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ItemNo Preview AvailableA Long Neglected World Malaria Map: Plasmodium vivax Endemicity in 2010(PUBLIC LIBRARY SCIENCE, 2012-09)BACKGROUND: Current understanding of the spatial epidemiology and geographical distribution of Plasmodium vivax is far less developed than that for P. falciparum, representing a barrier to rational strategies for control and elimination. Here we present the first systematic effort to map the global endemicity of this hitherto neglected parasite. METHODOLOGY AND FINDINGS: We first updated to the year 2010 our earlier estimate of the geographical limits of P. vivax transmission. Within areas of stable transmission, an assembly of 9,970 geopositioned P. vivax parasite rate (PvPR) surveys collected from 1985 to 2010 were used with a spatiotemporal Bayesian model-based geostatistical approach to estimate endemicity age-standardised to the 1-99 year age range (PvPR(1-99)) within every 5×5 km resolution grid square. The model incorporated data on Duffy negative phenotype frequency to suppress endemicity predictions, particularly in Africa. Endemicity was predicted within a relatively narrow range throughout the endemic world, with the point estimate rarely exceeding 7% PvPR(1-99). The Americas contributed 22% of the global area at risk of P. vivax transmission, but high endemic areas were generally sparsely populated and the region contributed only 6% of the 2.5 billion people at risk (PAR) globally. In Africa, Duffy negativity meant stable transmission was constrained to Madagascar and parts of the Horn, contributing 3.5% of global PAR. Central Asia was home to 82% of global PAR with important high endemic areas coinciding with dense populations particularly in India and Myanmar. South East Asia contained areas of the highest endemicity in Indonesia and Papua New Guinea and contributed 9% of global PAR. CONCLUSIONS AND SIGNIFICANCE: This detailed depiction of spatially varying endemicity is intended to contribute to a much-needed paradigm shift towards geographically stratified and evidence-based planning for P. vivax control and elimination.
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ItemNo Preview AvailableAnalysis of Plasmodium falciparum diversity in natural infections by deep sequencing(NATURE PUBLISHING GROUP, 2012-07-19)Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.
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ItemThe Plasmodium falciparum Erythrocyte Invasion Ligand Pfrh4 as a Target of Functional and Protective Human Antibodies against Malaria(PUBLIC LIBRARY SCIENCE, 2012-09-20)BACKGROUND: Acquired antibodies are important in human immunity to malaria, but key targets remain largely unknown. Plasmodium falciparum reticulocyte-binding-homologue-4 (PfRh4) is important for invasion of human erythrocytes and may therefore be a target of protective immunity. METHODS: IgG and IgG subclass-specific responses against different regions of PfRh4 were determined in a longitudinal cohort of 206 children in Papua New Guinea (PNG). Human PfRh4 antibodies were tested for functional invasion-inhibitory activity, and expression of PfRh4 by P. falciparum isolates and sequence polymorphisms were determined. RESULTS: Antibodies to PfRh4 were acquired by children exposed to P. falciparum malaria, were predominantly comprised of IgG1 and IgG3 subclasses, and were associated with increasing age and active parasitemia. High levels of antibodies, particularly IgG3, were strongly predictive of protection against clinical malaria and high-density parasitemia. Human affinity-purified antibodies to the binding region of PfRh4 effectively inhibited erythrocyte invasion by P. falciparum merozoites and antibody levels in protected children were at functionally-active concentrations. Although expression of PfRh4 can vary, PfRh4 protein was expressed by most isolates derived from the cohort and showed limited sequence polymorphism. CONCLUSIONS: Evidence suggests that PfRh4 is a target of antibodies that contribute to protective immunity to malaria by inhibiting erythrocyte invasion and preventing high density parasitemia. These findings advance our understanding of the targets and mechanisms of human immunity and evaluating the potential of PfRh4 as a component of candidate malaria vaccines.
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ItemEstimating the Numbers of Malaria Infections in Blood Samples Using High-Resolution Genotyping Data(PUBLIC LIBRARY SCIENCE, 2012-08-29)People living in endemic areas often habour several malaria infections at once. High-resolution genotyping can distinguish between infections by detecting the presence of different alleles at a polymorphic locus. However the number of infections may not be accurately counted since parasites from multiple infections may carry the same allele. We use simulation to determine the circumstances under which the number of observed genotypes are likely to be substantially less than the number of infections present and investigate the performance of two methods for estimating the numbers of infections from high-resolution genotyping data. The simulations suggest that the problem is not substantial in most datasets: the disparity between the mean numbers of infections and of observed genotypes was small when there was 20 or more alleles, 20 or more blood samples, a mean number of infections of 6 or less and where the frequency of the most common allele was no greater than 20%. The issue of multiple infections carrying the same allele is unlikely to be a major component of the errors in PCR-based genotyping. Simulations also showed that, with heterogeneity in allele frequencies, the observed frequencies are not a good approximation of the true allele frequencies. The first method that we proposed to estimate the numbers of infections assumes that they are a good approximation and hence did poorly in the presence of heterogeneity. In contrast, the second method by Li et al estimates both the numbers of infections and the true allele frequencies simultaneously and produced accurate estimates of the mean number of infections.
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ItemDefining the Antigenic Diversity of Plasmodium falciparum Apical Membrane Antigen 1 and the Requirements for a Multi-Allele Vaccine against Malaria(PUBLIC LIBRARY SCIENCE, 2012-12-05)Apical Membrane Antigen 1 (AMA1) is a leading malaria vaccine candidate and a target of naturally-acquired human immunity. Plasmodium falciparum AMA1 is polymorphic and in vaccine trials it induces strain-specific protection. This antigenic diversity is a major roadblock to development of AMA1 as a malaria vaccine and understanding how to overcome it is essential. To assess how AMA1 antigenic diversity limits cross-strain growth inhibition, we assembled a panel of 18 different P. falciparum isolates which are broadly representative of global AMA1 sequence diversity. Antibodies raised against four well studied AMA1 alleles (W2Mef, 3D7, HB3 and FVO) were tested for growth inhibition of the 18 different P. falciparum isolates in growth inhibition assays (GIA). All antibodies demonstrated substantial cross-inhibitory activity against different isolates and a mixture of the four different AMA1 antibodies inhibited all 18 isolates tested, suggesting significant antigenic overlap between AMA1 alleles and limited antigenic diversity of AMA1. Cross-strain inhibition by antibodies was only moderately and inconsistently correlated with the level of sequence diversity between AMA1 alleles, suggesting that sequence differences are not a strong predictor of antigenic differences or the cross-inhibitory activity of anti-allele antibodies. The importance of the highly polymorphic C1-L region for inhibitory antibodies and potential vaccine escape was assessed by generating novel transgenic P. falciparum lines for testing in GIA. While the polymorphic C1-L epitope was identified as a significant target of some growth-inhibitory antibodies, these antibodies only constituted a minor proportion of the total inhibitory antibody repertoire, suggesting that the antigenic diversity of inhibitory epitopes is limited. Our findings support the concept that a multi-allele AMA1 vaccine would give broad coverage against the diversity of AMA1 alleles and establish new tools to define polymorphisms important for vaccine escape.
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ItemOwnership and usage of mosquito nets after four years of large-scale free distribution in Papua New Guinea(BMC, 2012-06-10)BACKGROUND: Papua New Guinea (PNG) is a highly malaria endemic country in the South-West Pacific with a population of approximately 6.6 million (2009). In 2004, the country intensified its malaria control activities with support from the Global Fund. With the aim of achieving 80% ownership and usage, a country-wide campaign distributed two million free long-lasting insecticide-treated nets (LLINs). METHODS: In order to evaluate outcomes of the campaign against programme targets, a country-wide household survey based on stratified multi-stage random sampling was carried out in 17 of the 20 provinces after the campaign in 2008/09. In addition, a before-after assessment was carried out in six purposively selected sentinel sites. A structured questionnaire was administered to the heads of sampled households to elicit net ownership and usage information. RESULTS: After the campaign, 64.6% of households owned a LLIN, 80.1% any type of mosquito net. Overall usage by household members amounted to 32.5% for LLINs and 44.3% for nets in general. Amongst children under five years, 39.5% used a LLIN and 51.8% any type of net, whereas 41.3% of pregnant women used a LLIN and 56.1% any net. Accessibility of villages was the key determinant of net ownership, while usage was mainly determined by ownership. Most (99.5%) of the household members who did not sleep under a net did not have access to a (unused) net in their household. In the sentinel sites, LLIN ownership increased from 9.4% to 88.7%, ownership of any net from 52.7% to 94.1%. Usage of LLINs increased from 5.5% to 55.1%, usage of any net from 37.3% to 66.7%. Among children under five years, usage of LLINs and of nets in general increased from 8.2% to 67.0% and from 44.6% to 76.1%, respectively (all p ≤ 0.001). CONCLUSIONS: While a single round of free distribution of LLINs significantly increased net ownership, an insufficient number of nets coupled with a heterogeneous distribution led to overall low usage rates. Programme targets were missed mainly as a result of the distribution mechanism itself and operational constraints in this very challenging setting.
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ItemSevere Anemia in Papua New Guinean Children from a Malaria-Endemic Area: A Case-Control Etiologic Study(PUBLIC LIBRARY SCIENCE, 2012-12)BACKGROUND: There are few detailed etiologic studies of severe anemia in children from malaria-endemic areas and none in those countries with holoendemic transmission of multiple Plasmodium species. METHODOLOGY/PRINCIPAL FINDINGS: We examined associates of severe anemia in 143 well-characterized Papua New Guinean (PNG) children aged 0.5-10 years with hemoglobin concentration <50 g/L (median [inter-quartile range] 39 [33]-[44] g/L) and 120 matched healthy children (113 [107-119] g/L) in a case-control cross-sectional study. A range of socio-demographic, behavioural, anthropometric, clinical and laboratory (including genetic) variables were incorporated in multivariate models with severe anemia as dependent variable. Consistent with a likely trophic effect of chloroquine or amodiaquine on parvovirus B19 (B19V) replication, B19V PCR/IgM positivity had the highest odds ratio (95% confidence interval) of 75.8 (15.4-526), followed by P. falciparum infection (19.4 (6.7-62.6)), vitamin A deficiency (13.5 (5.4-37.7)), body mass index-for-age z-score <2.0 (8.4 (2.7-27.0)) and incomplete vaccination (2.94 (1.3-7.2)). P. vivax infection was inversely associated (0.12 (0.02-0.47), reflecting early acquisition of immunity and/or a lack of reticulocytes for parasite invasion. After imputation of missing data, iron deficiency was a weak positive predictor (6.4% of population attributable risk). CONCLUSIONS/SIGNIFICANCE: These data show that severe anemia is multifactorial in PNG children, strongly associated with under-nutrition and certain common infections, and potentially preventable through vitamin A supplementation and improved nutrition, completion of vaccination schedules, and intermittent preventive antimalarial treatment using non-chloroquine/amodiaquine-based regimens.
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ItemMalaria case management in Papua New Guinea prior to the introduction of a revised treatment protocol(BMC, 2012-05-07)BACKGROUND: This study aimed to document malaria case management practices in Papua New Guinea prior to the introduction of a revised national malaria treatment protocol. The revised protocol stipulates routine testing of malaria infection by rapid diagnostic test or microscopy, anti-malarial prescription to test positive cases only, and the introduction of a new artemisinin-based first-line anti-malarial. Findings presented in this paper primarily focus on diagnostic, prescription and treatment counselling practices. METHODS: In a national cross-sectional survey of 79 randomly selected health facilities, data were collected via non-participant observation of the clinical case management of patients presenting with fever or a recent history of fever. Data were recorded on a structured clinical observation instrument. RESULTS: Overall, 15% of observed fever patients (n=468) were tested for malaria infection by rapid diagnostic test and a further 3.6% were tested via microscopy. An anti-malarial prescription was made in 96.4% (451/468) of cases, including 100% (17/17) of test positive cases and 82% (41/50) of test negative cases. In all, 79.8% of anti-malarial prescriptions conformed to the treatment protocol current at the time of data collection. The purpose of the prescribed medication was explained to patients in 63.4% of cases, dosage/regimen instructions were provided in 75.7% of cases and the possibility of adverse effects and what they might look like were discussed in only 1.1% of cases. CONCLUSION: The revised national malaria treatment protocol will require a substantial change in current clinical practice if it is to be correctly implemented and adhered to. Areas that will require the most change include the shift from presumptive to RDT/microscopy confirmed diagnosis, prescribing (or rather non-prescribing) of anti-malarials to patients who test negative for malaria infection, and the provision of thorough treatment counselling. A comprehensive clinician support programme, possibly inclusive of 'booster' training opportunities and regular clinical supervision will be needed to support the change.
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ItemRapid Antigen Detection Tests for Malaria Diagnosis in Severely Ill Papua New Guinean Children: A Comparative Study Using Bayesian Latent Class Models(PUBLIC LIBRARY SCIENCE, 2012-11-05)BACKGROUND: Although rapid diagnostic tests (RDTs) have practical advantages over light microscopy (LM) and good sensitivity in severe falciparum malaria in Africa, their utility where severe non-falciparum malaria occurs is unknown. LM, RDTs and polymerase chain reaction (PCR)-based methods have limitations, and thus conventional comparative malaria diagnostic studies employ imperfect gold standards. We assessed whether, using Bayesian latent class models (LCMs) which do not require a reference method, RDTs could safely direct initial anti-infective therapy in severe ill children from an area of hyperendemic transmission of both Plasmodium falciparum and P. vivax. METHODS AND FINDINGS: We studied 797 Papua New Guinean children hospitalized with well-characterized severe illness for whom LM, RDT and nested PCR (nPCR) results were available. For any severe malaria, the estimated prevalence was 47.5% with RDTs exhibiting similar sensitivity and negative predictive value (NPV) to nPCR (≥96.0%). LM was the least sensitive test (87.4%) and had the lowest NPV (89.7%), but had the highest specificity (99.1%) and positive predictive value (98.9%). For severe falciparum malaria (prevalence 42.9%), the findings were similar. For non-falciparum severe malaria (prevalence 6.9%), no test had the WHO-recommended sensitivity and specificity of >95% and >90%, respectively. RDTs were the least sensitive (69.6%) and had the lowest NPV (96.7%). CONCLUSIONS: RDTs appear a valuable point-of-care test that is at least equivalent to LM in diagnosing severe falciparum malaria in this epidemiologic situation. None of the tests had the required sensitivity/specificity for severe non-falciparum malaria but the number of false-negative RDTs in this group was small.
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ItemReduced Risk of Plasmodium vivax Malaria in Papua New Guinean Children with Southeast Asian Ovalocytosis in Two Cohorts and a Case-Control Study(PUBLIC LIBRARY SCIENCE, 2012-09)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, p<0.0001) in a cohort of infants aged 3-21 months and a significant 52% reduction in P. vivax (blood-stage) reinfection diagnosed by PCR (95% CI 22-71, p = 0.003) and 55% by light microscopy (95% CI 13-77, p = 0.014), respectively, in a cohort of children aged 5-14 years. SAO was also associated with a reduction in risk of P. vivax parasitaemia in children 3-21 months (1,111/µl versus 636/µl, p = 0.011) and prevalence of P. vivax infections in children 15-21 months (odds ratio [OR] = 0.39, 95% CI 0.23-0.67, p = 0.001). In a case-control study of children aged 0.5-10 years, no child with SAO was found among 27 cases with severe P. vivax or mixed P. falciparum/P. vivax malaria (OR = 0, 95% CI 0-1.56, p = 0.11). SAO was associated with protection against severe P. falciparum malaria (OR = 0.38, 95% CI 0.15-0.87, p = 0.014) but no effect was seen on either the risk of acquiring blood-stage infections or uncomplicated episodes with P. falciparum. Although Duffy antigen receptor expression and function were not affected on SAO erythrocytes compared to non-SAO children, high level (>90% 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.