importance of considering the quality of the response. In this study, we have evaluated the humoral immune response against Plasmodium falciparum blood stages of individuals naturally exposed to malaria who live in endemic areas of Brazil in order to assess the prevalence of different specific isotypes and their association with different malaria clinical expressions. Methods Different isotypes against P. falciparum blood stages, IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA, were determined by ELISA. The results were based on the analysis of different clinical expressions of malaria (complicated, uncomplicated and asymptomatic) and factors related to prior malaria exposure such as age and the number of previous clinical malaria attacks. The occurrence of the H131 polymorphism of the FcγIIA receptor was also investigated in part of the studied population. Results The highest levels of IgG, IgG1, IgG2 and IgG3 antibodies were observed in individuals with asymptomatic and uncomplicated malaria, while highest levels of IgG4, IgE and IgM antibodies were predominant among individuals with complicated malaria. Individuals reporting more than five previous clinical malaria attacks presented a predominance of IgG1, IgG2 and IgG3 antibodies, while IgM, IgA and IgE antibodies predominated among individuals reporting five or less previous clinical malaria attacks. Among individuals with uncomplicated and asymptomatic malaria, there was a predominance of high-avidity IgG, IgG1, IgG2 antibodies and low-avidity IgG3 antibodies. The H131 polymorphism was found in 44.4% of the individuals, and the highest IgG2 levels were observed among asymptomatic individuals with this allele, suggesting the protective role of IgG2 in this population. Conclusion Together, the results suggest a differential regulation in the anti-P. falciparum antibody pattern in different clinical expressions of malaria and showed that even in unstable transmission areas, protective immunity against malaria can be observed, when the appropriated antibodies are produced.

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Abstract Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients' plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2(DF=1) = 9.26/p = 0.0047) and MSP5 (X2(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2(DF=1) = 6.41/p = 0.0206, Fisher's exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney's U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95% = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.

Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients

Abstract Background Plasmodium vivax is the most widely distributed human malaria, responsible for 70–80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected. Methods A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10-30 was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology Results A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them. Conclusion These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.

Amazonian malaria: Asymptomatic human reservoirs, diagnostic challenges, environmentally driven changes in mosquito vector populations, and the mandate for sustainable control strategies

Across the Americas and the Caribbean, nearly 561,000 slide-confirmed malaria infections were reported officially in 2008. The nine Amazonian countries accounted for 89% of these infections; Brazil and Peru alone contributed 56% and 7% of them, respectively. Local populations of the relatively neglected parasite Plasmodium vivax, which currently accounts for 77% of the regional malaria burden, are extremely diverse genetically and geographically structured. At a time when malaria elimination is placed on the public health agenda of several endemic countries, it remains unclear why malaria proved so difficult to control in areas of relatively low levels of transmission such as the Amazon Basin. We hypothesize that asymptomatic parasite carriage and massive environmental changes that affect vector abundance and behavior are major contributors to malaria transmission in epidemiologically diverse areas across the Amazon Basin. Here we review available data supporting this hypothesis and discuss their implications for current and future malaria intervention policies in the region. Given that locally generated scientific evidence is urgently required to support malaria control interventions in Amazonia, we briefly describe the aims of our current field-oriented malaria research in rural villages and gold-mining enclaves in Peru and a recently opened agricultural settlement in Brazil. (C) 2011 Elsevier B.V. All rights reserved.

Vesicles as carriers of virulence factors in parasitic protozoan diseases

Different types of shed vesicles as, for example, exosomes, plasma-membrane-derived vesicles or microparticles, are the focus of intense research in view of their potential role in cell cell communication and under the perspective that they might be good tools for immunotherapy, vaccination or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism. Trypanosomatids are compared with Apicomplexa, which circulate parasite antigens bound to vesicles shed by host cells. The knowledge of the origin and chemical composition of these different vesicles might lead to the understanding of the mechanisms that determine their biological function. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

CD8(+) T-cell-mediated immunity against malaria: a novel heterologous prime-boost strategy

Evaluation of: Rodriguez D, Gonzalez-Aseguinolaza G, Rodriguez JR et al. Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium. PLoS ONE 7(4), e34445 (2012). Recently, a vaccine against malaria was successfully tested in a human Phase III trial. The efficacy of this vaccine formulation, based on the Plasmodium falciparum circumsporozoite protein, was approximately 50% and correlated with the presence of antibodies specific to the infective stages of the malaria parasites. Different strategies are being pursued to improve vaccine efficacy levels. One such strategy is the induction of specific cytotoxic T cells that can destroy the intracellular hepatocyte stages of the malaria parasite. In this study, a novel vaccination protocol was developed to elicit strong immune responses mediated by CD8(+) cytotoxic cells specific to the circumsporozoite protein. As proof-of-concept, the authors used the rodent malaria Plasmodium yoelii parasite. The vaccination strategy consisted of a heterologous prime-boost vaccination regimen involving porcine parvovirus-like particles for priming and the modified vaccinia virus Ankara for the booster immunization, both of which expressed the immunodominant CD8 epitope of the P. yoelii circumsporozoite protein. Results from this experimental model were extremely meaningful. This vaccination strategy led to a significant T-cell immune response mediated by CD8(+) multifunctional T effector and effector-memory cells. However, most importantly for the malaria vaccine development was the fact that following a sporozoite challenge, immunized mice eliminated more than 97% of the malaria parasites during the hepatocyte stages. These results confirm and extend a vast body of knowledge showing that a heterologous prime-boost vaccination strategy can elicit strong CD8(+) T-cell-mediated protective immunity and may increase the efficacy of malaria vaccines.

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

Abstract Background Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. Methods Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. Results Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). Conclusions Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Assessing malaria transmission in a low endemicity area of north-western Peru

Background Where malaria endemicity is low, control programmes need increasingly sensitive tools for monitoring malaria transmission intensity (MTI) and to better define health priorities. A cross-sectional survey was conducted in a low endemicity area of the Peruvian north-western coast to assess the MTI using both molecular and serological tools. Methods Epidemiological, parasitological and serological data were collected from 2,667 individuals in three settlements of Bellavista district, in May 2010. Parasite infection was detected using microscopy and polymerase chain reaction (PCR). Antibodies to Plasmodium vivax merozoite surface protein-119 (PvMSP119) and to Plasmodium falciparum glutamate-rich protein (PfGLURP) were detected by ELISA. Risk factors for exposure to malaria (seropositivity) were assessed by multivariate survey logistic regression models. Age-specific antibody prevalence of both P. falciparum and P. vivax were analysed using a previously published catalytic conversion model based on maximum likelihood for generating seroconversion rates (SCR). Results The overall parasite prevalence by microscopy and PCR were extremely low: 0.3 and 0.9%, respectively for P. vivax, and 0 and 0.04%, respectively for P. falciparum, while seroprevalence was much higher, 13.6% for P. vivax and 9.8% for P. falciparum. Settlement, age and occupation as moto-taxi driver during previous year were significantly associated with P. falciparum exposure, while age and distance to the water drain were associated with P. vivax exposure. Likelihood ratio tests supported age seroprevalence curves with two SCR for both P. vivax and P. falciparum indicating significant changes in the MTI over time. The SCR for PfGLURP was 19-fold lower after 2002 as compared to before (λ1 = 0.022 versus λ2 = 0.431), and the SCR for PvMSP119 was four-fold higher after 2006 as compared to before (λ1 = 0.024 versus λ2 = 0.006). Conclusion Combining molecular and serological tools considerably enhanced the capacity of detecting current and past exposure to malaria infections and related risks factors in this very low endemicity area. This allowed for an improved characterization of the current human reservoir of infections, largely hidden and heterogeneous, as well as providing insights into recent changes in species specific MTIs. This approach will be of key importance for evaluating and monitoring future malaria elimination strategies.

Assessing the gain of biological data integration in gene networks inference

Background: A current challenge in gene annotation is to define the gene function in the context of the network of relationships instead of using single genes. The inference of gene networks (GNs) has emerged as an approach to better understand the biology of the system and to study how several components of this network interact with each other and keep their functions stable. However, in general there is no sufficient data to accurately recover the GNs from their expression levels leading to the curse of dimensionality, in which the number of variables is higher than samples. One way to mitigate this problem is to integrate biological data instead of using only the expression profiles in the inference process. Nowadays, the use of several biological information in inference methods had a significant increase in order to better recover the connections between genes and reduce the false positives. What makes this strategy so interesting is the possibility of confirming the known connections through the included biological data, and the possibility of discovering new relationships between genes when observed the expression data. Although several works in data integration have increased the performance of the network inference methods, the real contribution of adding each type of biological information in the obtained improvement is not clear. Methods: We propose a methodology to include biological information into an inference algorithm in order to assess its prediction gain by using biological information and expression profile together. We also evaluated and compared the gain of adding four types of biological information: (a) protein-protein interaction, (b) Rosetta stone fusion proteins, (c) KEGG and (d) KEGG+GO. Results and conclusions: This work presents a first comparison of the gain in the use of prior biological information in the inference of GNs by considering the eukaryote (P. falciparum) organism. Our results indicates that information based on direct interaction can produce a higher improvement in the gain than data about a less specific relationship as GO or KEGG. Also, as expected, the results show that the use of biological information is a very important approach for the improvement of the inference. We also compared the gain in the inference of the global network and only the hubs. The results indicates that the use of biological information can improve the identification of the most connected proteins.

Defibrotide Interferes With Several Steps of the Coagulation-Inflammation Cycle and Exhibits Therapeutic Potential to Treat Severe Malaria

Objective-The coagulation-inflammation cycle has been implicated as a critical component in malaria pathogenesis. Defibrotide (DF), a mixture of DNA aptamers, displays anticoagulant, anti-inflammatory, and endothelial cell (EC)-protective activities and has been successfully used to treat comatose children with veno-occlusive disease. DF was investigated here as a drug to treat cerebral malaria. Methods and Results-DF blocks tissue factor expression by ECs incubated with parasitized red blood cells and attenuates prothrombinase activity, platelet aggregation, and complement activation. In contrast, it does not affect nitric oxide bioavailability. We also demonstrated that Plasmodium falciparum glycosylphosphatidylinositol (Pf-GPI) induces tissue factor expression in ECs and cytokine production by dendritic cells. Notably, dendritic cells, known to modulate coagulation and inflammation systemically, were identified as a novel target for DF. Accordingly, DF inhibits Toll-like receptor ligand-dependent dendritic cells activation by a mechanism that is blocked by adenosine receptor antagonist (8-p-sulfophenyltheophylline) but not reproduced by synthetic poly-A, -C, -T, and -G. These results imply that aptameric sequences and adenosine receptor mediate dendritic cells responses to the drug. DF also prevents rosetting formation, red blood cells invasion by P. falciparum and abolishes oocysts development in Anopheles gambiae. In a murine model of cerebral malaria, DF affected parasitemia, decreased IFN-gamma levels, and ameliorated clinical score (day 5) with a trend for increased survival. Conclusion-Therapeutic use of DF in malaria is proposed. (Arterioscler Thromb Vasc Biol. 2012; 32:786-798.)

Synthesis and Antiplasmodial Activity of Betulinic Acid and Ursolic Acid Analogues

More than 40% of the World population is at risk of contracting malaria, which affects primarily poor populations in tropical and subtropical areas. Antimalarial pharmacotherapy has utilised plant-derived products such as quinine and artemisinin as well as their derivatives. However, worldwide use of these antimalarials has caused the spread of resistant parasites, resulting in increased malaria morbidity and mortality. Considering that the literature has demonstrated the antimalarial potential of triterpenes, specially betulinic acid (1) and ursolic acid (2), this study investigated the antimalarial activity against P. falciparum chloroquine-sensitive 3D7 strain of some new derivatives of 1 and 2 with modifications at C-3 and C-28. The antiplasmodial study employed flow cytometry and spectrofluorimetric analyses using YOYO-1, dihydroethidium and Fluo4/AM for staining. Among the six analogues obtained, compounds 1c and 2c showed excellent activity (IC50 = 220 and 175 nM, respectively) while 1a and b demonstrated good activity ( IC50 = 4 and 5 mu M, respectively). After cytotoxicity evaluation against HEK293T cells, 1a was not toxic, while 1c and 2c showed IC50 of 4 mu M and a selectivity index (SI) value of 18 and 23, respectively. Moreover, compound 2c, which presents the best antiplasmodial activity, is involved in the calcium-regulated pathway(s).

Effect of artesunate-mefloquine fixed-dose combination in malaria transmission in amazon basin communities

Background: Studies in South-East Asia have suggested that early diagnosis and treatment with artesunate (AS) and mefloquine (MQ) combination therapy may reduce the transmission of Plasmodium falciparum malaria and the progression of MQ resistance. Methods: The effectiveness of a fixed-dose combination of AS and MQ (ASMQ) in reducing malaria transmission was tested in isolated communities of the Jurua valley in the Amazon region. Priority municipalities within the Brazilian Legal Amazon area were selected according to pre-specified criteria. Routine national malaria control programmatic procedures were followed. Existing health structures were reinforced and health care workers were trained to treat with ASMQ all confirmed falciparum malaria cases that match inclusion criteria. A local pharmacovigilance structure was implemented. Incidence of malaria and hospitalizations were recorded two years before, during, and after the fixed-dose ASMQ intervention. In total, between July 2006 and December 2008, 23,845 patients received ASMQ. Two statistical modelling approaches were applied to monthly time series of P. falciparum malaria incidence rates, P. falciparum/Plasmodium vivax infection ratio, and malaria hospital admissions rates. All the time series ranged from January 2004 to December 2008, whilst the intervention period span from July 2006 to December 2008. Results: The ASMQ intervention had a highly significant impact on the mean level of each time series, adjusted for trend and season, of 0.34 (95% CI 0.20 - 0.58) for the P. falciparum malaria incidence rates, 0.67 (95% CI 0.50 - 0.89) for the P. falciparum/P. vivax infection ratio, and 0.53 (95% CI 0.41 - 0.69) for the hospital admission rates. There was also a significant change in the seasonal (or monthly) pattern of the time series before and after intervention, with the elimination of the malaria seasonal peak in the rainy months of the years following the introduction of ASMQ. No serious adverse events relating to the use of fixed-dose ASMQ were reported. Conclusions: In the remote region of the Jurua valley, the early detection of malaria by health care workers and treatment with fixed-dose ASMQ was feasible and efficacious, and significantly reduced the incidence and morbidity of P. falciparum malaria.