The epidemiology of Plasmodium falciparum and Plasmodium vivax in East Sepik Province, Papua New Guinea, pre- and post-implementation of national malaria control efforts.

BACKGROUND: In the past decade, national malaria control efforts in Papua New Guinea (PNG) have received renewed support, facilitating nationwide distribution of free long-lasting insecticidal nets (LLINs), as well as improvements in access to parasite-confirmed diagnosis and effective artemisinin-combination therapy in 2011-2012. METHODS: To study the effects of these intensified control efforts on the epidemiology and transmission of Plasmodium falciparum and Plasmodium vivax infections and investigate risk factors at the individual and household level, two cross-sectional surveys were conducted in the East Sepik Province of PNG; one in 2005, before the scale-up of national campaigns and one in late 2012-early 2013, after 2 rounds of LLIN distribution (2008 and 2011-2012). Differences between studies were investigated using Chi square (χ2), Fischer's exact tests and Student's t-test. Multivariable logistic regression models were built to investigate factors associated with infection at the individual and household level. RESULTS: The prevalence of P. falciparum and P. vivax in surveyed communities decreased from 55% (2005) to 9% (2013) and 36% to 6%, respectively. The mean multiplicity of infection (MOI) decreased from 1.8 to 1.6 for P. falciparum (p = 0.08) and from 2.2 to 1.4 for P. vivax (p < 0.001). Alongside these reductions, a shift towards a more uniform distribution of infections and illness across age groups was observed but there was greater heterogeneity across the study area and within the study villages. Microscopy positive infections and clinical cases in the household were associated with high rate infection households (> 50% of household members with Plasmodium infection). CONCLUSION: After the scale-up of malaria control interventions in PNG between 2008 and 2012, there was a substantial reduction in P. falciparum and P. vivax infection rates in the studies villages in East Sepik Province. Understanding the extent of local heterogeneity in malaria transmission and the driving factors is critical to identify and implement targeted control strategies to ensure the ongoing success of malaria control in PNG and inform the development of tools required to achieve elimination. In household-based interventions, diagnostics with a sensitivity similar to (expert) microscopy could be used to identify and target high rate households.

Differential impact of malaria control interventions on P. falciparum and P. vivax infections in young Papua New Guinean children.

INTRODUCTION: As malaria transmission declines, understanding the differential impact of intensified control on Plasmodium falciparum relative to Plasmodium vivax and identifying key drivers of ongoing transmission is essential to guide future interventions. METHODS: Three longitudinal child cohorts were conducted in Papua New Guinea before (2006/2007), during (2008) and after scale-up of control interventions (2013). In each cohort, children aged 1-5 years were actively monitored for infection and illness. Incidence of malaria episodes, molecular force of blood-stage infections (molFOB) and population-averaged prevalence of infections were compared across the cohorts to investigate the impact of intensified control in young children and the key risk factors for malaria infection and illness in 2013. RESULTS: Between 2006 and 2008, P. falciparum infection prevalence, molFOB, and clinical malaria episodes reduced by 47%, 59% and 69%, respectively, and a further 49%, 29% and 75% from 2008 to 2013 (prevalence 41.6% to 22.1% to 11.2%; molFOB: 3.4 to 1.4 to 1.0 clones/child/year; clinical episodes incidence rate (IR) 2.6 to 0.8 to IR 0.2 episodes/child/year). P. vivax clinical episodes declined at rates comparable to P. falciparum between 2006, 2008 and 2013 (IR 2.5 to 1.1 to 0.2), while P. vivax molFOB (2006, 9.8; 2008, 12.1) and prevalence (2006, 59.6%; 2008, 65.0%) remained high in 2008. However, in 2013, P. vivax molFOB (1.2) and prevalence (19.7%) had also substantially declined. In 2013, 89% of P. falciparum and 93% of P. vivax infections were asymptomatic, 62% and 47%, respectively, were sub-microscopic. Area of residence was the major determinant of malaria infection and illness. CONCLUSION: Intensified vector control and routine case management had a differential impact on rates of P. falciparum and P. vivax infections but not clinical malaria episodes in young children. This suggests comparable reductions in new mosquito-derived infections but a delayed impact on P. vivax relapsing infections due to a previously acquired reservoir of hypnozoites. This demonstrates the need to strengthen implementation of P. vivax radical cure to maximise impact of control in co-endemic areas. The high heterogeneity of malaria in 2013 highlights the importance of surveillance and targeted interventions to accelerate towards elimination.

Efficacy of artemether-lumefantrine and dihydroartemisinin-piperaquine for the treatment of uncomplicated malaria in Papua New Guinea.

BACKGROUND: In 2009, the Papua New Guinea (PNG) Department of Health adopted artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DHA-PPQ) as the first- and second-line treatments for uncomplicated malaria, respectively. This study was conducted to assess the efficacy of both drugs following adoption of the new policy. METHODS: Between June 2012 and September 2014, a therapeutic efficacy study was conducted in East Sepik and Milne Bay Provinces of PNG in accordance with the standard World Health Organization (WHO) protocol for surveillance of anti-malarial drug efficacy. Patients ≥ 6 months of age with microscopy confirmed Plasmodium falciparum or Plasmodium vivax mono-infections were enrolled, treated with AL or DHA-PPQ, and followed up for 42 days. Study endpoints were adequate clinical and parasitological response (ACPR) on days 28 and 42. The in vitro efficacy of anti-malarials and the prevalence of selected molecular markers of resistance were also determined. RESULTS: A total of 274 P. falciparum and 70 P. vivax cases were enrolled. The day-42 PCR-corrected ACPR for P. falciparum was 98.1% (104/106) for AL and 100% (135/135) for DHA-PPQ. The day-42 PCR-corrected ACPR for P. vivax was 79.0% (15/19) for AL and 92.3% (36/39) for DHA-PPQ. Day 3 parasite clearance of P. falciparum was 99.2% with AL and 100% with DHA-PPQ. In vitro testing of 96 samples revealed low susceptibility to chloroquine (34% of samples above IC50 threshold) but not to lumefantrine (0%). Molecular markers assessed in a sub-set of the study population indicated high rates of chloroquine resistance in P. falciparum (pfcrt SVMNT: 94.2%, n = 104) and in P. vivax (pvmdr1 Y976F: 64.8%, n = 54). CONCLUSIONS: AL and DHA-PPQ were efficacious as first- and second-line treatments for uncomplicated malaria in PNG. Continued in vivo efficacy monitoring is warranted considering the threat of resistance to artemisinin and partner drugs in the region and scale-up of artemisinin-based combination therapy in PNG.

IgG antibodies to synthetic GPI are biomarkers of immune-status to both Plasmodium falciparum and Plasmodium vivax malaria in young children.

BACKGROUND: Further reduction in malaria prevalence and its eventual elimination would be greatly facilitated by the development of biomarkers of exposure and/or acquired immunity to malaria, as well as the deployment of effective vaccines against Plasmodium falciparum and Plasmodium vivax. A better understanding of the acquisition of immunity in naturally-exposed populations is essential for the identification of antigens useful as biomarkers, as well as to inform rational vaccine development. METHODS: ELISA was used to measure total IgG to a synthetic form of glycosylphosphatidylinositol from P. falciparum (PfGPI) in a cohort of 1-3 years old Papua New Guinea children with well-characterized individual differences in exposure to P. falciparum and P. vivax blood-stage infections. The relationship between IgG levels to PfGPI and measures of recent and past exposure to P. falciparum and P. vivax infections was investigated, as well as the association between antibody levels and prospective risk of clinical malaria over 16 months of follow-up. RESULTS: Total IgG levels to PfGPI were low in the young children tested. Antibody levels were higher in the presence of P. falciparum or P. vivax infections, but short-lived. High IgG levels were associated with higher risk of P. falciparum malaria (IRR 1.33-1.66, P = 0.008-0.027), suggesting that they are biomarkers of increased exposure to P. falciparum infections. Given the cross-reactive nature of antibodies to PfGPI, high IgG levels were also associated with reduced risk of P. vivax malaria (IRR 0.65-0.67, P = 0.039-0.044), indicating that these antibodies are also markers of acquired immunity to P. vivax. CONCLUSIONS: This study highlights that in young children, IgG to PfGPI might be a useful marker of immune-status to both P. falciparum and P. vivax infections, and potentially useful to help malaria control programs to identify populations at-risk. Further functional studies are necessary to confirm the potential of PfGPI as a target for vaccine development.

Acquisition of antibodies against Plasmodium falciparum merozoites and malaria immunity in young children and the influence of age, force of infection, and magnitude of response.

Individuals in areas of Plasmodium falciparum endemicity develop immunity to malaria after repeated exposure. Knowledge of the acquisition and nature of protective immune responses to P. falciparum is presently limited, particularly for young children. We examined antibodies (IgM, IgG, and IgG subclasses) to merozoite antigens and their relationship to the prospective risk of malaria in children 1 to 4 years of age in a region of malaria endemicity in Papua New Guinea. IgG, IgG1, and IgG3 responses generally increased with age, were higher in children with active infection, and reflected geographic heterogeneity in malaria transmission. Antigenic properties, rather than host factors, appeared to be the main determinant of the type of IgG subclass produced. High antibody levels were not associated with protection from malaria; in contrast, they were typically associated with an increased risk of malaria. Adjustment for malaria exposure, using a novel molecular measure of the force of infection by P. falciparum, accounted for much of the increased risk, suggesting that the antibodies were markers of higher exposure to P. falciparum. Comparisons between antibodies in this cohort of young children and in a longitudinal cohort of older children suggested that the lack of protective association was explained by lower antibody levels among young children and that there is a threshold level of antibodies required for protection from malaria. Our results suggest that in populations with low immunity, such as young children, antibodies to merozoite antigens may act as biomarkers of malaria exposure and that, with increasing exposure and responses of higher magnitude, antibodies may act as biomarkers of protective immunity.

Significant geographical differences in prevalence of mutations associated with Plasmodium falciparum and Plasmodium vivax drug resistance in two regions from Papua New Guinea.

BACKGROUND: Drug resistance remains a major obstacle to malaria treatment and control. It can arise and spread rapidly, and vary substantially even at sub-national level. National malaria programmes require cost-effective and timely ways of characterizing drug-resistance at multiple sites within their countries. METHODS: An improved multiplexed post-PCR ligase detection reaction-fluorescent microsphere assay (LDR-FMA) was used to simultaneously determine the presence of mutations in chloroquine resistance transporter (crt), multidrug resistance 1 (mdr1), dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes in Plasmodium falciparum (n = 727) and Plasmodium vivax (n = 574) isolates collected in 2006 from cross-sectional community population surveys in two geographically distinct regions (Madang and East Sepik) of Papua New Guinea (PNG) where strong regional differences in in vivo aminoquinoline and antifolate therapeutic efficacy had previously been observed. Data were compared to those of a follow-up survey conducted in 2010. RESULTS: Despite some very low parasite densities, the assay successfully amplified all P. falciparum and P. vivax loci in 77 and 69 % of samples, respectively. In 2006, prevalences of pfdhfr (59R-108 N) double mutation/wild type pfdhps haplotype, pfcrt SVMNT haplotype (72S-76T double mutation), and 86Y pfmdr1 mutation all exceeded 90 %. For P. vivax, 65 % carried at least two pvdhfr mutations, 97 % the 647P pvdhps mutation and 54 % the 976F pvmdr1 mutation. Prevalence of mutant haplotypes was higher in Madang than East Sepik for pfcrt SVMNT (97.4 vs 83.3 %, p = 0.001), pfdhfr (59R-108 N) (100 vs 90.6 %, p = 0.001), pvdhfr haplotypes (75.8 vs 47.6 %, p = 0.001) and pvmdr1 976F (71.2 vs 26.2 %, p < 0.001). Data from a subsequent Madang survey in 2010 showed that the prevalence of pfdhps mutations increased significantly from <5 % to >30 % (p < 0.001) as did the prevalence of pvdhfr mutant haplotypes (from 75.8 to 97.4 %, p = 0.012). CONCLUSIONS: This LDR-FMA multiplex platform shows feasibility for low-cost, high-throughput, rapid characterization of a broad range of drug-resistance markers in low parasitaemia infections. Significant geographical differences in mutation prevalence correlate with previous genotyping surveys and in vivo trials and may reflect variable drug pressure and differences in health-care access in these two PNG populations.

Force of infection is key to understanding the epidemiology of Plasmodium falciparum malaria in Papua New Guinean children.

Genotyping Plasmodium falciparum parasites in longitudinal studies provides a robust approach to estimating force of infection (FOI) in the presence of superinfections. The molecular parameter (mol)FOI, defined as the number of new P. falciparum clones acquired over time, describes basic malaria epidemiology and is suitable for measuring outcomes of interventions. This study was designed to test whether (mol)FOI influenced the risk of clinical malaria episodes and how far (mol)FOI reflected environmental determinants of transmission, such as seasonality and small-scale geographical variation or effects of insecticide-treated nets (ITNs). Two hundred sixty-four children 1-3 y of age from Papua New Guinea were followed over 16 mo. Individual parasite clones were tracked longitudinally by genotyping. On average, children acquired 5.9 (SD 9.6) new P. falciparum infections per child per y. (mol)FOI showed a pronounced seasonality, was strongly reduced in children using ITNs (incidence rate ratio, 0.49; 95% confidence interval, [0.38, 0.61]), increased with age, and significantly varied within villages (P = 0.001). The acquisition of new parasite clones was the major factor determining the risk of clinical illness (incidence rate ratio, 2.12; 95% confidence interval, [1.93, 2.31]). Adjusting for individual differences in (mol)FOI completely explained spatial variation, age trends, and the effect of ITN use. This study highlights the suitability of (mol)FOI as a measure of individual exposure and its central role in malaria epidemiology. It has substantial advantages over entomological measures in studies of transmission patterns, and could be used in analyses of host variation in susceptibility, in field efficacy trials of novel interventions or vaccines, and for evaluating intervention effects.

Relapses contribute significantly to the risk of Plasmodium vivax infection and disease in Papua New Guinean children 1-5 years of age.

BACKGROUND: Plasmodium vivax forms long-lasting hypnozoites in the liver. How much they contribute to the burden of P. vivax malaria in children living in highly endemic areas is unknown. METHODS: In this study, 433 Papua New Guinean children aged 1-5 years were Randomized to receive artesunate (7 days) plus primaquine (14 days), artesunate alone or no treatment and followed up actively for recurrent Plasmodium infections and disease for 40 weeks. RESULTS: Treatment with artesunate-primaquine reduced the risk of P. vivax episodes by 28% (P = .042) and 33% (P = .015) compared with the artesunate and control arms, respectively. A significant reduction was observed only in the first 3 months of follow-up (artesunate-primaquine vs control, -58% [P = .004]; artesunate-primaquine vs artesunate, -49% [P = .031]) with little difference thereafter. Primaquine treatment also reduced the risk of quantitative real-time polymerase chain reaction- and light microscopy-positive P. vivax reinfections by 44% (P < .001) and 67% (P < .001), respectively. Whereas primaquine treatment did not change the risk of reinfection with Plasmodium falciparum, fewer P. falciparum clinical episodes were observed in the artesunate-primaquine arm. CONCLUSIONS: Hypnozoites are an important source of P. vivax infection and contribute substantially to the high burden of P. vivax disease observed in young Papua New Guinean children. Even in highly endemic areas with a high risk of reinfection, antihypnozoite treatment should be given to all cases with parasitologically confirmed P. vivax infections.

Identification of novel Plasmodium vivax proteins associated with protection against clinical malaria.

As progress towards malaria elimination continues, the challenge posed by the parasite species Plasmodium vivax has become more evident. In many regions co-endemic for P. vivax and Plasmodium falciparum, as transmission has declined the proportion of cases due to P. vivax has increased. Novel tools that directly target P. vivax are thus warranted for accelerated elimination. There is currently no advanced vaccine for P. vivax and only a limited number of potential candidates in the pipeline. In this study we aimed to identify promising P. vivax proteins that could be used as part of a subunit vaccination approach. We screened 342 P. vivax protein constructs for their ability to induce IgG antibody responses associated with protection from clinical disease in a cohort of children from Papua New Guinea. This approach has previously been used to successfully identify novel candidates. We were able to confirm previous results from our laboratory identifying the proteins reticulocyte binding protein 2b and StAR-related lipid transfer protein, as well as at least four novel candidates with similar levels of predicted protective efficacy. Assessment of these P. vivax proteins in further studies to confirm their potential and identify functional mechanisms of protection against clinical disease are warranted.

Tolerability and safety of primaquine in Papua New Guinean children 1 to 10 years of age.

Primaquine is currently the only drug available for radical cure of Plasmodium vivax and P. ovale liver infection stages, but limited safety data exist for children <10 years of age. Detailed daily assessments of side effects in glucose-6-phosphate dehydrogenase (G6PD)-normal children treated with 14 days of primaquine plus chloroquine (3 days; n = 252) or artesunate (7 days; n = 141) (0.5 mg/kg of body weight) showed that both treatments are well tolerated, do not lead to reductions in hemoglobin levels, and can thus safely be used in children 1 to 10 years of age.

Co-infection of the four major Plasmodium species: Effects on densities and gametocyte carriage.

BACKGROUND: Co-infection of the four major species of human malaria parasite Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale sp. (Po) is regularly observed, but there is limited understanding of between-species interactions. In particular, little is known about the effects of multiple Plasmodium species co-infections on gametocyte production. METHODS: We developed molecular assays for detecting asexual and gametocyte stages of Pf, Pv, Pm, and Po. This is the first description of molecular diagnostics for Pm and Po gametocytes. These assays were implemented in a unique epidemiological setting in Papua New Guinea with sympatric transmission of all four Plasmodium species permitting a comprehensive investigation of species interactions. FINDINGS: The observed frequency of Pf-Pv co-infection for asexual parasites (14.7%) was higher than expected from individual prevalence rates (23.8%Pf x 47.4%Pv = 11.3%). The observed frequency of co-infection with Pf and Pv gametocytes (4.6%) was higher than expected from individual prevalence rates (13.1%Pf x 28.2%Pv = 3.7%). The excess risk of co-infection was 1.38 (95% confidence interval (CI): 1.09, 1.67) for all parasites and 1.37 (95% CI: 0.95, 1.79) for gametocytes. This excess co-infection risk was partially attributable to malaria infections clustering in some villages. Pf-Pv-Pm triple infections were four times more frequent than expected by chance alone, which could not be fully explained by infections clustering in highly exposed individuals. The effect of co-infection on parasite density was analyzed by systematic comparison of all pairwise interactions. This revealed a significant 6.57-fold increase of Pm density when co-infected with Pf. Pm gametocytemia also increased with Pf co-infection. CONCLUSIONS: Heterogeneity in exposure to mosquitoes is a key epidemiological driver of Plasmodium co-infection. Among the four co-circulating parasites, Pm benefitted most from co-infection with other species. Beyond this, no general prevailing pattern of suppression or facilitation was identified in pairwise analysis of gametocytemia and parasitemia of the four species. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov, Trial ID: NCT02143934.

Assessment of IgG3 as a serological exposure marker for Plasmodium vivax in areas with moderate-high malaria transmission intensity.

A more sensitive surveillance tool is needed to identify Plasmodium vivax infections for treatment and to accelerate malaria elimination efforts. To address this challenge, our laboratory has developed an eight-antigen panel that detects total IgG as serological markers of P. vivax exposure within the prior 9 months. The value of these markers has been established for use in areas with low transmission. In moderate-high transmission areas, there is evidence that total IgG is more long-lived than in areas with low transmission, resulting in poorer performance of these markers in these settings. Antibodies that are shorter-lived may be better markers of recent infection for use in moderate-high transmission areas. Using a multiplex assay, the antibody temporal kinetics of total IgG, IgG1, IgG3, and IgM against 29 P. vivax antigens were measured over 36 weeks following asymptomatic P. vivax infection in Papua New Guinean children (n = 31), from an area with moderate-high transmission intensity. IgG3 declined faster to background than total IgG, IgG1, and IgM. Based on these kinetics, IgG3 performance was then assessed for classifying recent exposure in a cohort of Peruvian individuals (n = 590; age 3-85 years) from an area of moderate transmission intensity. Using antibody responses against individual antigens, the highest performance of IgG3 in classifying recent P. vivax infections in the prior 9 months was to one of the Pv-fam-a proteins assessed (PVX_125728) (AUC = 0.764). Surprisingly, total IgG was overall a better marker of recent P. vivax infection, with the highest individual classification performance to RBP2b1986-2653 (PVX_094255) (AUC = 0.838). To understand the acquisition of IgG3 in this Peruvian cohort, relevant epidemiological factors were explored using a regression model. IgG3 levels were positively associated with increasing age, living in an area with (relatively) higher transmission intensity, and having three or more PCR-detected blood-stage P. vivax infections within the prior 13 months. Overall, we found that IgG3 did not have high accuracy for detecting recent exposure to P. vivax in the Peruvian cohort, with our data suggesting that this is due to the high levels of prior exposure required to acquire high IgG3 antibody levels.

Multilocus haplotypes reveal variable levels of diversity and population structure of Plasmodium falciparum in Papua New Guinea, a region of intense perennial transmission.

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 (FST) 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.

Comparison of Plasmodium falciparum allelic frequency distribution in different endemic settings by high-resolution genotyping.

BACKGROUND: The diversity of genotyping markers of Plasmodium falciparum depends on transmission intensity. It has been reported that the diversity of the merozoite surface protein 2 (msp2) is greater in areas of high compared to low endemicity, however, results for msp1 were inconsistent. These previous reports relied on low resolution genotyping techniques. METHODS: In the present study, a high-resolution capillary electrophoresis-based technique was applied to genotype samples from areas of different endemicity in Papua New Guinea and Tanzania. For both endemic settings, the diversity of msp1 and msp2 was investigated; the mean multiplicity of infection (MOI) and the FST values were determined to investigate whether more accurate sizing generates different results. RESULTS AND CONCLUSION: The results of the present study confirmed previous reports of a higher mean MOI for both marker genes and increased genetic diversity in areas of higher endemicity as estimated by the total number of distinct alleles for msp2. For msp1 a minor increase in diversity was observed. Measures of between population variance in allele frequencies (FST) indicated little genetic differentiation for both marker genes between the two populations from different endemic settings. MOI adjusted for the probability of multiple infections sharing the same allele was estimated by using the msp2 allele frequency distribution and the distribution of observed numbers of concurrent infections. For the high-resolution typing technique applied in this study, this adjustment made little difference to the estimated mean MOI compared to the observed mean MOI.

Three different Plasmodium species show similar patterns of clinical tolerance of malaria infection.

BACKGROUND: In areas where malaria endemicity is high, many people harbour blood stage parasites without acute febrile illness, complicating the estimation of disease burden from infection data. For Plasmodium falciparum the density of parasitaemia that can be tolerated is low in the youngest children, but reaches a maximum in the age groups at highest risk of infection. There is little data on the age dependence of tolerance in other species of human malaria. METHODS: Parasite densities measured in 24,386 presumptive malaria cases at two local health centres in the Wosera area of Papua New Guinea were compared with the distributions of parasite densities recorded in community surveys in the same area. We then analyse the proportions of cases attributable to each of Plasmodium falciparum, P. vivax, and P. malariae as functions of parasite density and age using a latent class model. These attributable fractions are then used to compute the incidence of attributable disease. RESULTS: Overall 33.3%, 6.1%, and 0.1% of the presumptive cases were attributable to P. falciparum, P. vivax, and P. malariae respectively. The incidence of attributable disease and parasite density broadly follow similar age patterns. The logarithm of the incidence of acute illness is approximately proportion to the logarithm of the parasite density for all three malaria species, with little age variation in the relationship for P. vivax or P. malariae. P. falciparum shows more age variation in disease incidence at given levels of parasitaemia than the other species. CONCLUSION: The similarities between Plasmodium species in the relationships between parasite density and risk of attributable disease are compatible with the hypothesis that pan-specific mechanisms may regulate tolerance to different human Plasmodia. A straightforward mathematical expression might be used to project disease burden from parasite density distributions assessed in community-based parasitological surveys.

High sensitivity detection of Plasmodium species reveals positive correlations between infections of different species, shifts in age distribution and reduced local variation in Papua New Guinea.

BACKGROUND: When diagnosed by standard light microscopy (LM), malaria prevalence can vary significantly between sites, even at local scale, and mixed species infections are consistently less common than expect in areas co-endemic for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. The development of a high-throughput molecular species diagnostic assay now enables routine PCR-based surveillance of malaria infections in large field and intervention studies, and improves resolution of species distribution within and between communities. METHODS: This study reports differences in the prevalence of infections with all four human malarial species and of mixed infections as diagnosed by LM and post-PCR ligase detection reaction-fluorescent microsphere (LDR-FMA) assay in 15 villages in the central Sepik area of Papua New Guinea. RESULTS: Significantly higher rates of infection by P. falciparum, P. vivax, P. malariae and Plasmodium ovale were observed in LDR-FMA compared to LM diagnosis (p < 0.001). Increases were particularly pronounced for P. malariae (3.9% vs 13.4%) and P. ovale (0.0% vs 4.8%). In contrast to LM diagnosis, which suggested a significant deficit of mixed species infections, a significant excess of mixed infections over expectation was detected by LDR-FMA (p < 0.001). Age of peak prevalence shifted to older age groups in LDR-FMA diagnosed infections for P. falciparum (LM: 7-9 yrs 47.5%, LDR-FMA: 10-19 yrs 74.2%) and P. vivax (LM: 4-6 yrs 24.2%, LDR-FMA: 7-9 yrs 50.9%) but not P. malariae infections (10-19 yrs, LM: 7.7% LDR-FMA: 21.6%). Significant geographical variation in prevalence was found for all species (except for LM-diagnosed P. falciparum), with the extent of this variation greater in LDR-FMA than LM diagnosed infections (overall, 84.4% vs. 37.6%). Insecticide-treated bednet (ITN) coverage was also the dominant factor linked to geographical differences in Plasmodium species infection prevalence explaining between 60.6% - 74.5% of this variation for LDR-FMA and 81.8% - 90.0% for LM (except P. falciparum), respectively. CONCLUSION: The present study demonstrates that application of molecular diagnosis reveals patterns of malaria risk that are significantly different from those obtained by standard LM. Results provide insight relevant to design of malaria control and eradication strategies.

Protective Immunity against Severe Malaria in Children Is Associated with a Limited Repertoire of Antibodies to Conserved PfEMP1 Variants.

Extreme diversity of the major Plasmodium falciparum antigen, PfEMP1, poses a barrier to identifying targets of immunity to malaria. Here, we used protein microarrays containing hundreds of variants of the DBLα domain of PfEMP1 to cover the diversity of Papua New Guinean (PNG) parasites. Probing the plasma of a longitudinal cohort of malaria-exposed PNG children showed that group 2 DBLα antibodies were moderately associated with a lower risk of uncomplicated malaria, whereas individual variants were only weakly associated with clinical immunity. In contrast, antibodies to 85 individual group 1 and 2 DBLα variants were associated with a 70%-100% reduction in severe malaria. Of these, 17 variants were strong predictors of severe malaria. Analysis of full-length PfEMP1 sequences from PNG samples shows that these 17 variants are linked to pathogenic CIDR domains. This suggests that whereas immunity to uncomplicated malaria requires a broad repertoire of antibodies, immunity to severe malaria targets a subset of conserved variants. These findings provide insights into antimalarial immunity and potential antibody biomarkers for disease risk.

Antibody responses to Plasmodium vivax Duffy binding and Erythrocyte binding proteins predict risk of infection and are associated with protection from clinical Malaria.

BACKGROUND: The Plasmodium vivax Duffy Binding Protein (PvDBP) is a key target of naturally acquired immunity. However, region II of PvDBP, which contains the receptor-binding site, is highly polymorphic. The natural acquisition of antibodies to different variants of PvDBP region II (PvDBPII), including the AH, O, P and Sal1 alleles, the central region III-V (PvDBPIII-V), and P. vivax Erythrocyte Binding Protein region II (PvEBPII) and their associations with risk of clinical P. vivax malaria are not well understood. METHODOLOGY: Total IgG and IgG subclasses 1, 2, and 3 that recognize four alleles of PvDBPII (AH, O, P, and Sal1), PvDBPIII-V and PvEBPII were measured in samples collected from a cohort of 1 to 3 year old Papua New Guinean (PNG) children living in a highly endemic area of PNG. The levels of binding inhibitory antibodies (BIAbs) to PvDBPII (AH, O, and Sal1) were also tested in a subset of children. The association of presence of IgG with age, cumulative exposure (measured as the product of age and malaria infections during follow-up) and prospective risk of clinical malaria were evaluated. RESULTS: The increase in antigen-specific total IgG, IgG1, and IgG3 with age and cumulative exposure was only observed for PvDBPII AH and PvEBPII. High levels of total IgG and predominant subclass IgG3 specific for PvDBPII AH were associated with decreased incidence of clinical P. vivax episodes (aIRR = 0.56-0.68, P≤0.001-0.021). High levels of total IgG and IgG1 to PvEBPII correlated strongly with protection against clinical vivax malaria compared with IgGs against all PvDBPII variants (aIRR = 0.38, P<0.001). Antibodies to PvDBPII AH and PvEBPII showed evidence of an additive effect, with a joint protective association of 70%. CONCLUSION: Antibodies to the key parasite invasion ligands PvDBPII and PvEBPII are good correlates of protection against P. vivax malaria in PNG. This further strengthens the rationale for inclusion of PvDBPII in a recombinant subunit vaccine for P. vivax malaria and highlights the need for further functional studies to determine the potential of PvEBPII as a component of a subunit vaccine for P. vivax malaria.

Mass drug administration trial to eliminate lymphatic filariasis in Papua New Guinea: changes in microfilaremia, filarial antigen, and Bm14 antibody after cessation.

Laboratory tools to monitor infection burden are important to evaluate progress and determine endpoints in programs to eliminate lymphatic filariasis. We evaluated changes in Wuchereria bancrofti microfilaria, filarial antigen and Bm14 antibody in individuals who participated in a five-year mass drug administration trial in Papua New Guinea. Comparing values before treatment and one year after four annual treatments, the proportion of microfilaria positive individuals declined to the greatest degree, with less marked change in antibody and antigen rates. Considering children as sentinel groups who reflect recent transmission intensity, children surveyed before the trial were more frequently microfilaria and antibody positive than those examined one year after the trial stopped. In contrast, antigen positive rates were similar in the two groups. All infection indicators continued to decline five years after cessation of mass drug administration; Bm14 antibody persisted in the greatest proportion of individuals. These data suggest that Bm14 antibody may be a sensitive test to monitor continuing transmission during and after mass drug administration aimed at eliminating transmission of lymphatic filariasis.

Seroprevalence of antibodies to parvovirus B19 among children in Papua New Guinea.

Parvovirus B19 (B19) is a common childhood infection that has recently been found to be associated with severe anemia in Papua New Guinean children. Population surveys were performed in 15 villages in Maprik district, East Sepik Province, Papua New Guinea in 2005. Plasma samples collected from children less than 10 years of age were tested for IgM and IgG antibodies to B19 by enzyme immunoassay. The prevalence of IgG antibody to B19 was 53.8% and ranged from 20% in those less than one year of age to 85.5% in those nine years of age. Considerable variation in IgG prevalence was observed between study areas, indicating complex patterns of transmission. Prevalence of IgM antibody to B19 was 1.5%. This study confirms that B19 infection is common among children in this tropical area. With 19.5% of children one year of age showing evidence of previous infection, any preventive measures should be targeted at the very young.