Glutathione transferase from Plasmodium falciparum – Interaction with malagashanine and selected plant natural products

A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC₅₀ values of 8.5, 12, 50 and 69 μM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The K_i for GSH and CDNB were ?0.015?μM and 0.011?μM, respectively. Malagashanine (100?µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t_1/2 of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5?mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds.     

Modelling the Incidence of Plasmodium vivax and Plasmodium falciparum Malaria in Afghanistan 2006–2009

Background

Identifying areas that support high malaria risks and where populations lack access to health care is central to reducing the burden in Afghanistan. This study investigated the incidence of Plasmodium vivax and Plasmodium falciparum using routine data to help focus malaria interventions.

Methods

To estimate incidence, the study modelled utilisation of the public health sector using fever treatment data from the 2012 national Malaria Indicator Survey. A probabilistic measure of attendance was applied to population density metrics to define the proportion of the population within catchment of a public health facility. Malaria data were used in a Bayesian spatio-temporal conditional-autoregressive model with ecological or environmental covariates, to examine the spatial and temporal variation of incidence.

Findings

From the analysis of healthcare utilisation, over 80% of the population was within 2 hours’ travel of the nearest public health facility, while 64.4% were within 30 minutes’ travel. The mean incidence of P. vivax in 2009 was 5.4 (95% Crl 3.2–9.2) cases per 1000 population compared to 1.2 (95% Crl 0.4–2.9) cases per 1000 population for P. falciparum. P. vivax peaked in August while P. falciparum peaked in November. 32% of the estimated 30.5 million people lived in regions where annual incidence was at least 1 case per 1,000 population of P. vivax; 23.7% of the population lived in areas where annual P. falciparum case incidence was at least 1 per 1000.

Conclusion

This study showed how routine data can be combined with household survey data to model malaria incidence. The incidence of both P. vivax and P. falciparum in Afghanistan remain low but the co-distribution of both parasites and the lag in their peak season provides challenges to malaria control in Afghanistan. Future improved case definition to determine levels of imported risks may be useful for the elimination ambitions in Afghanistan.     

The P-glycoprotein homologues of Plasmodium falciparum: Are they involved in chloroquine resistance?

Chloroquine has been the mainstay of antimalarial chemotherapy but the rapid spread of resistance to this important drug has now compromised its efficacy. The mechanism of chloroquine resistance has not been known but recent evidence from Plasmodium falciparum, the causative agent of the most severe form of human malaria, suggested similarities to the multidrug resistance phenotype (MDR) of mammalian tumour cells which is mediated by a protein molecule termed P-glycoprotein. Two mdr genes (pfmdr1 and pfmdr2) encoding P-glycoprotein homologues have been identified in P. falciparum and one of these (pfmdr1) has several alleles that have been linked to the chloroquine resistance phenotype. In contrast analysis of a genetic cross between chloroquine-resistant and -sensitive P. falciparum has suggested that the genes encoding the known P-glycoprotein homologues are not linked. This review outlines the similarities of the chloroquine resistance phenotype with the MDR phenotype of mammalian tumour cells and explores the possible role of the pfmdr genes.     

Nosocomial Plasmodium falciparum infections confirmed by molecular typing in Medellín, Colombia

Three cases of nosocomial malaria are reported from patients of the Internal Medicine Ward of a tertiary University teaching hospital in Medellin, Colombia. Epidemiological research, based on entomological captures, medical records review and interviews of nursery staff about patient care practices potentially involving contact with blood, were carried out. Molecular characterization of Plasmodium falciparum was based on the amplification of MSP1, MSP2 and GLURP genes. This method enabled confirmation of the same P. falciparum genotype in all three patients as well as in a fourth one (index case). The presence of nosocomial malaria was confirmed and it was concluded that the most likely source of transmission was through multi-dose preparations of heparin applied to heparin locks.     

Nosocomial Plasmodium falciparum infections confirmed by molecular typing in Medellín, Colombia

Background

Malaria is a serious, sometimes fatal, disease caused by Plasmodium infection of human red blood cells. The host-parasite co-evolutionary processes are well understood by the association of coding variations such as G6PD, Duffy blood group receptor, HLA, and beta-globin gene variants with malaria resistance. The profound genetic diversity in host is attributed to polymorphic microsatellites loci. The microsatellite alleles in bacterial species are known to have aided their survival in fatal environmental conditions. The fascinating question is whether microsatellites are genomic cushion in the human genome to combat disease stress and has cause-effect relationships with infections.

Presentation of the hypothesis

It is hypothesized that repeat units or alleles of microsatellites TH01 and D5S818, located in close proximity to beta-globin gene and immune regulatory region in human play a role in malaria predisposition. Association of alleles at aforesaid microsatellites with malaria infection was analysed. To overrule the false association in unrecognized population stratification, structure analysis and AMOVA were performed among the sampled groups.

Testing of hypothesis

Associations of microsatellite alleles with malaria infection were verified using recombination rate, Chi-square, and powerful likelihood tests. Further investigation of population genetic structure, and AMOVA was done to rule out the confounding effects of population stratification in interpretation of association studies.

Implication of the hypothesis

Lower recombination rate (θ) between microsatellites and genes implicated in host fitness; positive association between alleles -13 (D5S818), 9 (TH01) and strong susceptibility to Plasmodium falciparum; and alleles-12 (D5S818) and 6 (TH01) rendering resistance to human host were evident. The interesting fact emerging from the study was that while predisposition to malaria was a prehistoric attribute, among TH01 alleles; evolution of resistant allele-6 was a recent phenomenon, which could conceivably be driven by infection related selective forces. The host's microsatellite allelic associations with malaria infection were valid in the light of low genetic variance between sampled groups and no population stratification.     

Characterization of Plasmodium falciparum adenylyl cyclase-β and its role in erythrocytic stage parasites

The most severe form of human malaria is caused by the parasite Plasmodium falciparum. The second messenger cAMP has been shown to be important for the parasite's ability to infect the host's liver, but its role during parasite growth inside erythrocytes, the stage responsible for symptomatic malaria, is less clear. The P. falciparum genome encodes two adenylyl cyclases, the enzymes that synthesize cAMP, PfACα and PfACβ. We now show that one of these, PfACβ, plays an important role during the erythrocytic stage of the P. falciparum life cycle. Biochemical characterization of PfACβ revealed a marked pH dependence, and sensitivity to a number of small molecule inhibitors. These inhibitors kill parasites growing inside red blood cells. One particular inhibitor is selective for PfACβ relative to its human ortholog, soluble adenylyl cyclase (sAC); thus, PfACβ represents a potential target for development of safe and effective antimalarial therapeutics.     

HipA-Triggered Growth Arrest and β-Lactam Tolerance in Escherichia coli Are Mediated by RelA-Dependent ppGpp Synthesis

Persistence is a phenomenon whereby a subpopulation of bacterial cells enters a transient growth-arrested state that confers antibiotic tolerance. While entrance into persistence has been linked to the activities of toxin proteins, the molecular mechanisms by which toxins induce growth arrest and the persistent state remain unclear. Here, we show that overexpression of the protein kinase HipA in Escherichia coli triggers growth arrest by activating synthesis of the alarmone guanosine tetraphosphate (ppGpp) by the enzyme RelA, a signal typically associated with amino acid starvation. We further demonstrate that chemically suppressing ppGpp synthesis with chloramphenicol relieves inhibition of DNA replication initiation and RNA synthesis in HipA-arrested cells and restores vulnerability to β-lactam antibiotics. HipA-arrested cells maintain glucose uptake and oxygen consumption and accumulate amino acids as a consequence of translational inhibition. We harness the active metabolism of HipA-arrested cells to provide a bacteriophage-resistant platform for the production of biotechnologically relevant compounds, which may represent an innovative solution to the costly problem of phage contamination in industrial fermentations.     

Asymptomatic infection with Plasmodium falciparum and Plasmodium vivax in the Brazilian Amazon Basin: to treat or not to treat?

In this study, we determined whether the treatment of asymptomatic parasites carriers (APCs), which are frequently found in the riverside localities of the Brazilian Amazon that are highly endemic for malaria, would decrease the local malaria incidence by decreasing the overall pool of parasites available to infect mosquitoes. In one village, the treatment of the 19 Plasmodium falciparum-infected APCs identified among the 270 residents led to a clear reduction (Z = -2.39, p = 0.017) in the incidence of clinical cases, suggesting that treatment of APCs is useful for controlling falciparum malaria. For vivax malaria, 120 APCs were identified among the 716 residents living in five villages. Comparing the monthly incidence of vivax malaria in two villages where the APCs were treated with the incidence in two villages where APCs were not treated yielded contradictory results and no clear differences in the incidence were observed (Z = -0.09, p = 0.933). Interestingly, a follow-up study showed that the frequency of clinical relapse in both the treated and untreated APCs was similar to the frequency seen in patients treated for primary clinical infections, thus indicating that vivax clinical immunity in the population is not species specific but only strain specific.     

Are extensive T cell epitope polymorphisms in the Plasmodium falciparum circumsporozoite antigen, a leading sporozoite vaccine candidate, selected by immune pressure?

Protective cellular immune responses depend on MHC presentation of pathogen-derived Ag fragments. MHC diversity renders this process sensitive to point mutations coding for altered amino acid sequence of the short target Ag-derived peptides epitopes. Thus, in a given host, a pathogen with an altered epitope sequence will be more likely to escape detection and elimination by the immune system. At a population level, selection by immune pressure will increase the likelihood of polymorphism in important pathogen antigenic epitopes. This mechanism of immune evasion is found in viruses and other pathogens. The detection of polymorphic hot spots in an Ag is often taken as a strong indication of its role in protective immunity. We provide evidence that polymorphisms in the T cell epitopes of a malaria vaccine candidate are unlikely to have been selected by immune pressure in the human host.     

Haemoglobin C and S in natural selection against Plasmodium falciparum malaria: a plethora or a single shared adaptive mechanism?

Conclusive evidence exists on the protective role against clinical Plasmodium falciparum malaria of Haemoglobin S (beta 6Glu-->Val) and HbC (HbC; beta 6Glu-->Lys), both occurring in sub-Saharan Africa. However, the mechanism/s of the protection exerted remain/s debated for both haemoglobin variants, HbC and HbS. Recently, an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, was reported on HbAC and HbCC infected erythrocytes that showed reduced cytoadhesion and impaired rosetting in vitro. On this basis it has been proposed that HbC protection might be attributed to the reduced PfEMP1-mediated adherence of parasitized erythrocytes in the microvasculature. Furthermore, impaired cytoadherence was observed in HbS carriers suggesting for the first time a convergence in the protection mechanism of these two haemoglobin variants. We investigated the impact of this hypothesis on the development of acquired immunity against P. falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC and HbS carriers in comparison with HbA of Burkina Faso. Higher immune response against a VSA panel and several malaria antigens were observed in all adaptive genotypes containing at least one allelic variant HbC or HbS in the low transmission urban area whereas no differences were detected in the high transmission rural area. In both contexts the response against tetanus toxoid was not influenced by the beta-globin genotype. Thus, these findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. We reviewed the hypothesized mechanisms so far proposed in light of these recent results.     

Impact of Glutathione S-Transferase M1 and T1 on Anti-Tuberculosis Drug–Induced Hepatotoxicity in Chinese Pediatric Patients

Background

Anti-tuberculosis drug induced hepatotoxicity (ATDH) is a major adverse drug reaction associated for anti-tuberculosis therapy. The glutathione S-transferases (GST) plays a crucial role in the detoxification of hepatotoxic metabolites of anti-tuberculosis drugs.An association between GSTM1/GSTT1 null mutations and increased risk of ATDH has been demonstrated in adults. Given the ethnic differences and developmental changes, our study aims to investigate the potential impacts of GSTM1/GSTT1genotypes on the development of ATDH in Han Chinese children treated with anti-tuberculosis therapy.

Methods

Children receiving anti-tuberculosis therapy with or without evidence of ATDH were considered as the cases or controls, respectively. The GSTM1 and GSTT1 genotyping were performed using the polymerase chain reaction.

Results

One hundred sixty-three children (20 cases and 143 controls) with a mean age of 4.7 years (range: 2 months-14.1 years) were included. For the GSTM1, 14 (70.0%) cases and 96 (67.1%) controls had homozygous null mutations. For the GSTT1, 13 (65.0%) cases and 97 (67.8%) controls had homozygous null mutations. Neither the GSTM1, nor the GSTT1 polymorphism was significantly correlated with the occurrence of ATHD.

Conclusion

Ourresults did not support the GSTM1 and GSTT1 polymorphisms as the predictors of ADTH in Chinese Han children treated with anti-tuberculosis drugs. An age-related association between pharmacogenetics and ATHD need to be confirmed in the further study.     

Antibodies against PfEMP1, RIFIN,MSP3 and GLURP Are Acquired during Controlled Plasmodium falciparum Malaria Infections in Na?ve Volunteers

Antibodies to polymorphic antigens expressed during the parasites erythrocytic stages are important mediators of protective immunity against P. falciparum malaria. Therefore, polymorphic blood stage antigens like MSP3, EBA-175 and GLURP and variant surface antigens PfEMP1 and RIFIN are considered vaccine candidates. However, to what extent these antibodies to blood stage antigens are acquired during naive individuals'' first infections has not been studied in depth. Using plasma samples collected from controlled experimental P. falciparum infections we show that antibodies against variant surface antigens, PfEMP1 and RIFIN as well as MSP3 and GLURP, are acquired during a single short low density P. falciparum infection in non-immune individuals including strain transcendent PfEMP1 immune responses. These data indicate that the immunogenicity of the variant surface antigens is similar to the less diverse merozoite antigens. The acquisition of a broad and strain transcendent repertoire of PfEMP1 antibodies may reflect a parasite strategy of expressing most or all PfEMP1 variants at liver release optimizing the likelihood of survival and establishment of chronic infections in the new host.     

Plasmodium falciparum Erythrocyte Membrane Protein 1 Diversity in Seven Genomes – Divide and Conquer

The var gene encoded hyper-variable Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates cytoadhesion of infected erythrocytes to human endothelium. Antibodies blocking cytoadhesion are important mediators of malaria immunity acquired by endemic populations. The development of a PfEMP1 based vaccine mimicking natural acquired immunity depends on a thorough understanding of the evolved PfEMP1 diversity, balancing antigenic variation against conserved receptor binding affinities. This study redefines and reclassifies the domains of PfEMP1 from seven genomes. Analysis of domains in 399 different PfEMP1 sequences allowed identification of several novel domain classes, and a high degree of PfEMP1 domain compositional order, including conserved domain cassettes not always associated with the established group A–E division of PfEMP1. A novel iterative homology block (HB) detection method was applied, allowing identification of 628 conserved minimal PfEMP1 building blocks, describing on average 83% of a PfEMP1 sequence. Using the HBs, similarities between domain classes were determined, and Duffy binding-like (DBL) domain subclasses were found in many cases to be hybrids of major domain classes. Related to this, a recombination hotspot was uncovered between DBL subdomains S2 and S3. The VarDom server is introduced, from which information on domain classes and homology blocks can be retrieved, and new sequences can be classified. Several conserved sequence elements were found, including: (1) residues conserved in all DBL domains predicted to interact and hold together the three DBL subdomains, (2) potential integrin binding sites in DBLα domains, (3) an acylation motif conserved in group A var genes suggesting N-terminal N-myristoylation, (4) PfEMP1 inter-domain regions proposed to be elastic disordered structures, and (5) several conserved predicted phosphorylation sites. Ideally, this comprehensive categorization of PfEMP1 will provide a platform for future studies on var/PfEMP1 expression and function.     

Homo-oligomerization and Activation of AMP-activated Protein Kinase Are Mediated by the Kinase Domain ��G-Helix

AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain αG-helix. Mutation of the corresponding AMPK α-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the αG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (α1^−/−, α2^−/−) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the αG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Cα was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic αG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic αG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.The maintenance of energy homeostasis is a basic requirement of all living organisms. The AMP-activated protein kinase (AMPK)² is crucially involved in this essential process by playing a central role in sensing and regulating energy metabolism on the cellular and whole body level (1–6). AMPK is also participating in several signaling pathways associated with cancer and metabolic diseases, like type 2 diabetes mellitus, obesity, and other metabolic disorders (7–9).Mammalian AMPK belongs to a highly conserved family of serine/threonine protein kinases with homologs found in all eukaryotic organisms examined (1, 3, 10). Its heterotrimeric structure includes a catalytic α-subunit and regulatory β- and γ-subunits. These subunits exist in different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3) and splice variants (for γ2 and γ3) and can thus assemble to a broad variety of heterotrimeric isoform combinations. The α- and β-subunits possess multiple autophosphorylation sites, which have been implicated in regulation of subcellular localization and kinase activation (11–15). The most critical step of AMPK activation, however, is phosphorylation of Thr-172 within the activation segment of the α-subunit kinase domain. At least two AMPK upstream kinases (AMPKKs) have been identified so far, namely the tumor suppressor kinase LKB1 in complex with MO25 and STRAD (16) and Ca²⁺/calmodulin-dependent protein kinase kinase-2 (CamKK2) (17). Furthermore, the transforming growth factor-β-activated kinase 1 was also shown to activate AMPK using a variety of in vitro approaches (18), but the physiological relevance of these findings remains unclear. Besides direct phosphorylation of Thr-172, AMPK activity is stimulated by the allosteric activator AMP, which can bind to two Bateman domains formed by two pairs of CBS domains within the γ-subunit (19–22). Hereby bound AMP not only allosterically stimulates AMPK but also protects Thr-172 from dephosphorylation by protein phosphatase 2Cα (PP2Cα) and thus hinders inactivation of the kinase (19, 22, 23). Consequently, on the cellular level, AMPK is activated upon metabolic stress increasing the AMP/ATP ratio. Furthermore, AMPK activation can also be induced by several chemical compounds, like nucleoside 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (24) and the anti-diabetic drug Metformin (25–28). In addition, the small molecule compound A-769662 was recently developed as a direct allosteric activator of AMPK (29, 30).Previous work in our groups proposed a model of AMPK regulation by AMP, which incorporates the major functional features and the latest structural information (31). The latter mainly included truncated core complexes of AMPK from different species (32–35). Further valuable structural information is provided by the x-ray structures of the isolated catalytic domains, in particular of the human AMPK α2-subunit (Protein Data Bank code 2H6D) and its yeast ortholog SNF1 (36, 37). The kinase domain of SNF1 is capable of forming homodimers in the protein crystal, as well as in vitro in solution, in a unique way, which has not been observed previously in any other kinase (36). The dimer interface is predominantly formed by hydrophobic interactions of the loop-αG region, also known as subdomain X situated on the large kinase lobe (36, 38, 39), and it mainly involves Ile-257 and Phe-261. Because the T-loop activation segment was buried within the dimer interface, it was suggested that the dimeric state of the SNF1 catalytic domain represents the inactive form of the kinase. Intriguingly, it was shown in our groups by small angle x-ray scattering that AMPK self-organizes in a concentration-dependent manner to form homo-oligomers in solution (31). However, the interface responsible for oligomerization of the AMPK heterotrimer has remained elusive.Here we further investigate the distinct oligomeric states of the AMPK heterotrimer and suggest a possible regulatory function for this process. Most importantly, we provide conclusive evidence for participation of αG-helix residues in the recognition of AMPK by its upstream kinases LKB1 and CamKK2.     

Are Tree Species Diversity and Genotypic Diversity Effects on Insect Herbivores Mediated by Ants?

Plant diversity can influence predators and omnivores and such effects may in turn influence herbivores and plants. However, evidence for these ecological feedbacks is rare. We evaluated if the effects of tree species (SD) and genotypic diversity (GD) on the abundance of different guilds of insect herbivores associated with big-leaf mahogany (Swietenia macrophylla) were contingent upon the protective effects of ants tending extra-floral nectaries of this species. This study was conducted within a larger experiment consisting of mahogany monocultures and species polycultures of four species and –within each of these two plot types– mahogany was represented by either one or four maternal families. We selected 24 plots spanning these treatment combinations, 10 mahogany plants/plot, and within each plot experimentally reduced ant abundance on half of the selected plants, and surveyed ant and herbivore abundance. There were positive effects of SD on generalist leaf-chewers and sap-feeders, but for the latter group this effect depended on the ant reduction treatment: SD positively influenced sap-feeders under ambient ant abundance but had no effect when ant abundance was reduced; at the same time, ants had negative effects on sap feeders in monoculture but no effect in polyculture. In contrast, SD did not influence specialist stem-borers or leaf-miners and this effect was not contingent upon ant reduction. Finally, GD did not influence any of the herbivore guilds studied, and such effects did not depend on the ant treatment. Overall, we show that tree species diversity influenced interactions between a focal plant species (mahogany) and ants, and that such effects in turn mediated plant diversity effects on some (sap-feeders) but not all the herbivores guilds studied. Our results suggest that the observed patterns are dependent on the combined effects of herbivore identity, diet breadth, and the source of plant diversity.     

Characterization of the Third Glutathione S-Transferase Gene Involved in Enantioselective Cleavage of the β-Aryl Ether by Sphingobium sp. Strain SYK-6

The glutathione S-transferases, LigF and LigE, of Sphingobium sp. strain SYK-6 respectively play a role in cleavage of the β-aryl ether of (+)-(βS)-α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) and (?)-(βR)-MPHPV. The ligP gene, which showed 59% similarity to ligE at the amino acid level, was isolated from SYK-6. LigP produced in Escherichia coli revealed enantioselectivity for (?)-(βR)-MPHPV, and ligE and ligP alone contributed to the degradation of (?)-(βR)-MPHPV in SYK-6.     

Efficacy of amodiaquine and sulfadoxine/pyrimethamine in the treatment of malaria not complicated by Plasmodium falciparum in Nariño,Colombia, 1999-2002

The resurgence and spread of antimalarial drug resistance is one of the causes of the worldwide increase of malaria. In Colombia, uncomplicated Plasmodium falciparum malaria has been treated with a combination of amodiaquine (AQ) and sulfadoxine/pyrimethamine (SP) since 2000. The efficacy of these two antimalarials was evaluated after the implementation of the new malaria treatment scheme. In the municipalities of El Charco and Tumaco (Nari?o) on the Pacific Coast region, the standard PAHO protocol was used to evaluate antimalarial efficacy in areas of low to moderate malaria transmission. Patients were randomly allocated to treatment regime in two cities of Nari?o, El Charco (n = 48) and Tumaco (n = 50). After 14 days none of El Charco patients presented therapeutic failure to either antimalarial. However, in Tumaco after 28 days, 12 of 24 (95% CI: 30.6-69.4) patients presented AQ treatment failure while 4 of 26 (95% CI: 5.1-33.1) patients had SP treatment failure. The high level of AQ treatment failure in Tumaco was unexpected because it had been introduced only recently as an antimalarial treatment in Colombia. The results suggest that the use of the current dose of AQ in combination with SP will be therapeutically useful for less time than expected. Use of combined therapies is a key strategy to delay antimalarial resistance. Unfortunately, its success depends on the efficacy of antimalarial drugs individually.