Optimal health and disease management using spatial uncertainty: a geographic characterization of emergent artemisinin-resistant Plasmodium falciparum distributions in Southeast Asia.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum malaria parasites are now present across much of mainland Southeast Asia, where ongoing surveys are measuring and mapping their spatial distribution. These efforts require substantial resources. Here we propose a generic 'smart surveillance' methodology to identify optimal candidate sites for future sampling and thus map the distribution of artemisinin resistance most efficiently. METHODS: The approach uses the 'uncertainty' map generated iteratively by a geostatistical model to determine optimal locations for subsequent sampling. RESULTS: The methodology is illustrated using recent data on the prevalence of the K13-propeller polymorphism (a genetic marker of artemisinin resistance) in the Greater Mekong Subregion. CONCLUSION: This methodology, which has broader application to geostatistical mapping in general, could improve the quality and efficiency of drug resistance mapping and thereby guide practical operations to eliminate malaria in affected areas.

Observing in real time the evolution of artemisinin resistance in Plasmodium falciparum.

Simple genetic changes that correlate with drug resistance are used routinely to identify resistant pathogens. These "molecular markers" have usually been defined long after the phenotype of resistance was noted. The molecular changes at the "end game" reflect a long and complex evolution of genetic changes, but once a solidly resistant set of changes assembles under drug selection, that genotype is likely to become fixed, and resistant pathogens will spread widely. Artemisinins are currently used worldwide to treat malaria caused by Plasmodium falciparum, but parasite response has diminished rapidly in the Mekong region of Southeast Asia. Should artemisinins lose potency completely and this effect spread worldwide, effective malaria treatment would be almost impossible. The full range of modern methods has been applied to define rapidly the genetic changes responsible. Changes associated with artemisinin resistance are complex and seem to be evolving rapidly, especially in Southeast Asia. This is a rare chance to observe the early stages in evolution of resistance, and to develop strategies to reverse or mitigate the trend and to protect these key medicines.

Spatiotemporal mathematical modelling of mutations of the dhps gene in African Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum has repeatedly evolved resistance to first-line anti-malarial drugs, thwarting efforts to control and eliminate the disease and in some period of time this contributed largely to an increase in mortality. Here a mathematical model was developed to map the spatiotemporal trends in the distribution of mutations in the P. falciparum dihydropteroate synthetase (dhps) gene that confer resistance to the anti-malarial sulphadoxine, and are a useful marker for the combination of alleles in dhfr and dhps that is highly correlated with resistance to sulphadoxine-pyrimethamine (SP). The aim of this study was to present a proof of concept for spatiotemporal modelling of trends in anti-malarial drug resistance that can be applied to monitor trends in resistance to components of artemisinin combination therapy (ACT) or other anti-malarials, as they emerge or spread. METHODS: Prevalence measurements of single nucleotide polymorphisms in three codon positions of the dihydropteroate synthetase (dhps) gene from published studies of dhps mutations across Africa were used. A model-based geostatistics approach was adopted to create predictive surfaces of the dhps540E mutation over the spatial domain of sub-Saharan Africa from 1990-2010. The statistical model was implemented within a Bayesian framework and hence quantified the associated uncertainty of the prediction of the prevalence of the dhps540E mutation in sub-Saharan Africa. CONCLUSIONS: The maps presented visualize the changing prevalence of the dhps540E mutation in sub-Saharan Africa. These allow prediction of space-time trends in the parasite resistance to SP, and provide probability distributions of resistance prevalence in places where no data are available as well as insight on the spread of resistance in a way that the data alone do not allow. The results of this work will be extended to design optimal sampling strategies for the future molecular surveillance of resistance, providing a proof of concept for similar techniques to design optimal strategies to monitor resistance to ACT.

The impact of phenotypic and genotypic G6PD deficiency on risk of plasmodium vivax infection: a case-control study amongst Afghan refugees in Pakistan.

BACKGROUND: The most common form of malaria outside Africa, Plasmodium vivax, is more difficult to control than P. falciparum because of the latent liver hypnozoite stage, which causes multiple relapses and provides an infectious reservoir. The African (A-) G6PD (glucose-6-phosphate dehydrogenase) deficiency confers partial protection against severe P. falciparum. Recent evidence suggests that the deficiency also confers protection against P. vivax, which could explain its wide geographical distribution in human populations. The deficiency has a potentially serious interaction with antirelapse therapies (8-aminoquinolines such as primaquine). If the level of protection was sufficient, antirelapse therapy could become more widely available. We therefore tested the hypothesis that G6PD deficiency is protective against vivax malaria infection. METHODS AND FINDINGS: A case-control study design was used amongst Afghan refugees in Pakistan. The frequency of phenotypic and genotypic G6PD deficiency in individuals with vivax malaria was compared against controls who had not had malaria in the previous two years. Phenotypic G6PD deficiency was less common amongst cases than controls (cases: 4/372 [1.1%] versus controls 42/743 [5.7%]; adjusted odds ratio [AOR] 0.18 [95% confidence interval (CI) 0.06-0.52], p = 0.001). Genetic analysis demonstrated that the G6PD deficiency allele identified (Mediterranean type) was associated with protection in hemizygous deficient males (AOR = 0.12 [95% CI 0.02-0.92], p = 0.041). The deficiency was also protective in females carrying the deficiency gene as heterozygotes or homozygotes (pooled AOR = 0.37 [95% CI 0.15-0.94], p = 0.037). CONCLUSIONS: G6PD deficiency (Mediterranean type) conferred significant protection against vivax malaria infection in this population whether measured by phenotype or genotype, indicating a possible evolutionary role for vivax malaria in the selective retention of the G6PD deficiency trait in human populations. Further work is required on the genotypic protection associated with other types of G6PD deficiency and on developing simple point-of-care technologies to detect it before administering antirelapse therapy.

Functional analysis of the Mycobacterium tuberculosis FAD-dependent thymidylate synthase, ThyX, reveals new amino acid residues contributing to an extended ThyX motif.

A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX(7-8)S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a delta thyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X(24)H69X(25)R95HRX(7)S105XRYX(90)R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

A network to monitor antimalarial drug resistance: a plan for moving forward.

The spread of resistance to antimalarial drugs has required changes in the recommended first-line treatment for falciparum malaria in almost all regions. Most drugs recommended currently are combinations of a long-acting antimalarial and an artemisinin derivative. This article presents the rationale for establishing a web-based, open-access database of antimalarial drug resistance and efficacy: the World Antimalarial Resistance Network (WARN). The goal of this network is to assemble the tools and information that will enable the malaria community to collate, analyze and share contemporary information on antimalarial-drug efficacy in all endemic regions so that decisions on antimalarial-drug use are based on solid evidence.

Geographical distribution of amino acid mutations in Plasmodium vivax DHFR and DHPS from malaria endemic areas of Thailand.

Both malaria treatment and prophylaxis target the parasite dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes. Specific point mutations in these genes confer resistance to sulfadoxine-pyrimethamine (SP) in both Plasmodium falciparum and P. vivax. We used direct sequencing to examine the prevalence of point mutations in pvdhps and pvdhfr in 160 P. vivax isolates collected from areas along the international borders of Thailand. Results show that the majority of the isolates harbored a quadruple mutant allele of pvdhfr and a double mutant allele of pvdhps, but the distribution was not uniform. The highly mutant allele combination was especially prevalent along the Thai-Myanmar border, whereas the majority of the isolates from areas along the Thai-Cambodian and Thai-Malaysian borders carried double mutant alleles of pvdhfr and single mutant alleles of pvdhps. Novel mutations that have not been identified previously at codon 512 of pvdhps (K512M, K512E, K512T) were also found.

Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase.

BACKGROUND: In order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions. METHODS: The P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network. RESULTS: Synonymous and non-synonymous single nucleotide polymorphisms (SNPs) within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel). SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N) dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T) and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T) mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu. CONCLUSION: It was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts sharply with the selective sweep of rare antifolate resistant alleles observed in the P. falciparum populations in Asia and Africa. The finding of multiple origins of resistance-conferring mutations has important implications for drug policy.

Antifolates can have a role in the treatment of Plasmodium vivax.

Plasmodium vivax is a serious health concern in many regions and is sometimes inadvertently treated with sulfadoxine-pyrimethamine (SP). Mutations in the genes that encode dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) confer resistance to pyrimethamine and sulfadoxine, respectively. Numerous studies have examined the prevalence and diversity of mutations in P. vivax dhfr and some have assessed the relationship between dhfr genotype and clinical or in vitro response to pyrimethamine. Other studies have examined the impact of dhps genotype on response to sulfadoxine. These data indicate that, under certain circumstances, SP could be a valuable tool in the fight against P. vivax.

Pyrimethamine-resistant dihydrofolate reductase enzymes of Plasmodium falciparum are not enzymatically compromised in vitro.

Plasmodium falciparum, the protozoan that causes the most lethal form of human malaria, has been controlled principally by two safe, affordable drugs, chloroquine and sulfadoxine-pyrimethamine (SP). Studies in the laboratory and in the field have demonstrated that resistance to SP depends on non-synonymous point mutations in the dihydrofolate reductase (DHFR), and dihydropteroate synthase (DHPS) coding regions. Parasites that carry dhfr genes with 3 or 4 point mutations (51I/59R/108N triple mutation or 51I/59R/108N/164L quadruple mutation) are resistant to pyrimethamine in vitro and patients infected with these parasites respond poorly to SP treatment. The wide spread of these pyrimethamine-resistant alleles demonstrates the increased fitness over drug-sensitive alleles in the presence of the drug. However, it is not clear whether these alleles might reduce the fitness of parasites in the absence of drug pressure. As a first step, we compared the kinetic properties of the wild type, and three mutant alleles to determine whether the native DHFR-thymidylate synthase form of the mutant proteins showed compromised activity in vitro. The mutant enzymes had K(m) values for their substrate, dihydrofolate that were significantly lower than the wild type, k(cat) values in the same range as the wild type enzyme, and k(cat)/K(m) values higher than wild type. In contrast, the K(m) values for the NADPH cofactor were higher than wild type for the mutant enzymes. These observations suggest that the fitness of these parasites may not be compromised relative to those that carry the wild type allele, even without sustained SP drug pressure.

NADP+ expels both the co-factor and a substrate analog from the Mycobacterium tuberculosis ThyX active site: opportunities for anti-bacterial drug design.

The novel flavin-dependent thymidylate synthase, ThyX, is absent in humans but several pathogenic bacteria depend exclusively on ThyX activity to synthesize thymidylate. Reduction of the enzyme-bound FAD by NADPH is suggested to be the critical first step in ThyX catalysis. We soaked Mycobacterium tuberculosis ThyX-FAD-BrdUMP ternary complex crystals in a solution containing NADP+ to gain structural insights into the reductive step of the catalytic cycle. Surprisingly, the NADP+ displaced both FAD and BrdUMP from the active site. In the resultant ThyX-NADP+ binary complex, the AMP moiety is bound in a deep pocket similar to that of the same moiety of FAD in the ternary complex, while the nicotinamide part of NADP+ is engaged in a limited number of contacts with ThyX. The additional 2'-phosphate group attached to the AMP ribose of NADP+ could be accommodated with minor rearrangement of water molecules. The newly introduced 2'-phosphate groups are engaged in water-mediated interactions across the non-crystallographic 2-fold axis of the ThyX tetramer, suggesting possibilities for design of high-affinity bivalent inhibitors of this intriguing enzyme.

Sensitivity to antifolates and genetic analysis of Plasmodium vivax isolates from Thailand.

We investigated the association between the Plasmodium vivax dihydrofolate reductase (Pvdhfrtas) and the P. vivax dihydropteroate synthase (Pvdhps) genotype and in vitro sensitivity to the antifolates pyrimethamine, WR99210, chlorcycloguanil, sulfadoxine, and dapsone. Drug responses of 32 P. vivax isolates were assessed in two in vitro systems: schizont maturation inhibition and a yeast expression system. The geometric mean of 50% inhibition concentration (IC(50)) values for pyrimethamine, chlorcycloguanil, WR99210, sulfadoxine, and dapsone were 85 +/- 88, 784 +/- 662, 95 +/- 87, 2,424 +/- 2,784, and 1,625 +/- 1,801 nM, respectively, for the schizont maturation assay. Five different Pvdhfr alleles and four Pvdhps alleles were observed: 26 of 32 quadruple mutant alleles of Pvdhfr (F57I,L/S58R/T61M/S117T), four triple mutants (S58R/T61M/S117T, K49C/S58R/S117N), and two double mutant isolates (S58R/S117N). All isolates carried Pvdhps 585V. Twenty four isolates carried double mutant Pvdhps (A383G/A553G), six an additional mutation, S382A,C/A383G/A553G, and two a single mutation, A383G. Increasing geometric mean IC(50) values were observed with increased number of Pvdhfr mutations from double to quadruple. Results suggest that quadruple mutant alleles confer decreased sensitivity to pyrimethamine but retain sensitivity to WR99210.

The rationale and plan for creating a World Antimalarial Resistance Network (WARN).

Drug resistant malaria was a major factor contributing to the failure of a worldwide campaign to eradicate malaria in the last century, and now threatens the large investment being made by the global community in the rollout of effective new drug combinations to replace failed drugs. Four related papers in this issue of Malaria Journal make the case for creating the World Antimalarial Resistance Network (WARN), which will consist of four linked open-access global databases containing clinical, in vitro, molecular and pharmacological data, and networks of reference laboratories that will support these databases and related surveillance activities. WARN will serve as a public resource to guide antimalarial drug treatment and prevention policies and to help confirm and characterize the new emergence of new resistance to antimalarial drugs and to contain its spread.

World Antimalarial Resistance Network (WARN) II: in vitro antimalarial drug susceptibility.

Intrinsic resistance of Plasmodium falciparum is clearly a major determinant of the clinical failure of antimalarial drugs. However, complex interactions between the host, the parasite and the drug obscure the ability to define parasite drug resistance in vivo. The in vitro antimalarial drug susceptibility assay determines ex-vivo growth of parasite in the presence of serial drug concentrations and, thus, eliminates host effects, such as drug metabolism and immunity. Although the sensitivity of the parasite to various antimalarials provided by such a test provides an important indicator of intrinsic parasite susceptibility, there are fundamental methodological issues that undermine comparison of in vitro susceptibility both between laboratories and within a single laboratory over time. A network of laboratories is proposed that will agree on the basic parameters of the in vitro test and associated measures of quality control. The aim of the network would be to establish baseline values of sensitivity to commonly used antimalarial agents from key regions of the world, and create a global database, linked to clinical, molecular and pharmacology databases, to support active surveillance to monitor temporal trends in parasite susceptibility. Such a network would facilitate the rapid detection of strains with novel antimalarial resistance profiles and investigate suitable alternative treatments with retained efficacy.

Systematic review of clinical trials assessing the therapeutic efficacy of visceral leishmaniasis treatments: A first step to assess the feasibility of establishing an individual patient data sharing platform.

BACKGROUND: There are an estimated 200,000 to 400,000 cases of visceral leishmaniasis (VL) annually. A variety of factors are taken into account when considering the best therapeutic options to cure a patient and reduce the risk of resistance, including geographical area, malnourishment and HIV coinfection. Pooled analyses combine data from many studies to answer specific scientific questions that cannot be answered with individual studies alone. However, the heterogeneity of study design, data collection, and analysis often makes direct comparison difficult. Individual Participant Data (IPD) files can be standardised and analysed, allowing detailed analysis of this merged larger pool, but only a small fraction of systematic reviews and meta-analyses currently employ pooled analysis of IPD. We conducted a systematic literature review to identify published studies and studies reported in clinical trial registries to assess the feasibility of developing a VL data sharing platform to facilitate an IPD-based analysis of clinical trial data. Studies conducted between 1983 to 2015 that reported treatment outcome were eligible. PRINCIPAL FINDINGS: From the 2,271 documents screened, 145 published VL clinical trials were identified, with data from 26,986 patients. Methodologies varied for diagnosis and treatment outcomes, but overall the volume of data potentially available on different drugs and dose regimens identified hundreds or possibly thousands of patients per arm suitable for IPD pooled meta-analyses. CONCLUSIONS: A VL data sharing platform would provide an opportunity to maximise scientific use of available data to enable assessment of treatment efficacy, contribute to evidence-based clinical management and guide optimal prospective data collection.