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As the last part of a program to survey the extent of malaria transmission in the Papua New Guinea highlands, a series of rapid malaria surveys were conducted in 2003-2004 and 2005 in different parts of Southern Highlands Province. Malaria was found to be highly endemic in Lake Kutubu (prevalence rate (PR): 17-33%), moderate to highly endemic in Erave (PR: 10-31%) and moderately endemic in low-lying parts (1500 m) of Poroma and Kagua (PR: 12-17%), but was rare or absent elsewhere. A reported malaria epidemic prior to the 2004 surveys could be confirmed for the Poroma (PR: 26%) but not for the lower Kagua area. In Kutubu/Erave Plasmodium falciparum was the most common cause of infection (42%), followed by P. vivax (39%) and P. malariae (16%). In other areas most infections were due to P. vivax (63%). Most infections were of low density (72%500/ microl) and not associated with febrile illness. Overall, malaria was only a significant source of febrile illness when prevalence rates rose above 10%, or in epidemics. However, concurrent parasitaemia led to a significant reduction in haemoglobin (Hb) level (1.2 g/dl, CI95: [1.1-1.4.], p0.001) and population mean Hb levels were strongly correlated with overall prevalence of malarial infections (r = -0.79, p0.001). Based on the survey results, areas of different malaria epidemiology are delineated and options for control in each area are discussed.

Background The Plasmodium vivax Apical Membrane Antigen 1 (PvAMA1) is a promising malaria vaccine candidate, however it remains unclear which regions are naturally targeted by host immunity and whether its high genetic diversity will preclude coverage by a monovalent vaccine. To assess its feasibility as a vaccine candidate, we investigated the global population structure of PvAMA1. Methodology and Principal Findings New sequences from Papua New Guinea (PNG, n = 102) were analysed together with published sequences from Thailand (n = 158), India (n = 8), Sri Lanka (n = 23), Venezuela (n = 74) and a collection of isolates from disparate geographic locations (n = 8). A total of 92 single nucleotide polymorphisms (SNPs) were identified including 22 synonymous SNPs and 70 non-synonymous (NS) SNPs. Polymorphisms and signatures of balancing (positive Tajima's D and low FST values) selection were predominantly clustered in domain I, suggesting it is a dominant target of protective immune responses. To estimate global antigenic diversity, haplotypes comprised of (i) non-singleton (n = 40) and (ii) common (≥10% minor allele frequency, n = 23) polymorphic amino acid sites were then analysed revealing a total of 219 and 210 distinct haplotypes, respectively. Although highly diverse, the 210 haplotypes comprised of only common polymorphisms were grouped into eleven clusters, however substantial geographic differentiation was observed, and this may have implications for the efficacy of PvAMA1 vaccines in different malaria-endemic areas. The PNG haplotypes form a distinct group of clusters not found in any other geographic region. Vaccine haplotypes were rare and geographically restricted, suggesting potentially poor efficacy of candidate PvAMA1 vaccines. Conclusions It may be possible to cover the existing global PvAMA1 diversity by selection of diverse alleles based on these analyses however it will be important to first define the relationships between the genetic and antigenic diversity of this molecule., Author Summary Traditionally misclassified as benign and neglected as a research priority, it is now understood that P. vivax is an increasingly important cause of human malaria. This important human pathogen poses an enormous obstacle to malaria control and elimination efforts due its broad geographic distribution, ability to cause recurring episodes of malaria after long periods of inactivity and extreme biodiversity. Vaccines are an essential component of global malaria control and elimination campaigns but the diversity of malaria antigens is thought to be a major cause of vaccine failure. Furthermore, at present the majority of current vaccine research is directed toward P. falciparum. The aims of this study were to investigate the global diversity of the P. vivax vaccine candidate, Apical Membrane Antigen 1 (PvAMA1), to determine the feasibility of designing a globally effective PvAMA1 vaccine and to determine which region of PvAMA1 is targeted by host immune responses, in order to identify the most promising vaccine candidates. We report that PvAMA1 diversity is extremely high, and that PvAMA1 domain I is a dominant target of host immune responses. These analyses of PvAMA1 diversity from several geographic regions provide a framework to guide development of a broadly efficacious P. vivax vaccine.

High levels of genetic diversity in Plasmodium falciparum populations are an obstacle to malaria control. Here, we investigate the relationship between local variation in malaria epidemiology and parasite genetic diversity in Papua New Guinea (PNG). Cross-sectional malaria surveys were performed in 14 villages spanning four distinct malaria-endemic areas on the north coast, including one area that was sampled during the dry season. High-resolution msp2 genotyping of 2,147 blood samples identified 761 P. falciparum infections containing a total of 1,392 clones whose genotypes were used to measure genetic diversity. Considerable variability in infection prevalence and mean multiplicity of infection was observed at all of the study sites, with the area sampled during the dry season showing particularly striking local variability. Genetic diversity was strongly associated with multiplicity of infection but not with infection prevalence. In highly endemic areas, differences in infection prevalence may not translate into a decrease in parasite population diversity.

Adherence of Plasmodium falciparum-infected erythrocytes to cerebral postcapillary venular endothelium is believed to be a critical step in the development of cerebral malaria. Some of the possible receptors mediating adherence have been identified, but the process of adherence in vivo is poorly understood. We investigated the role of carbohydrate ligands in adherence, and we identified chondroitin sulfate (CS) as a specific receptor for P. falciparum-infected erythrocytes. Parasitized cells bound to Chinese hamster ovary (CHO) cells and C32 melanoma cells in a chondroitin sulfate-dependent manner, whereas glycosylation mutants lacking chondroitin sulfate A (CSA) supported little or no binding. Chondroitinase treatment of wild-type CHO cells reduced binding by up to 90%. Soluble CSA inhibited binding to CHO cells by 99.2 +/- 0.2% at 10 mg/ml and by 72.5 +/- 3.8% at 1 mg/ml, whereas a range of other glycosaminoglycans such as heparan sulfate had no effect. Parasite lines selected for increased binding to CHO cells and most patient isolates bound specifically to immobilized CSA. We conclude that P. falciparum can express or expose proteins at the surface of the infected erythrocyte that mediate specific binding to CSA. This mechanism of adherence may contribute to the pathogenesis of P. falciparum malaria, but has wider implications as an example of an infectious agent with the capacity to bind specifically to cell-associated or immobilized CS.

To conduct an in-depth investigation of the epidemiology of malaria in the Papua New Guinea (PNG) highlands and provide a basis for evidence-based planning and monitoring of intensified malaria control activities.Between December 2000 and July 2005, 153 household-based, rapid malaria population surveys were conducted in 112 villages throughout the central PNG highlands. The presence of malaria infections was determined by light microscopy and risk factors assessed using a structured questionnaire.The combined dataset from all individually published surveys was reanalysed.The prevalence of malaria infections in the different surveys ranged from 0.0% to 41.8%(median 4.3%) in non-epidemic surveys and 6.6% to 63.2% (median 21.2%, P0.001) during epidemics. Plasmodium falciparum was the predominant infection below 1400 m and during epidemics, Plasmodium vivax at altitudes1600 m. Outside epidemics, prevalence decreased significantly with altitude, was reduced in people using bed nets [odds ratio (OR) = 0.8, P0.001] but increased in those sleeping in garden houses (OR = 1.34, P0.001) and travelling to highly endemic lowlands (OR = 1.80, P0.001). Below 1400 m, malaria was a significant source of febrile illness. At higher altitudes, malaria was only a significant source of febrile illness during epidemic outbreaks, but asymptomatic malaria infections were common in non-epidemic times.Malaria is once again endemic throughout the PNG highlands in areas below 1400–1500 m of altitude with a significant risk of seasonal malaria outbreaks in most area between 1400–1650 m. Ongoing control efforts are likely to result in a substantial reduction in malaria transmission and may even result in local elimination of malaria in higher lying areas.

Antibiotic-resistant strains of Neisseria gonorrhoeae have the potential to undermine treatment and control of gonorrhoea, which remains a highly prevalent sexually transmitted infection (STI) in Papua New Guinea (PNG). The standard treatment regimen for gonorrhoea in PNG based on amoxycillin and clavulanic acid (amoxycillin-clavulanate) was introduced about 15 years ago and there is some concern that over time circulating strains may have developed resistance to this therapy. To investigate this, N. gonorrhoeae isolates (n = 52) were collected from STI clinics in geographically representative centres in PNG and tested for their in vitro susceptibility to a range of antibiotics. All 52 isolates tested were found susceptible to amoxycillin-clavulanate, despite 40% (n = 21) being penicillinase producers and thus resistant to penicillin. These findings indicate that amoxycillin-clavulanate therapy remains an effective treatment for gonococcal infections in PNG, and support the maintenance of the present standard treatment for gonorrhoea in PNG.

A broadly effective malaria vaccine is a missing component of the practical tools needed to control and eventually eliminate malaria. For more than 50 years malaria researchers have been searching for a strategy that provides long lasting protection against a broad range of parasite strains. Many different approaches have been tested including whole parasite and subunit vaccines composed of one or more parasite surface proteins that are naturally targeted by the host immune response.

Traditionally, infection with Plasmodium vivax was thought to be benign and self-limiting, however, recent evidence has demonstrated that infection with P. vivax can also result in severe illness and death. Research into P. vivax has been relatively neglected and much remains unknown regarding the biology, pathogenesis and epidemiology of this parasite. One of the fundamental factors governing transmission and immunity is parasite diversity. An understanding of parasite population genetic structure is necessary to understand the epidemiology, diversity, distribution and dynamics of natural P. vivax populations. In addition, studying the population structure of genes under immune selection also enables investigation of the dynamic interplay between transmission and immunity, which is crucial for vaccine development. A lack of knowledge regarding the transmission and spread of P. vivax has been particularly highlighted in areas where malaria control and elimination programmes have made progress in reducing the burden of Plasmodium falciparum, yet P. vivax remains as a substantial obstacle. With malaria elimination back on the global agenda, mapping of global and local P. vivax population structure is essential prior to establishing goals for elimination and the roll-out of interventions. A detailed knowledge of the spatial distribution, transmission and clinical burden of P. vivax is required to act as a benchmark against which control targets can be set and measured. This paper presents an overview of what is known and what is yet to be fully understood regarding P. vivax population genetics, as well as the importance and application of P. vivax population genetics studies.

Ivo Mueller and colleagues examined the association of Southeast Asian ovalocytosis with Plasmodium vivax infection by genotyping 1975 children enrolled in three independent epidemiological studies conducted in the Madang area of Papua New Guinea and assessing P. vivax infection and disease in the children., Background The erythrocyte polymorphism, Southeast Asian ovalocytosis (SAO) (which results from a 27-base pair deletion in the erythrocyte band 3 gene, SLC4A1Δ27) protects against cerebral malaria caused by Plasmodium falciparum; however, it is unknown whether this polymorphism also protects against P. vivax infection and disease. Methods and Findings The association between SAO and P. vivax infection was examined through genotyping of 1,975 children enrolled in three independent epidemiological studies conducted in the Madang area of Papua New Guinea. SAO was associated with a statistically significant 46% reduction in the incidence of clinical P. vivax episodes (adjusted incidence rate ratio [IRR] = 0.54, 95% CI 0.40–0.72, p90% binding inhibition) P. vivax Duffy binding protein–specific binding inhibitory antibodies were observed significantly more often in sera from SAO than non-SAO children (SAO, 22.2%; non-SAO, 6.7%; p = 0.008). Conclusions In three independent studies, we observed strong associations between SAO and protection against P. vivax malaria by a mechanism that is independent of the Duffy antigen. P. vivax malaria may have contributed to shaping the unique host genetic adaptations to malaria in Asian and Oceanic populations. Please see later in the article for the Editors' Summary., Editors' Summary Background Hereditary blood disorders are widely prevalent in different regions around the world and the type of disorder depends on the population gene pool. For example, sickle cell disease is indigenous to sub-Saharan Africa, and Southeast Asian ovalocytosis (SAO), as the name suggests, to Southeast Asia and the South West Pacific, particularly Malaysia and Papua New Guinea. In SAO, the red blood cells (erythrocytes) are a different shape (elliptical) from the usual biconcave disc shape due to a genetic defect (caused by band 3 deletion SLC4A1Δ27) in the red blood cell membrane. This defect is carried by up to 35% of people living on the coasts of Papua New Guinea, and as these areas match high malaria endemic areas, it is thought that carrying this defect is associated with improved survival against malaria in these populations—some studies have suggested that SAO is associated with complete protection against cerebral malaria but not other forms of malaria caused by the same type of parasite—P. falciparum. Why Was This Study Done? Although P. falciparum gains most attention by the international health community as it causes the most severe types of malaria, recent epidemiological studies suggest that malaria caused by P. vivax can also cause severe illness in some areas of the world where it is highly prevalent. Furthermore, detailed genetic and laboratory studies have suggested that the genetic defect associated with SAO may actually protect against infection from P. vivax malaria. So in this study, the researchers examined the relationship of the SAO genetic defect and P. vivax malaria by doing genetic tests on children in Papua New Guinea—an area in which both conditions are widely prevalent. What Did the Researchers Do and Find? The researchers performed genetic tests for the SAO defect in 1,975 children in the Madang area of Papua New Guinea who were participating in three separate malaria studies that were conducted over different time periods: (i) a cohort of 1,121 infants aged 3–21 months participating in a clinical drug trial of intermittent preventative treatment of malaria; (ii) a case-control study of 318 children with severe malaria aged 10 years and under; and (iii) a cohort of 206 children aged 5–14 years who took part in a prospective study to evaluate the time of reinfection with all forms of malaria. Given the different nature of these studies, for example, the effect of intermittent treatment for which the researchers had to make statistical adjustments, the researchers analysed the presence of the SAO genetic defect and the incidence of all forms of malaria separately to calculate the association with SAO and malaria in the participants in each study. The researchers found that the SAO genetic defect present in 130 infants (11.6%) in the first study and 27 (13.1%) children in the third study. In the case-control study, the researchers found that 28 of the 330 controls (8.5%) had the SAO genetic defect compared to eight of 236 (3.4%) in children with P. falciparum single infections. Overall, the researchers found that the SAO genetic defect was associated with a 43% reduction in risk of clinical P. vivax episodes in the infants in the first study and a 52%–55% reduction in P. vivax reinfection in children in the third study. Furthermore, from the limited data from the second study, the researchers found that none of the children with P. vivax or mixed P. falciparum/P. vivax malaria had the SAO defect, which may indicate a protective effect. What Do These Findings Mean? These findings suggest that the SAO genetic defect (SLC4A1Δ27) may have a protective effect against malaria caused by P. vivax in infants and children of different ages in Papua New Guinea. However, although it seems likely that SAO may alter the ability of the malarial parasite to develop within the red blood cell, this study sheds no further light on the way in which the SAO genetic defect may protect against P. vivax infection and disease and so further studies are needed to investigate possible mechanisms. Importantly, these findings suggest that future studies investigating genetic adaptation of diverse populations around the world, particularly in the Asian Pacific region, should include all forms of human malaria, such as P. vivax, and not exclusively focus on P. falciparum. Additional Information Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001305. Wikipedia has information on Southeast Asian Ovalocytosis (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages) WHO provides information on malaria The US Centers for Disease Control and Prevention provide information on malaria (in English and Spanish) MedlinePlus provides links to additional information on malaria and on ovalocytosis

A three-arm randomized trial conducted among infants in Papua New Guinea estimates the preventive effect against malaria episodes of intermittent preventive treatment, in an area where children are exposed to both falciparum and vivax malaria., Background Intermittent preventive treatment in infants (IPTi) has been shown in randomized trials to reduce malaria-related morbidity in African infants living in areas of high Plasmodium falciparum (Pf) transmission. It remains unclear whether IPTi is an appropriate prevention strategy in non-African settings or those co-endemic for P. vivax (Pv). Methods and Findings In this study, 1,121 Papua New Guinean infants were enrolled into a three-arm placebo-controlled randomized trial and assigned to sulfadoxine-pyrimethamine (SP) (25 mg/kg and 1.25 mg/kg) plus amodiaquine (AQ) (10 mg/kg, 3 d, n = 374), SP plus artesunate (AS) (4 mg/kg, 3 d, n = 374), or placebo (n = 373), given at 3, 6, 9 and 12 mo. Both participants and study teams were blinded to treatment allocation. The primary end point was protective efficacy (PE) against all episodes of clinical malaria from 3 to 15 mo of age. Analysis was by modified intention to treat. The PE (compared to placebo) against clinical malaria episodes (caused by all species) was 29% (95% CI, 10–43, p≤0.001) in children receiving SP-AQ and 12% (95% CI, −11 to 30, p = 0.12) in those receiving SP-AS. Efficacy was higher against Pf than Pv. In the SP-AQ group, Pf incidence was 35% (95% CI, 9–54, p = 0.012) and Pv incidence was 23% (95% CI, 0–41, p = 0.048) lower than in the placebo group. IPTi with SP-AS protected only against Pf episodes (PE = 31%, 95% CI, 4–51, p = 0.027), not against Pv episodes (PE = 6%, 95% CI, −24 to 26, p = 0.759). Number of observed adverse events/serious adverse events did not differ between treatment arms (p>0.55). None of the serious adverse events were thought to be treatment-related, and the vomiting rate was low in both treatment groups (1.4%–2.0%). No rebound in malaria morbidity was observed for 6 mo following the intervention. Conclusions IPTi using a long half-life drug combination is efficacious for the prevention of malaria and anemia in infants living in a region highly endemic for both Pf and Pv. Trial registration ClinicalTrials.gov NCT00285662 Please see later in the article for the Editors' Summary, Editors' Summary Background Malaria is a major global public health problem. Half the world's population is at risk of this parasitic disease, which kills about one million people (mainly young children in sub-Saharan Africa) every year. Most of these deaths are caused by Plasmodium falciparum but P. vivax, the commonest and most widely distributed malaria parasite, is a major cause of malaria-related morbidity (illness and death) in many of the tropical and subtropical regions of the world where malaria is endemic (always present). Malaria is transmitted to people through the bites of night-flying mosquitoes. It can be prevented by controlling the mosquitoes that spread the parasite and by sleeping under insecticide-treated nets to avoid mosquito bites. Prompt treatment of malaria with antimalarial drugs can also reduce malaria transmission. In addition, intermittent preventative treatment (IPT)—the treatment of symptom-free individuals with full therapeutic courses of antimalarial drugs at fixed intervals regardless of their infection status—has been shown to reduce malaria-related morbidity among pregnant women in malaria-endemic areas and among African infants living in areas of high P. falciparum transmission. Why Was This Study Done? The World Health Organization recently recommended that, in Africa, IPT should be given during infancy (called IPTi) at the same time as routine immunizations. Because the studies on which this recommendation is based were all carried out in sub-Saharan Africa, in populations where P. falciparum is the predominant parasite and P. vivax is uncommon, it is not known whether IPTi would be an appropriate prevention strategy in non-African settings or in regions where both P. falciparum and P. vivax are endemic. In this randomized placebo-controlled trial, the researchers investigate the efficacy of IPTi in infants living in an area of Papua New Guinea where P. falciparum and P. vivax are both highly endemic. In a randomized placebo-controlled trial, the effects of an intervention and of a placebo (dummy) intervention are compared in groups of individuals chosen through the play of chance. What Did the Researchers Do and Find? The researchers assigned more than 1,000 infants to receive sulfadoxine/pyrimethamine (SP) plus amodiaquine (AQ) (SP and AQ are long-lasting antimalarial drugs), SP plus artesunate (AS) (AS is a short-lasting antimalarial), or placebo at 3, 6, 9, and 12 months old. They recorded the number of malaria episodes that occurred among the children between the ages of 3 and 15 months. Then, by comparing the number of episodes occurring among the children receiving SP-AS or SP-AQ with the number occurring among the children receiving placebo, the researchers calculated the protective efficacy of the two drug combinations over the study period. The protective efficacy of IPTi against all clinical malaria episodes (P. falciparum and P. vivax combined) was 29% for SP-AQ, but SP-AS was not associated with a statistically significant reduction in all malaria episodes as compared to placebo. For the two species of malaria separately, the incidence of P. falciparum malaria was 35% lower among the children receiving SP-AQ than among the children receiving placebo, whereas the incidence of P. vivax was reduced by 23%; IPTi with SP-AS provided protection only against P. falciparum malaria (protective efficacy 31%). Importantly, the number of adverse events (possible drug side effects) was similar in all the treatment arms, none of the severe adverse events were treatment-related, and there was no rebound in malaria-related morbidity for six months following the end of the intervention. What Do These Findings Mean? These findings show that IPTi using a combination of long-lasting antimalarial drugs (SP-AQ) can effectively and safely prevent malaria in a non-African population living in a region where P. falciparum and P. vivax are both highly endemic. Importantly, they also show that IPTi with SP-AQ can prevent both P. falciparum and P. vivax malaria. For Papua New Guinea, these findings suggest that SP-AQ is an appropriate drug choice for IPTi, particularly since the replacement of SP-AQ by artemether-lumefantrine as the national first line treatment for malaria will reduce the selection pressure for resistance against SP and AQ. However, although these finding provide proof-of-principle evidence for the efficacy and safety of IPTi, further studies are needed to identify the most effective combinations of long-lasting antimalarial drugs for use in IPTi in other malaria-endemic regions. Additional Information Please access these web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001195. Information is available from the World Health Organization on malaria (in several languages); the 2011 World Malaria Report provides details of the current global malaria situation; and the WHO policy recommendation on IPTi for P. falciparum malaria control in Africa is available The US Centers for Disease Control and Prevention provide information on malaria (in English and Spanish), including a selection of personal stories about malaria Information is available from the Roll Back Malaria Partnership on the global control of malaria, including a fact sheet about malaria in children and information on malaria in Papua New Guinea The IPTi Consortium was established to evaluate IPTi and inform public health policy making The Malaria Vaccine Initiative has a fact sheet on P. vivax malaria Vivaxmalaria.com provides information about P. vivax MedlinePlus provides links to additional information on malaria (in English and Spanish)

Background The Plasmodium falciparum merozoite surface protein Pf38 is targeted by antibodies of malaria immune adults and has been shown to be under balancing (immune) selection in a Gambian parasite population, indicating potential as a malaria vaccine candidate. This study explores the population genetics of Pf38 in Papua New Guinea, to determine the extent and geographic distribution of diversity and to measure selective pressure along the length of the gene. Methods Using samples collected during community-based cross-sectional surveys in the Mugil and Wosera regions, the Pf38 genes of 59 P. falciparum isolates were amplified and sequenced. These sequences, along with previously sequenced Gambian and laboratory isolates, were then subjected to an array of population genetic analyses, examining polymorphisms, haplotype diversity and balancing selection. In addition to whole-gene analysis, the two 6-cys domains were considered separately, to investigate domain specific polymorphism and selection. Results Nineteen polymorphic sites were identified in the Pf 38 gene. Of these, 13 were found in the Gambia, 10 in Mugil and 8 in Wosera. Notably, the majority of common polymorphisms were confined to domain I. Although only moderate levels of nucleotide diversity were observed, the haplotype diversity was high in all populations, suggesting extensive recombination. Analyses of the full-length sequence provided only modest evidence for balancing selection. However, there was a strong contrast between domain I, which showed strong evidence for positive balancing selection, and domain II which was neutral. Analyses of the geographic distribution of Pf38 haplotypes showed that four haplotypes accounted for the majority of sequences found world-wide, but there were many more haplotypes unique to the African than the PNG populations. Conclusion This study confirmed previous findings that Pf38 is a polymorphic gene under balancing selection. However, analysing polymorphism and selection across the length of the gene painted a considerably different picture. Domain I is highly polymorphic and the target of significant balancing selection. In contrast, domain II is relatively conserved and does not show evidence of immune selective pressure. The findings have implications for future population genetic studies on vaccine candidates, showing that the biological context must also be considered as a framework for analysis.

The International Union Against Tuberculosis and Lung Disease (The Union), Medecins sans Frontieres (MSF) and the Special Programme for Research and Training in Tropical Diseases (TDR) hosted at the World Health Organization, have joined forces under a programme called the Structured Operational Research and Training Initiative (SORT IT) to improve health care delivery and public health through operational research. This is done through integrated operational research and training programmes, in which participants from low- and middle-income countries are enrolled and taken through a research project from protocol development to implementation, data collection and analysis to the writing and submission of a paper to a peer-reviewed journal.1 Between April 2009 and January 2014, we have undertaken 18 programmes (of which 14 are completed), enrolling a total of 212 participants. We carefully monitor outputs from each SORT IT programme. Two important outputs, which are relatively easy to monitor, are the number and percentage of enrolled participants who complete the programme and the number of scientific papers that are published in peer-reviewed journals within 18 months of submission. However, an additional, vital output that needs to be monitored is whether any action has been taken in response to the research findings and the subsequent impact of research on policy and practice. Thus, one of our SORT IT targets is to record whether this has been documented in over 80% of projects within 18 months of study completion. It is frequently stated that it takes 17 years for research evidence to reach clinical practice,2 although for operational research with a focus on health service delivery we would expect a much shorter time lag. We have recently completed an assessment of the impact on policy and practice for our first eight programmes through a questionnaire survey. The amount of work that went into obtaining the data was considerable, with repeated e-mails sent to course alumni over a period of several months, and personal calls to chase up non-responders. Without the help of a dedicated person allocated to this task, it is unlikely we would have succeeded in our task. We therefore propose an innovative role for a journal, namely that of tracking and publishing the impact of its articles. This fulfils a mandate of operational research, and if, for example, this was taken on by Public Health Action (PHA), it would place this journal at the forefront of the discipline. Papers in PHA are published quarterly. An automated e-mail could go out to the corresponding author of a paper published in the Journal 18 months after publication. The e-mail could have a website link that takes the author to a site in which he/she can answer whether the paper has had any influence on policy and practice (Yes/No). If the answer is no, then reasons can be mentioned such as the paper being a review (not applicable), a descriptive methodology or a specific local context. If the answer is yes, then reasons can be articulated for claiming that the research has had such an effect. Supporting material could be added. Authors could be given four weeks to respond and their collective responses then published in either the following quarterly issue of the Journal or annually, in a section entitled ‘Research to Policy and Practice’. The papers would be classified into No response; Not applicable; No effect on policy and practice; Positive effect on policy and practice. In this way, authors could communicate to a wide audience whether their research has made a difference, and this could be the start of a journal-based observatory that systematically tracks this important operational research outcome. In the January 2014 issues of the Lancet (January 11–17 and 18–24), there was an excellent five-paper series on increasing the value and reducing the waste of research. An editorial summarised some of the key problems and made useful recommendations.3 A consistent theme in the series was that research is considered a waste if it does not impact on health care, but there were no practical suggestions or recommendations to monitor whether research has made a difference to the public's health and whether, in particular, it has contributed to the optimal use of available resources or an improvement in the effectiveness of health care or health system interventions. We would like to advocate that PHA takes the lead in this valuable and exciting new venture, and we hope that other journals would then follow its example. In a December 2013 editorial, PHA was judged to be meeting its goals and targets and wanted to increasingly be seen as the home of operational research.4 We agree with this assessment and vision. However, we believe that the Journal must push the frontiers of operational research and become a vehicle for analysing whether what we do is truly providing health solutions for the poor. This means going beyond the published paper and becoming a forum for assessing whether the research has made a difference. It is a bold step for a journal to aspire to doing more than publish papers, but operational research is about more than just publishing – it is a step to better health care. Operational research is also about ‘learning while doing’. The proposal we make is in its concept phase, and it may require a task force to work out the finer details of how to translate the idea into practice and how to define impact. The monitoring process is likely to evolve over time, but this could be the first step in a journey to formally and systematically evaluate whether the research is delivering the goods.5 Can PHA cross this Rubicon?

Background The South West Pacific nation of Papua New Guinea has intense year round transmission of Plasmodium falciparum on the coast and in the low-lying inland areas. Local heterogeneity in the epidemiology of malaria suggests that parasites from multiple locations will need to be surveyed to define the population biology of P. falciparum in the region. This study describes the population genetics of P. falciparum in thirteen villages spread over four distinct catchment areas of Papua New Guinea. Methods Ten microsatellite loci were genotyped in 318 P. falciparum isolates from the parasite populations of two inland catchment areas, namely Wosera (number of villages (n) = 7) and Utu (n = 1) and; and two coastal catchments, Malala (n = 3) and Mugil (n = 3). Analysis of the resultant multilocus haplotypes was done at different spatial scales (2-336 km) to define the genetic diversity (allelic richness and expected heterozygosity), linkage disequilibrium and population structure throughout the study area. Results Although genetic diversity was high in all parasite populations, it was also variable with a lower allelic richness and expected heterozygosity for inland populations compared to those from the more accessible coast. This variability was not correlated with two proxy measures of transmission intensity, the infection prevalence and the proportion multiple infections. Random associations among the microsatellite loci were observed in all four catchments showing that a substantial degree of out-crossing occurs in the region. Moderate to very high levels of population structure were found but the amount of genetic differentiation (F ST ) did not correlate with geographic distance suggesting that parasite populations are fragmented. Population structure was also identified between villages within the Malala area, with the haplotypes of one parasite population clustering with the neighbouring catchment of Mugil. Conclusion The observed population genetics of P. falciparum in this region is likely to be a consequence of the high transmission intensity combined with the isolation of human and vector populations, especially those located inland and migration of parasites via human movement into coastal populations. The variable genetic diversity and population structure of P. falciparum has important implications for malaria control strategies and warrants further fine scale sampling throughout Papua New Guinea.

BACKGROUND: The hypothesis is that hemoglobin-based metrics are useful tools for estimating malaria endemicity and for monitoring malaria control strategies. The aim of this study is to compare population hemoglobin mean and anemia prevalence to established indicators of malaria endemicity, including parasite rates, rates of enlarged spleens in children, and records of (presumptive) malaria diagnosis among populations living with different levels of malaria transmission. METHODOLOGY/PRINCIPAL FINDINGS: Convenience sample, multisite cross-sectional household surveys conducted in Papua New Guinea. Correlations (r(2)) between population Hb mean and anemia prevalence and altitude, parasite rate, and spleen rate were investigated in children ages 2 to 10 years, and in the general population; 21,664 individuals from 156 different communities were surveyed. Altitude ranged from 5 to 2120 meters. In young children, correlations between altitude and parasite rate, population Hb mean, anemia prevalence, and spleen rate were high (r(2): -0.77, 0.73, -0.81, and -0.68; p1500 m (p

Besides young children, pregnant women are at high risk of malaria in highly endemic countries. This paper reviews evidence from studies conducted in Papua New Guinea (PNG) in the last 20 years on the burden and prevention of malaria in pregnancy and highlights gaps in our knowledge of malaria in pregnancy in PNG. Overall, primigravidae were found to be at higher risk than multigravidae, with up to 40% of primigravidae but only 10-25% of multigravidae infected with Plasmodium falciparum at delivery. Such infections were found to be associated with a 128-145 g decrease in birthweight. Mean birthweights reported between 1980 and 2003 range from 2.58 to 2.72 kg in primigravidae and 2.84 to 3.09 kg in multigravidae, with 21% to 48% and 9% to 19% of babies born to primigravidae and multigravidae, respectively, of low birthweight (2500 g). The negative impact of malaria in pregnancy is compounded by relatively low rates of antenatal coverage. The current PNG national treatment policy which prescribes a treatment course of first-line antimalarial treatment (currently chloroquine and sulphadoxine-pyrimethamine) at first antenatal clinic contact, followed by weekly chloroquine prophylaxis and iron and folate supplementation, may no longer be effective given the high levels of resistance to chloroquine in PNG and poor compliance. In order to reduce the burden of malaria in pregnancy in PNG, alternative methods of control such as insecticide-treated nets and intermittent preventive treatment in pregnancy (IPTp), as well as improved modes of delivery of maternal health interventions, are urgently needed.

The extensive diversity of Plasmodium falciparum antigens is a major obstacle to a broadly effective malaria vaccine but population genetics has rarely been used to guide vaccine design. We have completed a meta-population genetic analysis of the genes encoding ten leading P. falciparum vaccine antigens, including the pre-erythrocytic antigens csp, trap, lsa1 and glurp; the merozoite antigens eba175, ama1, msp's 1, 3 and 4, and the gametocyte antigen pfs48/45. A total of 4553 antigen sequences were assembled from published data and we estimated the range and distribution of diversity worldwide using traditional population genetics, Bayesian clustering and network analysis. Although a large number of distinct haplotypes were identified for each antigen, they were organized into a limited number of discrete subgroups. While the non-merozoite antigens showed geographically variable levels of diversity and geographic restriction of specific subgroups, the merozoite antigens had high levels of diversity globally, and a worldwide distribution of each subgroup. This shows that the diversity of the non-merozoite antigens is organized by physical or other location-specific barriers to gene flow and that of merozoite antigens by features intrinsic to all populations, one important possibility being the immune response of the human host. We also show that current malaria vaccine formulations are based upon low prevalence haplotypes from a single subgroup and thus may represent only a small proportion of the global parasite population. This study demonstrates significant contrasts in the population structure of P. falciparum vaccine candidates that are consistent with the merozoite antigens being under stronger balancing selection than non-merozoite antigens and suggesting that unique approaches to vaccine design will be required. The results of this study also provide a realistic framework for the diversity of these antigens to be incorporated into the design of next-generation malaria vaccines.

Although not strictly a highlands province, Morobe encompasses large highlands areas, the most important being Aseki, Menyamya and Wau-Bulolo. A series of rapid malaria surveys conducted in both the wet and dry seasons found malaria to be clearly endemic in areas below 1400 m in Menyamya and Wau-Bulolo, with overall prevalence rates in the wet season (25.5%, range: 9.1%-39.2%) greatly exceeding those in the dry season (8.3%, range: 2.4%-22.8%; p0.001). In the wet season surveys Plasmodium falciparum was the clearly predominant species, accounting for 63% of all infections. P. vivax increased in frequency in the dry season (from 27% to 46%, p0.001), while P. falciparum and P. malariae decreased. In line with past surveys a low prevalence of malaria was found in the Aseki area. Malaria was found to be the main source of febrile illness in the wet season with at least 60% of measured or reported fever associated with parasitaemia. Other causes of febrile illness dominated in the dry. In villages with parasite prevalence rates20% mean haemoglobin levels and prevalence of severe anaemia were strongly correlated with overall parasite prevalence. In addition concurrent malarial infections were associated with a strong reduction of individual haemoglobin levels (-1.2 g/dl) and there was increased risk of moderate-to-severe anaemia with concurrent malaria. Malarial infections are thus the most significant cause of febrile illness and anaemia in the highlands fringe populations in Morobe. As a consequence all villages below 1500-1600 m in Morobe Province should be included in malaria control activities.

Although predominantly a lowland province, Madang also includes highland areas such as Simbai and Bundi along the northern highland fringe. While the malaria situation in the coastal lowlands has been studied in great detail, the current malaria situation in the highland fringe communities has not been studied in depth since the 1960s. A series of recent malariological surveys found that the malaria situation has changed little over the last 40 years in both Simbai and Bundi. In the Simbai area there is little malaria transmission in villages above 1400 m, with a prevalence rate (PR) of 2.5-4.2%. Below 1400 m, however, there is moderate to high transmission (PR 8.6-24.7%) with surprisingly little difference in prevalence rates between survey villages, despite large differences in altitude. Prevalence rates of malaria infection were low in all Bundi villages (2.5-8.5%) with most infections occurring in adolescents and adults, which indicates limited acquisition of effective immunity to malaria and the possibility that many infections are acquired when travelling to the highly malarious lowlands area. Based on spleen rates the lower Simbai area would be regarded as mesoendemic, and the upper Simbai and Bundi areas as hypoendemic. Only in the lower Simbai area is malaria a major cause of febrile illness. However, in all areas village mean haemoglobin (Hb) levels were highly correlated with the prevalence of malaria infections, while concurrent parasitaemia reduced individual Hb levels by 1.3 g/dl (CI95 [1.0-1.5], p0.001) and significantly increased the risk for moderate-to-severe anaemia (Hb8 g/dl) (adjusted odds ratio 5.6, CI95 [3.6-8.6], p0.001). Based on the survey results, areas of different malaria epidemiology are delineated and options for control in each area are discussed.

We aimed to determine the proportion of the population in Madang (Papua New Guinea) immune to measles infection by age groups, with respect to immunization status and study location, using dried blood sampling technology. We performed a prospective cross-sectional sero-survey. Population immunity against measles was sub-optimal (77%) and reported measles vaccine coverage in children10 years of age was low (41%). The urban population was more susceptible to measles infection, compared with the rural population (66% vs 79% immune, aOR=0.6, p=0.05). Sero-conversion and long term protection rates appeared to be higher when at least one dose of vaccine was provided at or after 12 months of age (84% vs 59%, aOR=4.3, p=0.004). Such a dose is, however, not currently prescribed by the national immunization schedule.

Sixty-one cultures of Gram-negative anaerobic rods were isolated from deep periodontal pockets of patients with rapidly progressive periodontitis. Isolates were speciated as Bacteroides gingivalis (18 isolates), Bacteroides intermedius (8), Bacteroides oris (1), Bacteroides gracilis (17) and Fusobacterium nucleatum (17). Their susceptibilities, to seven antimicrobial agents, were determined in vitro using a plate dilution technique. Amoxycillin and amoxycillin with clavulanic acid were active against all isolates (MIC less than 1 mg/l) and proved the most effective agents tested. F. nucleatum and B. gracilis showed resistance to erythromycin; F. nucleatum had MIC values ranging from 0.03 mg/l up to 128 mg/l when tested with this, least effective agent. Metronidazole was effective against all isolates except for a few strains of B. gracilis (MIC less than 4 mg/l). Tetracycline hydrochloride and minocycline were active against all isolates except for a few strains of B. gracilis (MIC less than 2 mg/l with both minocycline and tetracycline hydrochloride). Penicillin proved less effective than amoxycillin with regard to inhibition of B. gracilis.

Large-scale studies of genomic variation could assist efforts to eliminate malaria. But there are scientific, ethical and practical challenges to carrying out such studies in developing countries, where the burden of disease is greatest. The Malaria Genomic Epidemiology Network (MalariaGEN) is now working to overcome these obstacles, using a consortial approach that brings together researchers from 21 countries. © 2008 Macmillan Publishers Limited. All rights reserved.

Background. Molecular markers for sulfadoxine-pyrimethamine (SP) resistance in Plasmodium vivax have been reported. However, data on the molecular correlates involved in the development of resistance to 4-aminoquinolines and their association with the in vivo treatment response are scarce. Methods. We assessed pvdhfr (F57L/I, S58R, T61M, S117T/N, and I173F/L) and pvmdrl (Y976F and F1076L) mutations in 94 patients who received amodiaquine (AQ) plus SP in Papua New Guinea (PNG). We then investigated the association between parasite genotype and treatment response. Results. The treatment failure (TF) rate reached 13%. Polymorphisms in pvdhfr F57L, S58R, T61M, and S117T/N and in pvmdrl Y976F were detected in 60%, 67%, 20%, 40%, and 39% of the samples, respectively. The single mutant pvdhfr 57 showed the strongest association with TF (odds ratio [OR], 9.04; P = .01). The combined presence of the quadruple mutant pvdhfr 57L+58R+61M+117T andpvmdrl mutation 976F was the best predictor of TF (OR, 8.56; P = .01). The difference in TF rates between sites was reflected in the genetic drug-resistance profile of the respective parasites. Conclusions. The present study identified a new molecular marker in pvmdrl that is associated with the in vivo response to AQ+SP. We suggest suitable marker sets with which to monitor P. vivax resistance against AQ+SP in countries where these drugs are used.

Background Severe malaria (SM) is classically associated with Plasmodium falciparum infection. Little information is available on the contribution of P. vivax to severe disease. There are some epidemiological indications that P. vivax or mixed infections protect against complications and deaths. A large morbidity surveillance conducted in an area where the four species coexist allowed us to estimate rates of SM among patients infected with one or several species. Methods and Findings This was a prospective cohort study conducted within the framework of the Malaria Vaccine Epidemiology and Evaluation Project. All presumptive malaria cases presenting at two rural health facilities over an 8-y period were investigated with history taking, clinical examination, and laboratory assessment. Case definition of SM was based on the World Health Organization (WHO) criteria adapted for the setting (i.e., clinical diagnosis of malaria associated with asexual blood stage parasitaemia and recent history of fits, or coma, or respiratory distress, or anaemia [haemoglobin < 5 g/dl]). Out of 17,201 presumptive malaria cases, 9,537 (55%) had a confirmed Plasmodium parasitaemia. Among those, 6.2% (95% confidence interval [CI] 5.7%–6.8%) fulfilled the case definition of SM, most of them in children, In a study carried out in Papua New Guinea, Blaise Genton and colleagues show thatPlasmodium vivax is associated with severe malaria., Editors' Summary Background. Malaria is a parasitic infection that is transmitted to people by infected mosquitoes. Four different parasites cause malaria—Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. Of these, P. vivax is the commonest and most widely distributed, whereas P. falciparum causes the most deaths. All these parasites enter their human host when an infected mosquito takes a blood meal. They then migrate to the liver where they replicate without causing any symptoms. Eight to nine days later, mature parasites are released from the liver cells and invade red blood cells. Here, they multiply rapidly before bursting out and infecting more red blood cells. The recurring flu-like symptoms of malaria are caused by this cyclical increase in parasitemia (parasites in the blood) and should be treated promptly with antimalarial drugs to prevent the development of potentially fatal complications. Infections with P. falciparum in particular can cause anemia by destroying the red blood cells and can damage vital organs (including the brain) by blocking the capillaries that supply them with blood. Why Was This Study Done? It is generally believed that P. vivax malaria is rarely fatal. There is even some evidence that infection with P. vivax alone (monoinfection) or with other malaria parasites (mixed infection) provides protection against malarial complications. Recently, however, there have been reports of severe disease and deaths associated with infection by P. vivax alone. Most of these reports do not indicate what proportion of severe malaria cases are caused by P. vivax infections, but if P. vivax is responsible for a significant proportion of malarial deaths, efforts to prevent these deaths will need to target P. vivax as well as P. falciparum. In this study, therefore, the researchers estimate the proportion of cases of severe malaria among patients infected with one or several Plasmodium species in Papua New Guinea, a country where all four species coexist. What Did the Researchers Do and Find? The researchers enrolled everyone attending two rural health facilities in the Wosera subdistrict of Papua New Guinea over an eight-year period with symptoms indicative of malaria but without symptoms of any other disease (presumptive malaria cases) into their prospective cohort study. They asked each patient about their symptoms, did a standard physical examination, looked for parasites in their blood, and measured their hemoglobin levels to see whether they were anemic. Out of 17,201 presumptive malaria cases, 483 had severe malaria (defined as parasitemia plus a recent history of fits, coma, breathing problems, or anemia). Most of the patients with severe malaria were less than five years old—children have little immunity to Plasmodium parasites. In this age group, 11.7% of patients infected with P. falciparum, 8.8% of patients infected with P. vivax, and 17.3% of patients infected with both parasites had severe malaria. Patients with severe malaria caused by P. vivax presented with breathing difficulties more often than those infected with P. falciparum, whereas anemia was more common among patients with severe malaria caused by P. falciparum than by P. vivax. What Do These Findings Mean? The researchers use these results and data on the numbers of infections with each parasite to calculate that, in this rural region of Papua New Guinea, P. vivax is responsible for one-fifth of severe malaria cases, P. falciparum is responsible for three-quarters of cases, and the rest involve mixed P. falciparum/P. vivax infections. Put another way, these findings suggest that about one in ten children under the age of five years infected with either P. vivax or P. falciparum may develop severe malaria. These findings provide no evidence, however, that mixed infections are protective. Because the diagnosis of severe malaria was not confirmed by outcome data (deaths or permanent disability), additional, more detailed studies are needed to confirm these results. Nevertheless, these findings (and those reported separately in a related article published at the same time in PLoS Medicine) suggest that a significant proportion of the illness associated with malaria may be caused by P. vivax infections. Thus, efforts to reduce or eliminate the malarial burden must target P. vivax as well as P. falciparum in regions where these species coexist. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050127. A PLoSMedicine Research in Translation article by Stephen Rogerson further discusses this study and a related paper on vivax malaria infection in patients attending a regional hospital in Papua, Indonesia The MedlinePlus encyclopedia has a page on malaria (in English and Spanish) The US Centers for Disease Control and Prevention provides information on malaria (in English and Spanish) Information is available from the Roll Back Malaria Partnership on global control of malaria and on malaria in Papua New Guinea Vivaxmalaria provides information for the malaria research community on topics related to Plasmodiumvivax The Malaria Vaccine Initiative also provides a fact sheet on Plasmodiumvivax malaria

Background In Papua New Guinea (PNG), combination therapy with amodiaquine (AQ) or chloroquine (CQ) plus sulphadoxine-pyrimethamine (SP) was introduced as first-line treatment against uncomplicated malaria in 2000. Methods We assessed in vivo treatment failure rates with AQ+SP in two different areas in PNG and twenty-four molecular drug resistance markers of Plasmodium falciparum were characterized in pre-treatment samples. The aim of the study was to investigate the association between infecting genotype and treatment response in order to identify useful predictors of treatment failure with AQ+SP. Results In 2004, Day-28 treatment failure rates for AQ+SP were 29% in the Karimui and 19% in the South Wosera area, respectively. The strongest independent predictors for treatment failure with AQ+SP were pfmdr1 N86Y (OR = 7.87, p < 0.01) and pfdhps A437G (OR = 3.44, p < 0.01). Mutations found in CQ/AQ related markers pfcrt K76T, A220S, N326D, and I356L did not help to increase the predictive value, the most likely reason being that these mutations reached almost fixed levels. Though mutations in SP related markers pfdhfr S108N and C59R were not associated with treatment failure, they increased the predictive value of pfdhps A437G. The difference in treatment failure rate in the two sites was reflected in the corresponding genetic profile of the parasite populations, with significant differences seen in the allele frequencies of mutant pfmdr1 N86Y, pfmdr1 Y184F, pfcrt A220S, and pfdhps A437G. Conclusion The study provides evidence for high levels of resistance to the combination regimen of AQ+SP in PNG and indicates which of the many molecular markers analysed are useful for the monitoring of parasite resistance to combinations with AQ+SP.

Of all Papua New Guinea provinces, Enga has the largest proportion of people living at altitudes that preclude malaria transmission. However, the first systematic surveys in 1979 showed that malaria was endemic in lower-lying valleys to the north and east of the province. A series of new surveys conducted in both wet and dry seasons showed that these areas remain the main malaria focus in Enga. However, over the last 25 years the risk of malarial infections has increased substantially in areas1200 m (from 10% to 37-41%). In these low-lying areas people acquire substantial antimalarial immunity and most infections are asymptomatic. However, people in villages in these areas had significantly lower mean haemoglobin levels (13.0 vs 14.0 g/dl, p0.001) than in areas above 1200 m, where overall prevalence rates (0-9%) have not changed much. In areas between 1200 and 1600 m epidemics with parasite prevalence rates in excess of 20% have been found to occur. Malaria was a significant cause of febrile illness only in endemic areas or during outbreaks. Although rarely used, sleeping under a bednet was associated with a significant reduction in risk of malaria infection (adjusted OR = 0.45, p = 0.01). On the other hand, sleeping in garden houses away from the main villages increased the risk of malaria infection (adjusted OR = 1.6, p = 0.03). Malaria control in outlying, malarious areas of Enga province could therefore be based on the distribution of long-lasting impregnated bednets, while at the same time addressing the additional risks posed by the high mobility of many of these populations through targeted health education.

Summary We describe here the results of an analysis of Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene copy number from 440 field isolates from Papua New Guinea. No multiple copies of the gene were found, which corresponds to the lack of usage of mefloquine. These data extend regional knowledge about the distribution of multidrug-resistant P. falciparum.

The 2015 Nobel Prize for Medicine marks an important milestone for research on infectious diseases of poverty. The award was shared by researchers identifying novel therapies for infections caused by roundworm parasites and malaria. This is important not only because of the excellent science that was conducted to achieve these discoveries, but because the prizes were given for diseases affecting the poorest and most vulnerable members of society. The Nobel Prize Committee said: “These two discoveries have provided humankind with powerful new means to combat these debilitating diseases that affect hundreds of millions of people annually. The consequences in terms of improved human health and reduced suffering are immeasurable.” Despite these gains, huge gaps remain. As we celebrate the Nobel Prizes, it’s also time to reflect on the lessons learnt and to highlight what is still needed for a more equitable approach to research and development. Chinese scientist Youyou Tu was recognized for the rediscovery of artemisinin, which led to the artemisinin-based combination treatments for malaria that have saved millions of lives. Satoshi Ōmura from Japan and William Campbell from the United States of America (USA) discovered avermectin, which led to the development of an effective treatment against river blindness and lymphatic filariasis, diseases that cause immense suffering and severe disfigurement. In both examples, these discoveries of the 1970s did not lead directly to public health impact. That required the further commitment of the United Nations, and the development of organizations dedicated to building research capacity for neglected diseases, such as the Special Programme for Research and Training in Tropical Diseases. In addition, partnerships were formed between international research organizations and pharmaceutical companies. Non-profit product development partnerships such as the Medicines for Malaria Venture and the Drugs for Neglected Diseases initiative were later formed, to focus on the development of drugs and diagnostics specifically in low-income countries. For Youyou Tu, linkages spanned China, Africa, Europe and the USA. There was critical support from international pharmaceutical companies to develop the new treatment to international standards and to run multi-country clinical drug trials. Scientists from low- and middle-income countries across Africa were trained and a critical generation of research capacity was begun. Once the treatments were found to be safe and effective, research on packaging was needed to ensure that illiterate people could understand the dosages. New policies, both by the World Health Organization (WHO) and the national health ministries, were required. Research identified new community-based approaches that ensured treatment was available to those who needed them, where they needed them. Today, artemisinin compounds are the mainstay of malaria treatment. Since 2000, more than 1 billion artemisinin-based treatment courses have been administered to over 600 million people,1 which has contributed to the impressive 54% fall in malaria cases in the African region.1 Treatment for river blindness and lymphatic filariasis benefited from a similar public–private research collaboration. Discovery of avermectin led to the development of the related drug ivermectin. Clinical trials were conducted in Africa by Africans. However, the key to widespread use was research on social systems, gender and economics showing how communities could lead the distribution of ivermectin. By 2013, more than 100 million people in sub-Saharan Africa were receiving annual treatments of ivermectin for onchocerciasis,2 ending the era when many people in Africa were blinded or severely disfigured from onchocerciasis. A new programme to eliminate onchocerciasis has now been created. Unfortunately, despite these successes, there are still gaps. New products to fight drug resistance and new distribution strategies are needed. On a positive note, nearly 500 product candidates are in the pipeline for neglected diseases, including treatments, diagnostics and vaccines.3 About three-quarters of these have been developed through public–private product development partnerships. The challenge now is to ensure that scientific and political momentum is not only sustained but expanded and, importantly, coordinated and financed.4 At a recent G7 Summit, science ministers committed to extending research on infectious diseases of poverty.5 New mechanisms, such as the WHO Global Observatory on Research and Development and the proposed Global Research and Development Fund, will be needed to define and fund public health priorities. Today, we need to sustain the research and development system that has been built from these prize-winning discoveries. Achieving impact required a full spectrum of research from discovery to product development to implementation. Innovative new partnerships, industry support and political will were necessary to fund the work (including the resulting treatments). Now, a globally coordinated response is needed to ensure that more great discoveries and innovation reach the people who need them, no matter where they live.

Because of increasing resistance to 4-aminoquinolines in Papua New Guinea, combination therapy of amodiaquine (AQ) or chloroquine (CQ) plus sulfadoxine-pyrimethamine (SP) was introduced as first-line treatment against uncomplicated malaria in 2000. The purpose of this study was to monitor in vivo efficacy of the current standard combination therapy against Plasmodium falciparum and P. vivax malaria. Studies were conducted between 2003 and 2005 in the Simbu, East Sepik, and Madang Provinces in Papua New Guinea according to the revised protocol of the World Health Organization (WHO) for assessment of antimalarial drug efficacy. Children between six months and seven years of age with clinically overt and parasitologically confirmed P. falciparum or P. vivax malaria were treated according to the new policy guidelines (i.e., AQ plus SP given to patients weighing < 14 kg and CQ plus SP given to patients weighing < 14 kg). Children were monitored up to day 28 and classified according to clinical and parasitological outcome as adequate clinical and parasitological response (ACPR), early treatment failure (ETF), late clinical failure (LCF), or late parasitological failure (LPF). For P. falciparum malaria, polymerase chain reaction (PCR)-corrected treatment failure rates up to day 28 ranged between 10.3% and 28.8% for AQ plus SP and between 5.6% and 28.6% for CQ plus SP, depending on the region and the year of assessment. Overall treatment failure rate with AQ or CQ plus SP for P. vivax malaria was 12%. Our results suggest that the current first-line treatment in Papua New Guinea is not sufficiently effective. According to the new WHO guidelines for the treatment of malaria, a rate of parasitological resistance greater than 10% in the two dominant malaria species in the country justifies a change in treatment policy.

During pregnancy, specific variants of Plasmodium falciparum-infected erythrocytes (IEs) can accumulate in the placenta through adhesion to chondroitin sulfate A (CSA) mediated by expression of PfEMP1 encoded by var2csa-type genes. Antibodies against these variants are associated with protection from maternal malaria. We evaluated antibodies among Kenyan, Papua New Guinean, and Malawian men and Kenyan children against two different CSA-binding P. falciparum isolates expressing var2csa variants. Specific IgG was present at significant levels among some men and children from each population, suggesting exposure to these variants is not exclusive to pregnancy. However, the level and prevalence of antibodies was substantially lower overall than exposed multigravidas. IgG-binding was specific and did not represent antibodies to subpopulations of non-CSA-binding IEs, and some sera inhibited IE adhesion to CSA. These findings have significant implications for understanding malaria pathogenesis and immunity and may be significant for understanding the acquisition of immunity to maternal malaria.

In a treatment re-infection study of 206 Papua New Guinean school children, we examined risk of reinfection and symptomatic malaria caused by different Plasmodium species. Although children acquired a similar number of polymerase chain reaction-detectable Plasmodium falciparum and P. vivax infections in six months of active follow-up (P. falciparum = 5.00, P. vivax = 5.28), they were 21 times more likely to develop symptomatic P. falciparum malaria (1.17/year) than P. vivax malaria (0.06/year). Children greater than nine years of age had a reduced risk of acquiring P. vivax infections of low-to-moderate (>150/microL) density (adjusted hazard rate [AHR] = 0.65 and 0.42), whereas similar reductions in risk with age of P. falciparum infection was only seen for parasitemias > 5,000/microL (AHR = 0.49) and symptomatic episodes (AHR = 0.51). Infection and symptomatic episodes with P. malariae and P. ovale were rare. By nine years of age, children have thus acquired almost complete clinical immunity to P. vivax characterized by a very tight control of parasite density, whereas the acquisition of immunity to symptomatic P. falciparum malaria remained incomplete. These observations suggest that different mechanisms of immunity may be important for protection from these malaria species.

Levels of C-reactive protein (CRP), a classic marker for the acute phase response (APR), were measured in children with asymptomatic malaria infection in the Amele region of Papua New Guinea (PNG). Despite the presence of parasitemia, the prevalence of CRP levels consistent with an APR (CRP10 microg/mL) was very low (10%). Splenomegaly was significantly associated with increased parasitemia (P0.001) and CRP levels (P0.001), highlighting the importance of splenomegaly as an indicator of recent high density infection in this population. Multivariate analysis showed that CRP levels were significantly associated with splenomegaly, fever, hemoglobin, and age (Por = 0.002). CRP levels also increased with increasing parasitemia (P0.001) but remained3.5 microg/mL. The low levels of CRP indicate that children in the Amele modulate inflammation associated with malaria.

With a renewed interest in large-scale malaria interventions, knowledge about the possible long-term effects of such interventions on the nature of malaria transmission is essential. We document complex changes in malaria epidemiology over the last 40 years associated with changing malaria control activities in Karimui, an isolated area in Papua New Guinea. An initially equal distribution of Plasmodium falciparum, P. vivax and P. malariae changed to currently 68% P. falciparum, after passing through a phase of transitory P. vivax dominance, when control started to fail. Initial drops in malaria prevalence proved difficult to sustain and present post-control levels are significantly higher than pre-control levels. The example of Karimui indicates that unsustained control can lead to changes in malaria patterns that may leave a population worse off.

Background Erythrocyte Duffy blood group negativity reaches fixation in African populations where Plasmodium vivax (Pv) is uncommon. While it is known that Duffy-negative individuals are highly resistant to Pv erythrocyte infection, little is known regarding Pv susceptibility among heterozygous carriers of a Duffy-negative allele (+/−). Our limited knowledge of the selective advantages or disadvantages associated with this genotype constrains our understanding of the effect that interventions against Pv may have on the health of people living in malaria-endemic regions. Methods and Findings We conducted cross-sectional malaria prevalence surveys in Papua New Guinea (PNG), where we have previously identified a new Duffy-negative allele among individuals living in a region endemic for all four human malaria parasite species. We evaluated infection status by conventional blood smear light microscopy and semi-quantitative PCR-based strategies. Analysis of a longitudinal cohort constructed from our surveys showed that Duffy heterozygous (+/−) individuals were protected from Pv erythrocyte infection compared to those homozygous for wild-type alleles (+/+) (log-rank tests: LM, p = 0.049; PCR, p = 0.065). Evaluation of Pv parasitemia, determined by semi-quantitative PCR-based methods, was significantly lower in Duffy +/− vs. +/+ individuals (Mann-Whitney U: p = 0.023). Overall, we observed no association between susceptibility to P. falciparum erythrocyte infection and Duffy genotype. Conclusions Our findings provide the first evidence that Duffy-negative heterozygosity reduces erythrocyte susceptibility to Pv infection. As this reduction was not associated with greater susceptibility to Pf malaria, our in vivo observations provide evidence that Pv-targeted control measures can be developed safely.

In Papua New Guinea (PNG), complex patterns of malaria commonly include single and mixed infections of Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. Here, we assess recent epidemiologic characteristics of Plasmodium blood-stage infections in the Wosera region through four cross-sectional surveys (August 2001 to June 2003). Whereas previous studies performed here have relied on blood smear/light microscopy (LM) for diagnosing Plasmodium species infections, we introduce a newly developed, post-polymerase chain reaction (PCR), semi- quantitative, ligase detection reaction-fluorescent microsphere assay (LDR-FMA). A direct comparison of the two methods for > 1,100 samples showed that diagnosis was concordant for > 80% of the analyses performed for P. falciparum (PF), P. vivax (PV), and P. malariae (PM). Greater sensitivity of the LDR-FMA accounted for 75% of the discordance between diagnoses. Based on LM, the prevalence of blood-stage PF, PV, and PM infections was found to be markedly reduced compared with an early 1990s survey. In addition, there were significant shifts in age distribution of infections, with PV becoming the most common parasite in children < 4 years of age. Consistent with previous studies, prevalence of all Plasmodium species infections increased significantly in samples analyzed by the PCR-based LDR- FMA. This increase was most pronounced for PM, PO, and mixed infections and in adolescent (10-19 years) and adult age groups, suggesting that LM may lead to under-reported prevalence of less common Plasmodium species, infection complexity, and a skewed distribution of infections towards younger age groups. This study shows that the application of LDR-FMA diagnosis in large epidemiologic studies or malaria control interventions is feasible and may contribute novel insights regarding the epidemiology of malaria.

Two very distinct malaria zones can be found within Simbu Province. The north of the province is characterized by the absence or very low level of local malaria transmission, but there is a considerable risk of epidemics prevalent in the lower-lying parts. During non-epidemic periods, parasite prevalence was usually under 5%, with similar frequencies for Plasmodium falciparum and P. vivax (47% each), and malaria was an only minor source of febrile illness. During epidemics, however, 13-36% of people were infected, predominantly with P. falciparum (64%), and high levels of severe morbidity were present. In south Simbu malaria is clearly endemic with an overall prevalence of 35%, combined with a strong age-dependence of infections, low haemoglobin levels, high rates of enlarged spleen and moderate to severe anaemia (haemoglobin level7.5 g/dl) in children. The malaria epidemiology in south Simbu is thus more similar to the lowlands than to other highlands areas. Epidemic prevention, surveillance and response in the north, and bednet distribution and strengthening of curative services in the south, are therefore the priorities for malaria control in Simbu Province.

Haptoglobin (Hp) is an acute phase protein that removes free hemoglobin (Hb) released during hemolysis. Hp has also been shown to be toxic for malaria parasites. alpha(+)-Thalassemia is a hemoglobinopathy that results in subclinical hemolytic anemia. alpha(+)-Thassemia homozygosity confers protection against severe malarial disease by an as yet unidentified mechanism. Hp levels were measured in a serial cross-sectional survey of children in Madang Province, Papua New Guinea (PNG). Hp levels were related to age, Hp genotype, Hb levels, parasitemia, splenomegaly, and alpha(+)-thalassemia genotype. Surprisingly, children who were homozygous for alpha(+) -thalassemia had significantly higher levels of Hp than did heterozygotes, after controlling for relevant confounders. We suggest that this is the result of either reduced mean cell Hb associated with alpha(+) -thalassemia homozygosity or an elevated IL-6-dependent acute phase response.

The epidemiology of malaria in Eastern Highlands Province (EHP) is characterized by generally very low-level or no local malaria transmission but a considerable risk of epidemics. In non-epidemic situations, parasite prevalence was under 5% in all but one area, Lufa Valley, where P. vivax was the most common parasite (prevalence rate of 58%). During epidemics, however, 23% (range 8-63%) of people were infected, with P. falciparum the predominant parasite. Mixed infections were very common (12%) but P. malariae and P. ovale were rare. Outside epidemics, malaria was only a minor source of febrile illness and enlarged spleens and anaemia (haemoglobin level7.5 g/dl) were virtually absent. However, epidemics were associated with a high incidence of relatively severe morbidity, both in terms of fevers and moderate to severe anaemia. Epidemic prevention, surveillance and response therefore are priorities in malaria control in EHP.

Despite a resurgence of malaria in many Papua New Guinea highlands and highlands fringe areas after the cessation of control activities in the early 1980s the malaria situation in these areas has received little attention. A series of cross-sectional surveys were therefore carried out to provide accurate and up-to-date information on the prevalence of malaria and the risk of epidemics and to propose adequate malaria control strategies. Studies in 24 villages in Western Highlands Province found the prevalence of malarial infections to be strongly correlated with altitude, ranging from 1.6% at altitudes of 1500-1800 m to over 30% in villages below 900 m. Malaria outbreaks were observed at the end of the rainy season. All four human malaria species were present with P. falciparum infections clearly dominating. The relative importance of P. vivax increased with altitude, while both P. malariae and P. ovale were rare. Many infections were of low density. While malaria is an important source of febrile illness in endemic areas below 1500 m altitude, only few observed or reported fevers are due to malarial infections in higher, nonendemic areas. Rates of enlarged spleens, mean haemoglobin levels and the prevalence of anaemia (Hb7.5 g/dl) were strongly linked to the level of malaria found in each community and were associated with both altitude and concurrent malarial infection. Based on the survey results, areas of different malaria epidemiology are delineated and options for control in each area are discussed.

Haptoglobin (Hp) is an acute phase protein that removes free hemoglobin (Hb) released during hemolysis. Hp has also been shown to be toxic for malaria parasites. + -Thalassemia is a hemoglobinopathy that results in subclinical hemolytic anemia. + -Thassemia homozygosity confers protection against severe malarial disease by an as yet unidentified mechanism. Hp levels were measured in a serial cross-sectional survey of children in Madang Province, Papua New Guinea (PNG). Hp levels were related to age, Hp genotype, Hb levels, parasitemia, splenomegaly, and + -thalassemia genotype. Surprisingly, children who were homozygous for + -thalassemia had significantly higher levels of Hp than did heterozygotes, after controlling for relevant confounders. We suggest that this is the result of either reduced mean cell Hb associated with + -thalassemia homozygosity or an elevated IL-6-dependent acute phase response. Haptoglobin (Hp) is an acute phase protein with an impor- tant protective role in hemolytic disease. It binds irreversibly to hemoglobin (Hb) and reduces the oxidative and peroxida- tive potential of free Hb. 1 The Hp-Hb complex is endocy- tosed by macrophages through the acute phase-regulated and signal-inducing macrophage protein CD163. 2 Hp exists in three phenotypic forms, Hp1-1, 2-1, and 2-2, encoded by two co-dominant alleles, Hp 1 and Hp 2 . 3 The Hp1 type may be further subdivided into two subtypes, Hp1S and Hp1F, coded by two alleles hp 1F and hp 1S. 4 Allele frequencies of types and subtypes vary according to geographical location. 4 The ability to bind Hb is phenotype-dependent and has been found to be in the order 1-1 > 2-1 > 2-2, the binding capacity reflecting the plasma levels of the three phenotypes. 5 Various factors play a role in determining Hp concentration (i.e., Hp phenotype, age, hemolysis, and the acute phase response). 6 Hemolysis is a pathologic feature of malaria infection, and decreased Hp levels are associated with clinical malaria. 7 Hp levels are reduced by both chronic, low-density, asymptom- atic parasitemia and possibly malaria-associated immune complex destruction of uninfected erythrocytes. 8 The preva- lence of ahaptoglobinemia (absence of measurable Hp) is common in malaria endemic areas and increases with increas- ing malaria transmission. 9 Available data on the protective effect of Hp polymorphisms from West Africa have been geo- graphically variable. 10-13 Interestingly, Hp is directly toxic to P. falciparum in vitro at concentrations that may occur during an acute phase re- sponse. 14 The exact mechanism of action is not known. Hp does not enter the infected erythrocyte but may act indirectly, disrupting normal parasite protein trafficking within the host cell. 14 An in vivo model of malaria infection in mice has shown that parasite burdens and peak parasite densities were higher in Hp knockout mice. 15 Hp has other anti-microbial effects; Hp has been shown to make iron unavailable to bac- teria using Hb (e.g., E. coli) and to cause agglutination of

The malaria parasite Plasmodium falciparum invades red blood cells and deposits the virulence factor PfEMP1 on their cell surface. This is the key to the parasite's ability to evade the immune system, since PfEMP1 is encoded by a family of 60 var genes, only one of which is transcribed at any one time. How Plasmodium brings about this antigenic variation is not clear. Voss et al. show that one active var promoter is sufficient to initiate the transcription of one gene while shutting off the others. This is explained by the existence of a unique, peri-nuclear compartment that aids in the transcription of a single var gene; switching var transcription, and thus changing PfEMP1 identity, would occur by competition of a silenced var promoter for occupancy of this space. Mono-allelic expression of gene families is used by many organisms to mediate phenotypic variation of surface proteins. In the apicomplexan parasite Plasmodium falciparum, responsible for the severe form of malaria in humans, this is exemplified by antigenic variation of the highly polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1)1,2. PfEMP1, encoded by the 60-member var gene family3,4,5,6, represents a major virulence factor due to its central role in immune evasion and intravascular parasite sequestration. Mutually exclusive expression of PfEMP1 is controlled by epigenetic mechanisms involving chromatin modification and perinuclear var locus repositioning7,8. Here we show that a var promoter mediates the nucleation and spreading of stably inherited silenced chromatin. Transcriptional activation of this promoter occurs at the nuclear periphery in association with chromosome-end clusters. Additionally, the var promoter sequence is sufficient to infiltrate a transgene into the allelic exclusion programme of var gene expression, as transcriptional activation of this transgene results in silencing of endogenous var gene transcription. These results show that a var promoter is sufficient for epigenetic silencing and mono-allelic transcription of this virulence gene family, and are fundamental for our understanding of antigenic variation in P. falciparum. Furthermore, the PfEMP1 knockdown parasites obtained in this study will be important tools to increase our understanding of P. falciparum-mediated virulence and immune evasion.

As part of a larger study into the epidemiology of malaria in the highlands of Papua New Guinea, outbreak investigations were carried out at the end of the 2002 rainy season in 11 villages situated between 1,400 and 1,700 meters above sea level that had reported epidemics. Locations and timing of these epidemics corresponded largely to those reported in the pre-control era of the 1960s and 1970s. On average, 28.8% (range = 10.3-63.2%) of people in each of the 11 villages were found to be infected with malaria. Plasmodium falciparum accounted for 59% of all identified infections and P. vivax for 34%. The majority (53%) of infections were symptomatic. Although symptomatic infections were most common in children 2-9 years of age (36%), even in adults a prevalence of 20% was observed. A comparison with earlier non-epidemic data in three of the villages without easy access to health care showed markedly increased levels of morbidity, with 6-10-fold increases in parasite prevalence, a 3-fold increase in both measured and reported fevers, and a 12-fold increase in enlarged spleens. The average hemoglobin levels were reduced by 2.3-3.5 g/dL, with a concurrent increase in moderate to severe anemia (hemoglobin level < 7.5 g/dL) from 0.0-3.3% to 3.8-18.4%. These massive increases in morbidity have devastating impact on the affected communities and highlight that malaria epidemics are a serious and increasing public health problem in the highlands of Papua New Guinea.

The extent of gene polymorphisms associated with resistance to chloroquine and sulfadoxine-pyrimethamine was examined in field isolates of Plasmodium falciparum from Indonesia. Eight malaria-endemic areas, representing a broad region of the western and eastern Indonesian Archipelago were surveyed. Blood from 20-50 patients was collected at each site, DNA was isolated, and the sequences of four different genes (dihydrofolate reductase [dhfr], dihydropteroate synthase [dhps], P. falciparum multidrug resistance 1 [pfmdr1], and P. falciparum chloroquine resistance transporter [pfcrt]) were analyzed using polymerase chain reaction and restriction fragment length polymorphisms to detect polymorphisms previously shown to be associated with resistance. This analysis identified polymorphisms in dhfr at 108-Asn/Thr, 16-Val, and 59-Arg. Polymorphisms in dhps were found less frequently, either 437-Gly alone or paired with 540-Glu. The pfcrt 76-Thr polymorphism was fixed in all parasite populations and pfmdr1 86-Tyr polymorphisms in all populations except in the most eastern regions. The pfmdr1 1042-Asp polymorphism occurred less frequently. These findings indicate that polymorphisms in genes associated with drug resistance in P. falciparum are found across a broad region of Indonesia.

The first report of Plasmodium falciparum chloroquine (CQ) resistance (CQR) in Papua New Guinea (PNG) appeared in 1974. Although the current prevalence of CQR-associated parasite gene polymorphisms has been documented for some regions, the spatial and temporal relationships that characterize CQ-resistant parasites in PNG are unknown. Insight into the evolution of CQ-resistant parasites could be provided by evaluating genetic markers in parasite populations. We compared pfcrt and pfmdr1 polymorphisms and flanking microsatellite (MS) polymorphisms between P. falciparum-infected placental tissue (early 1980s) and blood (late 1990s) samples collected throughout PNG. Consistent with the results of recent studies, pfcrt-SVMNT and pfmdr1-86Y were the only CQR-associated alleles observed in the placental tissue samples, and they were observed together in 79% of the samples. Results of analysis of MS flanking pfcrt (approximately 40 kb) suggested that there was less diversity in the samples collected during the 1980s than in those collected during the 1990s and that the 1990s parasites were significantly differentiated from the 1980s parasites. On the other hand, for MS flanking pfmdr1 (approximately 5 kb) and for 1 putatively neutral locus, diversity levels were similar, and the 2 parasite populations were not significantly differentiated. These results suggest that selection for CQR was operating on the pfcrt-SVMNT allele during the early 1980s. Thus, archival samples can provide novel insight into the dynamics of CQR.

The extent of gene polymorphisms associated with resistance to chloroquine and sulfadoxine-pyrimethamine was examined in field isolates of Plasmodium falciparum from Indonesia. Eight malaria-endemic areas, representing a broad region of the western and eastern Indonesian Archipelago were surveyed. Blood from 20-50 patients was collected at each site, DNA was isolated, and the sequences of four different genes (dihydrofolate reductase [dhfr], dihydropteroate synthase [dhps], P. falciparum multidrug resistance 1 [pfmdr1], and P. falciparum chloroquine resistance transporter [pfcrt]) were analyzed using polymerase chain reaction and restriction fragment length polymorphisms to detect polymorphisms previously shown to be associated with resistance. This analysis identified polymorphisms in dhfr at 108-Asn/Thr, 16-Val, and 59-Arg. Polymorphisms in dhps were found less frequently, either 437-Gly alone or paired with 540-Glu. The pfcrt 76-Thr polymorphism was fixed in all parasite populations and pfmdr1 86-Tyr polymorphisms in all populations except in the most eastern regions. The pfmdr1 1042-Asp polymorphism occurred less frequently. These findings indicate that polymorphisms in genes associated with drug resistance in P. falciparum are found across a broad region of Indonesia.

In plasmodia, the dihydrofolate reductase (DHFR) enzyme is the target of the pyrimethamine component of sulfadoxine-pyrimethamine (S/P). Plasmodium vivax infections are not treated intentionally with antifolates. However, outside Africa, coinfections with Plasmodium falciparum and P. vivax are common, and P. vivax infections are often exposed to S/P. Cloning of the P. vivax dhfr gene has allowed molecular comparisons of dhfr alleles from different regions. Examination of the dhfr locus from a few locations has identified a very diverse set of alleles and showed that mutant alleles of the vivax dhfr gene are prevalent in Southeast Asia where S/P has been used extensively. We have surveyed patient isolates from six locations in Indonesia and two locations in Papua New Guinea. We sequenced P. vivax dhfr alleles from 114 patient samples and identified 24 different alleles that differed from the wild type by synonymous and nonsynonymous point mutations, insertions, or deletions. Most importantly, five alleles that carried four or more nonsynonymous mutations were identified. Only one of these highly mutant alleles had been previously observed, and all carried the 57L and 117T mutations. P. vivax cannot be cultured continuously, so we used a yeast assay system to determine in vitro sensitivity to pyrimethamine for a subset of the alleles. Alleles with four nonsynonymous mutations conferred very high levels of resistance to pyrimethamine. This study expands significantly the total number of novel dhfr alleles now identified from P. vivax and provides a foundation for understanding how antifolate resistance arises and spreads in natural P. vivax populations.